Interaction of Corynebacterium diphtheriae

with host cells

(Interaktion von Corynebacterium diphtheriae mit Wirtszellen)

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von
Lisa Ott

aus Nürnberg
 Als Dissertation genehmigt von der Naturwissen-
schaftlichen Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg

Tag der mündlichen Prüfung: 19.12.2012

Vorsitzender
der Promotionskomission: Prof. Dr. Johannes Barth

Erstberichterstatter: Prof. Dr. Andreas Burkovski

Zweitberichterstatter: Prof. Dr. Uwe Sonnewald

 Table of contents

Table of contents

1 Introduction............................................................................................................4
1.1 The genus Corynebacterium .................................................................................................... 4
1.2 Pathogenicity of Corynebacterium diphtheriae ...................................................................... 7
1.2.1 Adhesion properties of C. diphtheriae ............................................................................. 7
1.2.2 Factors involved in the invasion process of C. diphtheriae ........................................ 12
1.2.3 C. diphtheriae and host immune response .................................................................... 15
1.2.4 Phagocytosis and induction of apoptosis ..................................................................... 17

2 Aim of the work .................................................................................................... 23

3 Publications ......................................................................................................... 24
3.1 Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell
surface organization, adhesion and internalization in epithelial cells (Ott et al, 2010a) ............. 24
3.2 Strain-specific differences in pili formation and the interaction of Corynebacterium
diphtheriae with host cells (Ott et al, 2010b).................................................................................... 34
3.3 The complete genome sequence of Corynebacterium pseudotuberculosis FRC41
isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-
regulatory networks contributing to virulence (Trost et al, 2010b) ............................................... 47
3.4 Impact of improved potassium accumulation on pH homeostasis, membrane potential
adjustment and survival of Corynebacterium glutamicum (Ochrombel et al, 2011) ................... 65
3.5 Evaluation of invertebrate infection models for pathogenic corynebacteria (Ott et al,
2012a) ................................................................................................................................................... 73
3.6 Contour and persistence length of Corynebacterium diphtheriae pili by atomic force
microscopy (Rheinlaender et al, 2012) ............................................................................................. 83
3.7 Induction  of  the  NFĸB  signal  transduction  pathway  in  response  to  Corynebacterium
diphtheriae infection (Ott et al, 2012b) ............................................................................................. 94

4 Summary/Zusammenfassung........................................................................... 122
4.1 Summary ................................................................................................................................. 122
4.2 Zusammenfassung ................................................................................................................ 124

5 References ......................................................................................................... 126

6 Curriculum Vitae ................................................................................................ 135

7 Danksagung ....................................................................................................... 139

3
 Introduction

1 Introduction

1.1 The genus Corynebacterium

Corynebacteria belong to the class of Actinobacteria (high G+C Gram-positive bacteria) and

comprise a collection of morphologically similar, irregular- or clubbed-shaped microorganisms

(Ventura et al, 2007; Zhi et al, 2009). Since 1970, corynebacteria are dedicated to the CMN

group (Corynebacterium, Mycobacterium, Nocardia), which is based on the molecular

similarity of cell wall components. Figure 1 shows the typical v-shaped cell dimers, which

occur during cell division of corynebacteria. This is also called snapping division (Barksdale,

1970).

Fig. 1. Differential staining of infection of non-phagocytic epithelial cells with non-

toxigenic C. diphtheriae. (A) extracellular C. diphtheriae are depicted in green; (B)
internalized bacteria are indicated in dark red; adherent bacteria in light red; (C) Detroit 562
cells appear in blue; extracellular bacteria in orange; intracellular bacteria in light red. Scale
bars: 20 µm (Ott et al, 2010b).

Almost all members of the genus are characterized by complex cell envelope architecture: in

addition to cytoplasmic membrane and peptidoglycan layer, an outer lipid layer is observable,

which is covalently linked to arabinogalactan and mainly composed of mycolic acids. In some

cases the outer membrane is additionally covered by an ordered S- (surface-) layer of proteins

(Bayan et al, 2003) or complex protein mixtures are found (Marienfeld et al, 1997) (Hermann

et al, 2000) (Hansmeier et al, 2007; Hansmeier et al, 2006a; Hansmeier et al, 2006b). Figure 2

presents the mycobacterial outer cell envelope, which is similar to that of corynebacteria.

4
 Introduction

Fig. 2. Cryo-electron microscopy of the mycobacterial cell envelope. (a) The

mycobacterial outer membrane is visualized by cryosectioning and (b) cryo-electron
tomography. The periplasmic space between the outer membrane and the cytoplasmic
membrane contains the layers of the arabinogalactan-peptidoglycan polymer (indicated in blue
in the 3-D representation). Scale bars: 50 nm (Niederweis et al, 2010).

To date, the genus Corynebacterium comprises 88 species (Bernard, 2012), including soil

bacteria with biotechnological importance (Corynebacterium glutamicum), commensals found

on skin of humans and animals as well as pathogens (Corynebacterium diphtheriae,

Corynebacterium ulcerans and Corynebacterium pseudotuberculosis). C. diphtheriae is the

classical etiological agent of diphtheria and the type species of the genus Corynebacterium

(Lehmann & Neumann 1896; Barksdale, 1970). Pathogenic corynebacteria are mostly not

highly virulent, and only immune-compromised patients get infected. However, virulence can

be   dramatically   increased   when   bacteria   are   lysogenized   by   β-corynebacteriophages, which

encode the diphtheria toxin and are integrated into the genome of toxigenic strains. C.

ulcerans and C. pseudotuberculosis are also able to produce the diphtheria toxin due to

infection with tox gene-carrying corynebacteriophages (Groman et al, 1984; Cianciotto &

Groman, 1996).

C. ulcerans is a commensal in domestic and wild animals (Schuhegger et al, 2009;

Dixon, 2010; Sykes et al, 2010) that may serve as reservoir for zoonotic infections with a

diphtheria-like illness. Transmission occurs from animals to humans and usually adults get

infected. High risk of infection is given by consuming raw milk (Bostock et al, 1984; Hart, 1984)

or close contact with domestic animals (Wagner et al, 2010); no person to person transmission

was reported up to now.

5
 Introduction

C. pseudotuberculosis is the etiological agent of caseous lymphadenitis in sheep and

goats throughout the world (Dorella et al, 2006; Baird & Fontaine, 2007). Infections due to C.

pseudotuberculosis are rare in humans, but there are some cases of suppurative

lymphadenitis in patients with close contact with sheep. Despite the putative presence of a

diphtheria toxin, classical diphtheria of respiratory tract or skin is not observed. It is important

to know that C. pseudotuberculosis is able to proliferate and persist in macrophages, thus

escaping the immune response of the host since it is a facultative intracellular pathogen

(McKean et al, 2005; Dorella et al, 2006) (Fig. 3).

Fig. 3. Transmission of toxigenic corynebacteria. Transmission of C. diphtheriae from

person to person occurs by close physical contact. Respiratory diphtheria is mainly
disseminated by respiratory droplets but also smear infections are possible, mostly in case of
cutaneous diphtheria. C. ulcerans infections occur via close physical contact with pets and
farm animals or animal bites, while man-to-man transmission was not reported. C.
pseudotuberculosis is transferred to humans from infected cattle, especially sheep and goats,
by close physical contact (Burkovski, 2012).

Based on its medical importance as etiological agent of diphtheria, C. diphtheriae was one of

the first Corynebacterium species for which genome sequences became available (Cerdeno-

Tarraga et al, 2003). With the development of high throughput sequencing techniques, to date

numerous genome sequences of pathogenic corynebacteria are available, including those of

Corynebacterium aurimucosum (Trost et al, 2010a), Corynebacterium bovis (Schröder et al,

6
 Introduction

2012a), various C. diphtheriae strains (Sangal et al, 2012a; Sangal et al, 2012b; Trost et al,

2012), C. jeikeium (Tauch et al, 2005), Corynebacterium kroppenstettii (Tauch et al, 2008a),

several C. pseudotuberculosis strains (Trost et al, 2010b; Cerdeira et al, 2011a; Cerdeira et al,

2011b; Lopes et al, 2012; Pethick et al, 2012a; Pethick et al, 2012b; Silva et al, 2011),

Corynebacterium resistens (Schröder et al, 2012b), two C. ulcerans strains (Trost et al, 2011)

and Corynebacterium urealyticum (Tauch et al, 2008b). This wealth of genome information

now provides a basis for the characterization of corynebacterial virulence factors and

corresponding genes at a molecular level.

1.2 Pathogenicity of Corynebacterium diphtheriae

C. diphtheriae causes diphtheria of the upper respiratory tract, which is characterized by sore

throat, low fever and malaise. Symptoms range from mild pharyngitis to severe hypoxia with

pseudomembrane formation due to the toxin. In advanced cases swelling of lymph nodes

results in strong swelling of the neck tissue (bull neck diphtheria) (Hadfield et al, 2000).

Diphtheria also occurs on skin (cutaneous diphtheria), which outnumbers the classical

respiratory diphtheria in tropical areas. Besides these infections, toxigenic and non-toxigenic

C. diphtheriae strains are also associated with systemic infections, e.g. endocarditis,

osteomyelitis, splenic abscesses, meningitis and septic arthritis (Hirata et al, 2008; Puliti et al,

2006), indicating that C. diphtheriae is able to colonize not only epithelia but also deeper parts

of the body.

1.2.1 Adhesion properties of C. diphtheriae

To date, experimental data concerning host pathogen interaction of eukaryotic cell with C.

diphtheriae are limited. Best investigated adhesion factors might be pili structures on the

surface of C. diphtheriae, which are covalently attached to the cell wall peptidoglycan (Navarre

& Schneewind, 1999). Pili formation requires enzymes with transpeptidase activity and sorting

signals of surface proteins (Fischetti et al, 1990; Schneewind et al, 1995; Schneewind et al,

1992). The genome of C. diphtheriae NCTC 13129 encodes three distinct pili clusters

7
 Introduction

(spaABC, spaDEF, spaGHI), which are organized together with their sortase encoding genes

(srt, srtB, srtC, srtD, srtE and srtF) (Ton-That & Schneewind, 2003). The corynebacterial pili

consist of a major pilus subunit SpaA (SpaD and SpaH, respectively), a minor subunit SpaB

(SpaE and SpaG, respectively) and the tip protein SpaC (SpaF and SpaI, respectively) (Ton-

That & Schneewind, 2003). SrtA specifically catalyzes the covalent crosslinking of single pilin

monomers, whereas the only housekeeping sortase SrtF is necessary for efficient anchoring of

pili to the cell wall.

Mandlik and co-workers showed (2007) that only the SpaA-type pili contribute to

corynebacterial adherence to human pharyngeal cells (Detroit 562), whereas the SpaD and

SpaH-type pili support adhesion to laryngeal (HEp-2) and lung cells (A549). Mutant strains that

lacked all sortases showed decreased adhesion rates to different epithelial cell lines. In

contrast, the deletion of srtA alone impaired binding to pharyngeal cells (Mandlik et al, 2007).

In addition, strains that express only the SpaA-type pili were able to adhere to pharyngeal cells

properly, but showed deceased adhesion to any other cell line compared to the wild type.

Furthermore, it was shown that the minor pilin subunits do not only decorate the pilus shaft but

are also distributed in clusters all over the bacterial surface. The minor pilins that are directly

anchored to the cell wall are sufficient to mediate specific host cell adhesion, which is in

accordance to data reported on streptococci (Dramsi et al, 2006), indicating that this

observation is a conserved feature in Gram-positive pathogens. The deletion of either spaB or

spaC resulted in significantly lower adhesion rates, and the spaBC double mutant revealed

marginal binding to Detroit 562 cells.

When Ott and co-workers investigated the surface structures of the different wild type

strains by atomic force microscopy, this approach revealed significant differences between

various isolates. Interestingly, not all strains studied by Ott and co-workers showed pili-like

structures on the surface and, on top of that, the pili-like structures observed by atomic force

microscopy differed in length and number (Ott et al, 2010b). Some strains lacked pili

completely, while others revealed short, spike-like pili or long, hair-like protrusions, which had

different biophysical properties (Rheinlaender et al, 2012). Furthermore, adhesion and pili

8
 Introduction

formation were not strictly coupled, as strain ISS4060, which lacked pili-like structures showed

adhesion rates comparable to those determined for strain ISS3319, which revealed spike-like

pili (Ott et al, 2010b). In accordance with the pili repertoire observed, RNA hybridization

experiments revealed distinct differences in the expression pattern of pili subunits for the

strains investigated. All detected pili subunits belonged to cluster spaABC or spaGHI. The lack

of PCR products of cluster spaDEF and the absence of SpaD signal in Western blotting

experiments suggested that the spaDEF cluster is absent in these strains and, furthermore,

that the genetic repertoire of C. diphtheriae is rather variable. This idea was further supported

by a recent genome study, in which 12 different C. diphtheriae genome sequences were

analysed. At least two pilus gene clusters in each of the sequenced genomes, with C.

diphtheriae HC04 harboring four pilus gene clusters, were found. Three strains lacked cluster

spaGHI and in one strain cluster spaABC was absent (Trost et al, 2012).

Besides different pili formation, Ott and co-workers observed that investigated C.

diphtheriae isolates differed significantly in their interaction with eukaryotic cell lines. While

around 0.3 to 7% of the inoculated bacteria adhere to the human pharyngeal carcinoma cell

line Detroit 562, up to 10 fold higher adhesion rates were observed for C. diphtheriae to HeLa

cells (Ott et al, 2012b), indicating that adhesion efficiency is not only strain-specific but also

cell line-specific. Interestingly, the toxigenic strain studied, DSM43989, showed always the

lowest adhesion rates irrespective of the host cell line. Furthermore, scanning electron

analysis of HeLa cells infected with dead toxigenic bacteria showed significantly more bacteria

attached to HeLa cells than after infection of HeLa cells with vital toxigenic bacteria. This leads

to the assumption that the toxin might have a detrimental effect on the cells, which itself

impairs pathogen-host interaction. In summary, the reasons for strain-specific differences in

adhesion rates besides pili remain unclear and the characterization of bacterial surface

molecules is necessary for better understanding of the adhesion and infection process.

Interestingly, C. diphtheriae strains display two distinct adherence patterns on HEp-2

cell monolayers, termed localized (LA) and diffuse (DA). Localized adherence is characterized

by small clusters of bacteria, so-called microcolonies, while diffuse adherence is expressed in

9
 Introduction

random distribution over the surface of the cells. This is further indicating that more than one

adhesion mechanism is existing.

One important adhesin of C. diphtheriae, which was already described, is the non-

fimbrial surface protein 67-72p (Colombo et al, 2001) that is encoded by a single gene

(dip0733) (Sabbadini et al, 2012). The 67-72p protein (DIP0733) enables C. diphtheriae to

recognize and bind specifically to human erythrocyte receptors, a process named

hemagglutination, which capacitates C. diphtheriae to spread throughout the body via the

blood. However, 67-72p protein is not only able to bind human erythrocytes but also HEp-2

cells (Moreira Lde et al, 2003), and binding to this epithelial cell line was effectively blocked by

anti-67-72p IgG antibodies (Hirata et al, 2004). Interestingly, a negative correlation between

bacterial adhesion properties to HEp-2 cells and erythrocytes of the investigated strains was

found: strains with low hemagglutinating activity showed high adhesion rates to HEp-2 cells,

and vice versa.

Besides proteins as adhesion factors, C. diphtheriae exhibits a 10 kDa lipoglycan on its

surface, termed CdiLAM. The basic structure of CdiLAM shows similarity to mycobacterial

LAM. Unlike the non-fimbrial adhesin 67-72p, CdiLAM does not bind to human erythrocytes,

but it binds HEp-2 cells, and pre-incubation of bacterial cells with anti-CdiLAM IgG antibodies

blocked the adherence of C. diphtheriae strain 241 to HEp-2 cells effectively (Moreira et al,

2008). Additionally, purified CdiLAM was able to inhibit the adherence of C. diphtheriae to

HEp-2 cells. Thus, the CdiLAM functioning as an adhesin to human respiratory cells can be

regarded as a potential virulence factor of C. diphtheriae.

While in the upper examples the pathogen factor is clear, this is not the case for some

other described interactions. One example is fibrinogen: fibrinogen is a major protein in human

plasma and it is predominantly involved in the coagulation cascade through its conversion into

insoluble fibrin. Inflammatory or systemic infections lead to upregulation of fibrinogen

synthesis, indicating its function in pathogen control (Sabbadini et al, 2010). The main role of

fibrinogen is in blood clot formation through its conversion into insoluble fibrin which forms the

matrix of the clot. The conversion of fibrinogen to fibrin is catalyzed by a bacterial enzyme

10
 Introduction

termed coagulase that reacts with a cofactor found in blood plasma, and it is produced in the

most virulent Staphylococcus strains (Duthie, 1954) and Yersinia pestis (Beesley et al, 1967;

Rivera et al, 2007). C. diphtheriae is also able to bind to fibrinogen and convert fibrinogen to

fibrin (Sabbadini et al, 2012). Non-toxigenic and toxigenic strains formed bacterial aggregates

in the presence of plasma and the fibrinogen binding occurred in varying intensities. The

fibrinogen polymerisation process seems also to build a fibrin layer on the bacterial cell wall,

and the binding of fibrin to the surface of C. diphtheriae may be an efficient method to avoid

phagocytosis.

Bacterial pathogens do not only decorate their surface with fibrin to hide from host

immune system, but they also use sugar moieties as camouflage and sialidases or

neuramidases play a role in virulence of many pathogenic bacteria. These enzymes belong to

a class of glycosyl hydrolases that catalyze the removal of terminal sialic acid residues from a

variety of glycoconjugates of the host surface (Vimr, 1992). C. diphtheriae exposes sialic acids

on its surface but, to date, no genes involved in sialic acid decoration or metabolism have

been detected. By a search of the GenBank sequence database a putative exo-sialidase

NanH (DIP0543) from C. diphtheriae NCTC 13129 was identified. NanH is able to hydrolyze

2´-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MU-Neu5Ac) as a substrate (Kim et al,

2010). Furthermore, C. diphtheriae NanH showed trans-sialylation activity using sialyl-α2,3-

lactose and sialyl-α2,6-lactose as donors. Additionally, it could catalyze the transfer of sialic

acis from other sialoconjugates to MU-Gal and the sialidase-mediated transglycosilation

reaction using sialic acid-conjugated free oligosaccharides and glycoproteins as donors.

Accordingly, C. diphtheriae may transport sialic acids hydrolysed by extracellular sialidases for

synthesis of sialic acid derivates. But it is still to be clarified wether NanH is involved in sialic

acid decoration or not (Kim et al, 2010). Moreover, no host cell molecules involved in

corynebacterial adherence were identified so far.

11
 Introduction

1.2.2 Factors involved in the invasion process of C. diphtheriae

C. diphtheriae was generally considered as an extracellular colonizer, with the diphtheria toxin

as the main virulence factor. The upper respiratory tract is the primary site of colonization by

C. diphtheriae, and penicillin treatment failure of severe pharyngitis/tonsillitis could only

partially be explained by penicillin tolerance (von Hunolstein et al, 2002). The occurrence of

diphtheria among vaccinated adults in Rio de Janeiro, as well as an increasing number of

cases of endocarditis caused by non-toxigenic strains, indicates the possibility of other

virulence strategies besides the toxin (Mattos-Guaraldi et al, 2000; Mattos-Guaraldi et al,

2001) and gives rise to the idea that the bacteria are not only able to adhere to host cells of

the respiratory tract, but are also able to gain access into deeper tissues.

Hirata and co-workers demonstrated invasion of HEp-2 cells at varying levels with a

range from ~0.2% to 2.6% for different isolates. Interestingly, there was no correlation

between adhesion and invasion rates of the strains, and the internalization process of the

different strains investigated was completely blocked by cytochalasin E, supporting the idea

that cytoskeletal rearrangements are required for bacterial entry. Furthermore, Hirata and co-

workers showed that actin polymerization, along with a phosphotyrosine signaling event,

seems to be a relevant mechanism during the invasion process in cultured respiratory cells

(Hirata et al, 2008).

Additionally, C. diphtheriae was examined as to its ability to adhere to and enter into

the pharyngeal carcinoma cell line Detroit 562 for better understanding of the kinetics of entry

of non-toxigenic isolates into epithelial cells (Bertuccini et al, 2004). The invasion efficiency of

C. diphtheriae in HEp-2 cells was compared with that observed in Detroit 562 cells by using

gentamicin protection assays. Interestingly, at start of infection, there were significantly more

viable bacteria associated with HEp-2 cells than with Detroit 562 cells. After 48 h, there were

still viable bacteria in Detroit 562 cells but not in HEp-2 cells. In conclusion, C. diphtheriae

shows lower adhesion rates to and invasion rates into Detroit 562 cells compared to HEp-2

cells but it is able to survive significantly longer within Detroit 562 cells. This might indicate

better adaption of C. diphtheriae to its target cell. Cytochalasin D showed a dose-dependent

12
 Introduction

inhibitory effect on the internalization process, pointing to the involvement of F-actin in the

bacterial entry. The bacterial binding seems to induce intracellular signal transduction

responsible for actin microfilament rearrangements (Rosenshine et al, 1992). Staurosporine,

an inhibitor of different classes of protein kinases, seemed to contribute to C. diphtheriae

internalization, indicating that the activation of protein kinases negatively controlled bacterial

uptake. However, the possibility to enter the host cell, to hide in epithelial cells, even at low

numbers, and to persist at least 48 h, gives the opportunity to escape from the hosts immune

response and antibiotic treatment.

Investigations of the invasiveness of six non-toxigenic strains as well as the tox+ strain

DSM43989 by Ott and co-workers were carried out by gentamicin protection assays. By this

method, Ott and co-workers demonstrated that the toxigenic strain DSM43989 has the lowest

invasion rate at 2 h post infection. Interestingly, the toxigenic strain showed, in general, lower

adhesion and invasion rates compared to non-toxigenic strains. As in adhesion assays, these

strains differed significantly in invasion rates, ranging from ~0.02% to 0.06% (Ott et al, 2010b).

As in the case of Hirata and co-workers (Hirata et al, 2002), the comparison of these strains in

respect to adhesion and internalization rates revealed that these processes were not strictly

coupled. Additionally, Ott and co-workers observed V-shaped C. diphtheriae dimers within the

host cell by immune-fluorescence microscopy indicating that C. diphtheriae is able to replicate

within the host cells and growth and elimination are parallel processes (Ott et al, 2010b). While

host cell receptors and invasion-associated proteins of the pathogen are still unknown,

bacterial virulence factors beside the toxin have been at least partially characterized on the

molecular level, as described before. In contrast, the molecular mechanism of invasion is more

or less unclear.

Consequently, Ott and co-workers focused their work on the surface-associated protein

DIP1281, a member of the NlpC/P60 family (Anantharaman & Aravind, 2003), which was

annotated as hypothetical invasion-associated protein, and was found as part of the C.

diphtheriae secretome (Hansmeier et al, 2006a). Two non-toxigenic wild type strains as well

as the corresponding DIP1281 mutant strains were tested regarding adhesion to and

13
 Introduction

internalization into Detroit 562 cells (Ott et al, 2010a). In these experiments, strain ISS3319

showed higher adhesion rates to Detroit 562 cells, compared to strain ISS4060, while no

significant differences of the number of intracellular bacteria were observed. DIP1281 mutant

strains lacked the ability to adhere to host cells almost completely and, in contrast to the

respective parental strains, no intracellular bacteria were detectable by using gentamicin

protection assays. Further characterization of the mutant strains revealed an increased size of

the single bacteria, an altered less club-like shape and formation of chains of bacteria rather

than the typical V-like cell division forms or palisades of clustered C. diphtheriae. Additionally,

the mutants showed an uneven speckled immuno-fluorescence staining, indicating an altered

surface structure compared to the wild type strains, which was confirmed by SDS-PAGE,

Western blot and 2D-PAGE of the surface proteomes. These clear differences between wild

type and mutant surface protein patterns were also found by using atomic force microscopy.

The wild type strains showed round elevations with defined structures, while the DIP1281

mutant strains lost this fine structure. The results of Ott and co-workers indicate that DIP1281

is predominantly involved in the organization of the outer protein layer of C. diphtheriae rather

than in the separation of the peptidoglycan of dividing bacteria. These rearrangements of the

outer membrane lead to the adhesion and invasion-negative phenotype of the corresponding

mutant strains (Ott et al, 2010a). Tsuge and co-workers reported similar observations in C.

glutamicum R, when the DIP1281 homolog cgR_1596 and other members of the NlpC/P60

protein familiy cgR_2070 were mutated (Tsuge et al, 2008).

Recent studies of Sabbadini and co-workers demonstrated the involvement of the

surface-exposed non-fimbrial 67-72p protein (DIP0733), previously described as

adhesin/hemagglutinin (Colombo et al, 2001; Hirata et al, 2004), in the internalization of C.

diphtheriae into HEp-2 cells (Sabbadini et al, 2012). Furthermore, the intracellular viability of

two different C. diphtheriae strains was significantly reduced after pretreatment of bacterial

cells with 67-72p antibodies, while incubation with 67-72p of HEp-2 cells or bacterial

suspensions also revealed decreased internalization rates. However, since the internalization

process was not completely blocked due to treatments with 67-72p antibodies, the

14
 Introduction

involvement of other bacterial factors in this process cannot be excluded (Sabbadini et al,

2012).

The identification of new, up to now unknown virulence factors might be fostered in

future, not only by the availability of various C. diphtheriae gene sequences (Cerdeno-Tarraga

et al, 2003; Sangal et al, 2012a; Sangal et al, 2012b; Trost et al, 2012), but also by the

establishment of easy to handle invertebrate infection model systems. The nematode

Caenorhabditis elegans and larvae of the greater wax moth Galleria mellonella were found to

be suitable in this respect (Ott et al, 2012a).

1.2.3 C. diphtheriae and host immune response

The innate immune system provides the first line of defense against infectious diseases;

anatomic barriers such as skin and optimized phagocytic cells (macrophages and neutrophils)

play a main role in innate immunity, together with a large battery of antimicrobial compounds

synthesized by the host cell. Thereby, most of the microorganisms infecting healthy

vertebrates are readily cleared within a few days after infection, before activating the adaptive

immune response. Invasion of microorganisms into vertebrate cells is initially recognized by

the innate immune system through a limited number of germline-encoded pattern-recognition

receptors (PRRs) (Akira et al, 2006), including the Toll-like receptors (TLRs) with the first

described member Toll. Members of the TLR family are able to recognize Gram-negative cell

wall components, such as lipopolysaccharide (LPS). Gram-positive bacterial cell walls do not

contain LPS but LTA (lipoteichoic acids), or in case of C. diphtheriae mycolic acids that might

also stimulate the innate immune system. The activation of intracellular signaling pathways

leads to the transcription of genes involved in antimicrobial host defense mechanisms. This

was first investigated for C. diphtheriae in respect to septic arthritis. The clinical pattern of

human diphtheroid arthritis can occur as an acute, purulent process or as a chronic arthritis

with plasmacytic or histiocytic inflammation. Furthermore, patients are in general bad condition

or have a preexisting disease that impairs the immune system (Tiley et al, 1993; Patey et al,

1997; Barakett et al, 1993; Guran et al, 1979). Interestingly, non-toxigenic C. diphtheriae

15
 Introduction

strains are able to induce lethal infections in mice by intravenous inoculation of CD-1 mice in a

dose-dependent and strain-specific manner (Puliti et al, 2006). Clinical signs of arthritis

occurred 3 days post infection, most frequently in ankles and wrists. By quantitative monitoring

of C. diphtheriae, Puliti and co-workers also demonstrated that C. diphtheriae bacteria were

cleared from the bloodstream within 5 days after infection, but the bacteria are able to persist

in kidneys and spleens for more than two weeks after infection. Additionally, Puliti and co-

workers brought the evidence that the investigated non-toxigenic strains induce systemic and

local IL-6, IL-1 and TNF secretion, which might contribute to articular damage in cases of

septic arthritis. Interestingly, induction of high and early IFN- production was only observed in

mice infected with ISS3319, indicating that IFN- production counterbalances the

proinflammatory response.

Induction of host response was investigated in vitro on a more molecular level by Ott

and co-workers, using luciferase expressing reporter cell lines, monitoring the induction of

various host signaling pathways (Table 1). Furthermore, Ott and co-workers demonstrated that

different non-toxigenic C. diphtheriae wild   type   strains   are   able   to   induce   the   NFĸB  

transduction pathway in epithelial cells in a strain-specific manner; induction only occurs

during incubation with living bacteria and requires invasion of the bacteria into the epithelial

cells (Ott et al, 2012b).

16
 Introduction

Table 1: Induction of eukaryotic inflammatory pathways. Infection of epithelial cells

lentiviral transfected with luciferase-coupled reporter systems specific for inflammatory
pathways with different C. diphtheriae wild type strains indicated by - (no signal) and plus
(signal), with number of +s indicating signal strength; n.t. not tested.
HeLa Detroit 562

NFB STAT5RE AP-1 NFAT SIE NFB AP-1

ISS3319 ++ ++ ++ ++ - ++ -

ISS4060 +++ ++ ++ ++ - - -

ISS4746 + - - - n.t. ++ +

ISS4749 + - - - n.t. - -

DSM33988 ++ + - +++ n.t. - +

DSM43989 - - n.t. +++ n.t. - -

DSM44123 + (+) n.t. + n.t. - -

(Ott et al, 2012b and unpublished observation)

1.2.4 Phagocytosis and induction of apoptosis

Non-opsonic phagocytosis is mediated by phagocyte receptors that recognize structures on

microbial surfaces. There are three mechanisms of induction of non-opsonic phagocytosis

discussed: carbohydrate-protein interactions, protein-protein interactions and hydrophobic

interactions (Ofek et al, 1995). It is known that the diphtheria toxin can fully activate human

macrophages and phagocytosis was significantly impaired after pro-longed incubation with

diphtheria toxin (Saelinger et al, 1975; D'Onofrio & Paradisi, 1983). Dos Santos and co-

workers investigated the ability of homologous toxigenic and non-toxigenic C. diphtheriae

strains to be phagocytosed by the human macrophage cell line U937 in the absence of

immune serum (opsonins) and found that human macrophages can act differently in the way

of killing toxigenic and non-toxigenic C. diphtheriae strain. The presence of diphtheria toxin

seems to enhance adhesion to as well as internalization into U937 cells and, therefore, favors

non-opsonic phagocytosis (dos Santos et al, 2010). Interestingly, the non-toxigenic strain

adhered weakly to U937 cells, and only a very low number of bacteria were internalized,

indicating that C. diphtheriae might be able to escape non-opsonic phagocytosis by human

17
 Introduction

macrophages. In conclusion, there must be other receptors responsible for binding and

internalization of the tox+ strain than those responsible for binding of diphtheria toxin. Non-

opsonic phagocytosis of both strains toxigenic and non-toxigenic induced cytopathogenicity in

U937 cells; more than 42% of U937 cells were killed during infection with the tox+ strain (3h

post infection), while about 18% were killed during infection with the non-toxigenic strain.

Furthermore, C. diphtheriae seems to induce apoptosis and necrosis in human macrophages,

irrespective of the presence of diphtheria toxin. The toxigenic strain caused higher levels of

DNA fragmentation in U937 macrophages, but also the tox- strain was also able to induce

apoptosis and necrosis.

The 67-72p protein is not only able to interact with macrophages but also with epithelial

cells. This protein seems to be involved in the induction of apoptosis and necrosis in non-

phagocytic epithelial cells. The incubation of HEp-2 cells with 67-72p adsorbed microspheres

and homologous C. diphtheriae strain CDC-E8392 obtained cytoskeletal changes with

accumulations of polymerized actin in HEp-2 cells (Sabbadini et al, 2012). These results

indicate that the 67-72p protein (DIP0733) is as a bacterial factor involved in the mechanism of

recruitment of host actin filaments. Vacuolization and DNA fragmentation was observed after

12 h, and a high occurrence of apoptotic bodies appeared after 24h post treatment with 67-

72p. DNA condensation and aggregation were observed by DAPI staining of 67-72p treated

cells. Furthermore, the incubation of HEp-2 cells with 67-72p caused a reduction in cell

volume, which is a ubiquitous aspect of apoptosis, termed apoptotic volume decrease (Bortner

& Cidlowski, 2002). The ability of C. diphtheriae to kill non-phagocytic cells might be beneficial

for its survival and invasion of deeper tissue and thus to spread over the whole body. In

addition, the induction of apoptosis and necrosis during C. diphtheriae infection is not directly

coupled to diphtheria toxin production. The fact that human macrophages are able to

internalize C. diphtheriae bacilli in the absence of immune serum indicates that there is an

antibody-independent mechanism as resistance to infection. On the other hand, the

observation that the non-toxigenic strain was able to survive longer periods within the

18
 Introduction

macrophages might indicate that human macrophages may be not effective in killing C.

diphtheriae in the absence of opsonins (dos Santos et al, 2010).

Potassium transporters seem to be involved in one of these resistance mechanisms.

When Ochrombel and co-workers investigated potassium uptake systems in corynebacteria

(Ochrombel et al, 2011), they found that pathogenic corynebacteria such as C. diphtheriae and

C. jeikeium exhibit more potassium transporters than non-pathogens, such as C. glutamicum.

Furthermore, C. glutamicum K+ transporter mutants were eliminated faster by macrophages

and less stress resistant. This is also true for C. diphtheriae K+ uptake mutants (Ott,

unpublished), which turn K+ uptake systems of pathogenic corynebacteria into potential

virulence factors with regard to survival in an acid environment, like the inside of

macrophages. Figure 4 gives an overview about all known corynebacterial K+ transporter

systems.

19
 Introduction

Fig. 4: Different types of potassium transporters in Actinobacteria. Proteins similar to

known potassium transporter subunits in Escherichia coli (Ecoli, Kch, KefB, TrkH, KdpA) or
Streptomyces coelicolor (Scoel, KcsA) were identified by genomicBlast searches against the
genomes of Corynebacterium aurimucosum (Cauri), Corynebacterium diphtheriae biotype
gravis NCTC13129 (Cdiph), Corynebacterium efficiens YS-314 (Ceffi), Corynebacterium
glutamicum ATCC 13032 (Cglut), Corynebacterium glutamicum R (CglutR), Corynebacterium
jeikeium K411 (Cjeik), Corynebacterium urealyticum DSM7109 (Curea), Corynebacterium
kroppenstedtii DSM44385 (Ckrop), Mycobacterium tuberculosis H37RV (Mtube),
Mycobacterium abscessus ATCC 19977 (Mabsc), Nocardia farcinica IFM 10152 (Nfarc),
Propionibacterium acnes KPA171202 (Pacne), Streptomyces avermitilis MA-4680 (Saver), S.
coelicolor A3(2) M145 (Scoel), Bifidobacterium longum NCC2705 (Blong) and Clavibacter
michiganensis subsp. michiganensis NCPPB 382 (Cmich) (Ochrombel et al, 2011).

20
 Introduction

In summary, the interaction of the Gram-positive pathogen C. diphtheriae with its host is much

more complex than initially expected. Up to now, several different interaction mechanisms

were elucidated (summarized in Figure 5), but there is still room for research, especially

regarding interactions at the molecular level. Based on the already described results and

ongoing research, C. diphtheriae might work as a model for other pathogenic, but molecular

biologically less addressable corynebacteria such as C. jeikeium.

macrophage

erythrocytes
phagocytosis

non-opsonic
phagocytosis
agglutination
hemagglutination

Fbn
fibrin fibrin
receptor
neuraminidase

67-72p adhesin / invasin

Corynebacterium diphtheriae

trans- AB toxin
SpaB
cdiLAM sialidase production
adhesin
SpaA
∆  DIP1281 sialidase
receptor
SpaC
adhesion toxin
SpaB receptor
receptor
SpaC
receptor signaling cascade

endocytosis

translocation
activation of inflammatory pathways
NFĸB,  NFAT,  AP-1, STAT5RE
protein synthesis

epithelial cell

21
 Introduction

Fig. 5. Infection properties of C. diphtheriae. The most prominent virulence factor of C.

diphtheriae is   the   diphtheria   toxin,   which   is   encoded   by   a   β-corynephage integrated in the
bacterial chromosome. This AB toxin is released by the bacterial cell and binds the toxin
receptor (HB-EGF) on the host´s surface. The toxin gets internalized by endocytosis, which
leads to translocation of the A-fragment and inhibition of the protein biosynthesis by binding of
the elongation factor 2. Best investigated adhesion factors of C. diphtheriae might be the pili
structures. Strain NCTC 13129 exhibits three different pilus types, the SpaABC type pilus is
depicted here exemplarily. SpaA forms the pilus shaft, SpaB is linked to SpaA via
transpeptidation and SpaC forms the tip of the pilus. SpaB and SpaC are able to bind
receptors on the surface of the host cell, which enables colonization of the cell. Besides pili
formation the non-fimbrial protein 67-72p (DIP0733) might be an important adhesin. Via this
protein C. diphtheriae is able to bind erythrocytes, a process called hemagglutination, which
facilitates C. diphtheriae to disseminate throughout the body via the blood. Furthermore, the
67-72p protein seems to be involved in the internalization process and might contribute to
apoptosis and necrosis of the host cell. Neuraminidase-secreting strains were also found to be
capable of agglutinating human erythrocytes with high titers. Neuraminidases or sialidases
belong to a class of glycosyl hydrolases that catalyze the removal of terminal sialic acid
residues from a variety of glycoconjugates of the host surface. The sialidase NanH is able to
hydrolyze 2´-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid as a substrate and showed
trans-sialylation activity using sialyl-α2,3-lactose and sialyl-α2,6-lactose as donors. But it is still
to be clarified wether NanH is involved in sialic acids decoration or not. Besides proteins as
adhesion factors C. diphtheriae exhibits a 10 kDa lipoglycan on its surface, the CdiLAM, which
binds HEp-2 cells and can be regarded as a potential virulence factor of C. diphtheriae.
Additionally, C. diphtheriae is able to bind to fibrinogen and convert fibrinogen to fibrin,
resulting in a fibrin layer on the bacterial surface that may be an efficient method to avoid
phagocytosis. Since C. diphtheriae was expected to be an extracellular colonizer it is very
important to know that C. diphtheriae is able to internalize into non-phagocytic cells in a strain-
specific manner. The invasion process seems to involve actin polymerization along with a
phosphotyrosine signaling event in cultured respiratory cells. Investigations of DIP1281
(annotated as invasion-associated protein) revealed the involvement in the organization of the
outer protein layer, which resulted in an adhesion- and invasion-negative phenotype of the
corresponding mutant strains. Furthermore, infection of epithelial cells leads to inflammatory
host  response,  especially  the  activation  of  the  NFĸB  transduction pathway. NFĸB  induction  is  
strictly dependent on intracellular bacteria, since bacterial adhesion was not sufficient to
activate inflammatory pathways. In contrast to the bacterial property to enter epithelial cells,
U937 human macrophages are able to ingest C. diphtheriae by non-opsonic phagocytosis. C.
diphtheriae is able to persist inside macrophages and to induce apoptosis and necrosis in the
cell.

22
 Aim of the work

2 Aim of the work

C. diphtheriae was generally considered as an extracellular pathogen colonizing the mucosal

surfaces on tonsils, larynx and pharynx, with the diphtheria toxin as the main virulence factor.

The fact that non-toxigenic C. diphtheriae strains can cause invasive and systemic infections

such as endocarditis, splenic abcesses, mycotic aneurysm, osteomyelitis, pneumonia and

septic arthritis (Isaac-Renton et al, 1981; Hirata et al, 2008; Puliti et al, 2006; Honma et al,

2009; Muttaiyah et al, 2011), indicates that there must be other virulence factors besides the

toxin. With the development of high throughput sequencing techniques, to date, numerous

genome sequences of pathogenic corynebacteria are available including various C.

diphtheriae strains (Sangal et al, 2012a; Sangal et al, 2012b; Trost et al, 2012). This gives new

opportunities of global analysis techniques in respect to genomics and proteomics and

background for experiments at the molecular level.

The aim of this study was to elucidate the interaction of C. diphtheriae with host cells with

regard to adhesion, invasion, pili formation and host cell response. To address this, infection

assays with eukaryotic cell lines were carried out, as well as insertions mutagenesis of

putative virulence factors, proteomics, immuno-fluorescence microscopy, scanning electron

microscopy and atomic force microscopy.

23
 Publications

3 Publications
3.1 Corynebacterium diphtheriae invasion-associated protein
(DIP1281) is involved in cell surface organization, adhesion and
internalization in epithelial cells (Ott et al, 2010a)

24
 Publications
Ott et al. BMC Microbiology 2010, 10:2
http://www.biomedcentral.com/1471-2180/10/2

RESEARCH ARTICLE Open Access

Corynebacterium diphtheriae invasion-associated

protein (DIP1281) is involved in cell surface
organization, adhesion and internalization
in epithelial cells
Lisa Ott1, Martina Höller1, Roman G Gerlach2,4, Michael Hensel2,5, Johannes Rheinlaender3, Tilman E Schäffer3,
Andreas Burkovski1*

Abstract
Background: Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin
production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we
investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-
associated protein.
Results: Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in
combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like
division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence
microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein
patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape
and surface structure but completely lack the ability to adhere to host cells and consequently invade these.
Conclusions: Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface
protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and
invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface.

Background diphtheria, but can cause other infections. Non-toxi-

Corynebacterium diphtheriae is the causative agent of genic strains have been increasingly documented [4-6]
diphtheria, a toxaemic localized infection of the respira- and found to be the cause of invasive diseases such as
tory tract. By vaccination diphtheria is well-controlled in endocarditis, bacteraemia, pneumonia, osteomyelitis,
e. g. Western Europe [1-3]; however, this disease is still spleen abscesses, and septic arthritis ([7] and references
a cause of morbidity and mortality in less developed therein). These systemic infections caused by C.
countries. While the production of diphtheria toxin has diphtheriae suggest that this pathogen is not only able
been well-established as a major virulence factor, little is to attach to host epithelial cells, but must be able to
known about C. diphtheriae factors crucial for coloniza- gain access to deeper tissues by unknown portals of
tion of the host and corresponding host receptors recog- entry and to persist in these tissues.
nized by these factors, although colonization is an A possible clue for the background of persistence of
essential step of pathogenicity. C. diphtheriae came from investigations of adherence
In the last decades it has become evident that and invasion of toxigenic and non-toxigenic strains.
C. diphtheriae is not only the aetiological agent of Using a combination of gentamicin protection assays
and thin-section electron microscopy, Hirata and co-
workers [8] showed that toxigenic C. diphtheriae were
* Correspondence: aburkov@biologie.uni-erlangen.de
1
Friedrich-Alexander-Universität Erlangen-Nürnberg, Lehrstuhl für not only able to adhere to laryngeal HEp-2 cells, but
Mikrobiologie, Staudtstr. 5, 91058 Erlangen, Germany also enter these cells and survive after internalization.
© 2010 Ott et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

25

Ott et al. BMC Microbiology 2010, 10:2
http://www.biomedcentral.com/1471-2180/10/2
Similar observations were made for non-toxigenic
strains [9] showing that also pharyngeal Detroit
562 cells can be invaded by C. diphtheriae. In this study,
living intracellular bacteria were detected up to 48 h
after infection.
While host cell receptors and invasion-associated pro-
teins of the pathogen are still unknown, bacterial adhe-
sion factors have been recently at least partially
characterized on the molecular level. C. diphtheriae is
able to assemble three distinct pili on its surface.
Mutant analyses showed that the SpaA-type pilus is suf-
ficient for adhesion to pharynx cells, shaft proteins are
not crucial for pathogen-host interaction, while adher-
ence to pharyngeal cells is greatly diminished when
minor pili proteins SpaB and SpaC are lacking [10]. The
results obtained in this study also indicated the exis-
tence of other proteins besides pili subunits involved in
adhesion to larynx, pharynx, and lung epithelial cells,
since a total loss of attachment to pharyngeal cells due
to mutagenesis of pili- and sortase-encoding genes
could not be observed and attachment to lung or larynx
cells was less affected by the mutations. This is in line
with a number of studies suggesting the multi-factorial
mechanism of adhesion (reviewed in [11]). Furthermore,
Hirata and co-workers [12] described two distinct pat-
terns of adherence to HEp-2 cells, a localized and a dif-
fuse form, an observation that hint also to the existence
of several adhesion factors. This idea is in accordance
with the situation in other bacteria such as Salmonella
enterica where a high number of different factors are
crucial for pathogenesis [13]. The involvement of differ-
ent C. diphtheriae proteins to adherence to distinct cell
types is further supported by work on adhesion to
human erythrocytes, showing that non-fimbrial surface
proteins 67p and 72p, which were up to now only char-
acterized by their mass, are involved in this process [14].
Interestingly, besides strain-specific differences in adher-
ences (see references cited above), also growth-depen-
dent effects were observed. In a study using two
toxigenic C. diphtheriae strains and erythrocytes as well
as HEp-2 cells, de Oliveira Moreira and co-workers [15]
showed an effect of iron supply on hemagglutination
and lectin binding properties of the microorganisms.
Also in this study, strain-specific differences in adher-
ence were detected.
While pathogen factors responsible for adhesion are at
least partially known, the molecular background of inva-
sion is more or less unclear. Since we were interested in
this process, we started a functional genetics approach
to identify proteins involved in invasion, based on a
recently published work presenting a comprehensive
analysis of proteins secreted by C. diphtheriae [16]. In
this study, we focused on one of these identified pro-
teins, the surface-associated protein DIP1281, a member
26
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Page 2 of 9
of the NlpC/P60 family [17]. NlpC/P60 proteins define a
large superfamily of several diverse groups of proteins
including putative proteases and probably invasion-asso-
ciated proteins. They are found in bacteria, bacterio-
phages, RNA viruses, and eukaryotes and various
members are highly conserved among non-pathogenic
and pathogenic corynebacteria [18]. C. diphtheriae pro-
tein DIP1281 was, as its homologs Ce1659, Cg1735, and
JK0967 in Corynebacterium efficiens, Corynebacterium
glutamicum, and Corynebacterium jeikeium, previously
annotated as hypothetical invasion-associated protein
and was therefore in the focus of this study.
Results
Adhesion and invasion of C. diphtheriae wild type
and mutant strains
As a basis for further analyses of DIP1281 mutants,
strains ISS3319 and ISS4060, which were already shown
to be adhesion- and invasion-competent [9], were tested
for adhesion to and internalization in Detroit562 (D562)
cells. Using a slightly modified protocol (compared to
[9]) with increased number of washing steps, we were
able to generate highly reproducible infection conditions
(Table 1). In these experiments, strain ISS3319 showed
a higher number of adherent bacteria compared to
strain ISS4060 (corresponding to adhesion rates of
2.66 ± 0.12% for ISS3319 and 2.16 ± 0.29% for ISS4060),
while statistically relevant differences of the number of
invaded epithelial cells were not observed (Table 1).
After establishing infection conditions for the wild-
type strains, dip1281 gene disruption mutants Lilo1
(ISS3319::pK18 mob’dip1281’’) and Lilo2 (ISS4060::pK18
mob’dip1281’’) were analyzed. DIP1281 mutant strains
lacked the ability to adhere to host cells almost comple-
tely (with adhesion rates of 0.03 ± 0.01% for Lilo1 and
0.04 ± 0.01% for Lilo2) and in contrast to the wild-type
no internalized bacteria were detectable for strain Lilo1
and Lilo2 (Table 1).
Table 1 Adhesion of C. diphtheriae to epithelial cells and
internalization. D562 cells (2 × 105 cells per well) were
infected with C. diphtheriae (4 × 107 cfu/ml) leading to a
multiplicity of infection (MOI) of 200.
Strain Viable bacteria (CFU/ml)a
adherentb internalizedc
ISS3319 5
10.1 × 10 ± 1.4 × 10 5
1.6 × 103 ± 1.0 × 102
ISS4060 5
3.5 × 10 ± 1.0 × 10 5
3.0 × 103 ± 1.4 × 103
Lilo1 2
1.6 × 10 ± 2.1 × 10 2
n. d.
Lilo2 9.3 ± 10.6 n. d.
a
values represent the means and standard deviations of three separate
experiments
b
c
average number of bacteria recovered on agar plates after 1.5 h of infection
average number of bacteria recovered on agar plates after 1.5 h of infection
and further 2 h of treatment with gentamicin
n. d.: not detectable

Ott et al. BMC Microbiology 2010, 10:2
http://www.biomedcentral.com/1471-2180/10/2
Basic characterization of DIP1281 mutant strains
Microscopic inspection of the mutant strains revealed
unexpectedly an increased size of the single bacteria in
combination with an altered less club-like shape and
formation of chains of bacteria rather than the typical
V-like division forms or palisades of clustered
C. diphtheriae. Immuno-fluorescence microscopy carried
out for control verified that observation (Figure 1).
Additionally, this approach showed an uneven, speckled
staining of the mutants, indication an altered surface
structure compared to the wild-type strains.
To analyse, if all bacteria within the observed chains
of mutants were still viable or if changes were correlated
with detrimental effects on survival of bacteria, we car-
ried out LIVE/DEAD staining. No significant differences
were observed between wild-type and mutants in respect
to viability, in all cases the majority of bacteria were
fully viable and exclusively stained by SYTO9 green and
not by propidium iodide (Figure 2). During manipula-
tion of bacteria (washing steps, resuspension of pellets),
we observed that chains of mutants were occasionally
broken down to smaller units. Using LIVE/DEAD
Figure 1 Immuno-fluorescence microscopy of C. diphtheriae wild-type and mutant strains. An antiserum directed against the surface
proteome of C. diphtheriae was used as primary antibody; Alexa Fluor 488 goat anti-rabbit was used as secondary antibody. A: ISS3319, B: Lilo1,
C: ISS4060, D: Lilo2.
27
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staining, we could show that disruption of chains by vig-
orous vortexing (5 min) was not detrimental to the bac-
teria (Figure 2C and 2F), indicating that mutant strains
have a fully functional and rigid peptidoglycan layer.
For all strains, ISS3319, ISS4060, Lilo1 and Lilo2,
identical doubling times of about 70 min were observed.
Interestingly, with a final optical density (OD 600 ) of
approx. 13, the mutants reached a more than fourfold
higher OD600 compared to the corresponding wild-type
strains, which reached final optical densities between 2.5
and 3. This observation corresponds nicely with the
increased colony size of the mutants (data not shown)
and suggests that the altered bacterial size and form has
no severe impact on light scattering and consequently
OD measurement.
Analysis of surface proteins
Since we assumed that the altered shape of the mutants
might be correlated with an altered cell surface, espe-
cially in the light of the immuno-fluorescence micro-
scopy approach (Figure 1), which showed a different
antibody binding compared to the wild-type, we isolated
the surface proteins of wild-type and mutant strains.

Ott et al. BMC Microbiology 2010, 10:2
http://www.biomedcentral.com/1471-2180/10/2
Figure 2 LIVE/DEAD staining of C. diphtheriae wild-type and mutant strains. Green fluorescent bacteria have a functional cytoplasmic
membrane and are stained green, red propidium iodide staining indicates non-viable cells. A: ISS3319, B-C: Lilo1, D: ISS4060, E-F: Lilo2, C and F:
cells subjected to 5 min of vigorous vortexing.
When these were subjected to SDS-PAGE and silver
staining, significant differences in protein patterns were
observed (Figure 3A). Compared to the protein bands of
the wild-type strains, DIP1281 mutants showed a
decrease of bands in the upper molecular weight range
accompanied by an increased number of bands in the
lower molecular weight range. These changes were con-
firmed, when Western blot experiments were carried
out (Figure 3B), which also showed a dramatic change
and decrease of immuno-reactive bands. As a third
experimental approach to analyse surface proteins, 2-D
PAGE was carried out (gels for strains ISS3319 and
Lilo1 are shown in Figure 3C; ISS4060 and Lilo2 gave
comparable results, data not shown). As in the SDS-
PAGE experiments, the mutant showed a decrease of
proteins in the upper molecular weight range and an
increased number of spots in the lower molecular
weight range. Furthermore, in comparison to the wild-
type, the mutant showed a dramatic increased number
of multiple spots. The molecular background of these
multiple protein forms is unclear.
Surface structure of wild-type and mutant strains
The altered immuno-staining of the mutant strain sur-
faces and the clear differences of wild-type and mutant
protein patterns revealed by SDS-PAGE and 2-D PAGE
prompted us to perform a more detailed investigation of
the cell surface of C. diphtheriae by atomic force micro-
scopy. Compared to the surface structure of C. glutami-
cum, which was investigated for several strains in great
detail by atomic force microscopy [19-21], C. diphtheriae
shows a more structured surface (Figure 4). Furthermore,
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striking differences were observed when the cell surface
of different C. diphtheriae strains was examined. In the
wild-type strain ISS3319 (Figure 4A) round elevations
with a lateral diameter of 10-40 nm and a height of 1-4
nm can be seen (Figure 4A, upper row). The complemen-
tary phase images, which reflect adhesive and elastic tip-
sample interactions, show a similar, highly structured
surface structure (Figure 4A, lower row). In the mutant
strain Lilo1 (Figure 4B), a loss of this fine structure was
observed: Elongated elevations can be seen with a width
of 50-100 nm (Figure 4B, upper row). Their height is
similar as in the case of the wild-type strain. Differences
in surface structure are especially obvious when compar-
ing the complementary phase images (lower rows in
Figure 4A and 4B). Analyses of strains ISS4060 and Lilo2
gave similar results (data not shown).
Discussion
In this study, the function of the surface-associated pro-
tein DIP1281, a member of the NlpC/P60 family was
investigated, which was annotated as hypothetical inva-
sion-associated protein. By fluorescence staining and
atomic force microscopy, we could show that DIP1281
mutations cause formation of chains of bacteria, rear-
rangements of cell surface structures, and dramatic
changes in protein patterns. Our data indicate that
DIP1281 is not crucial for the separation of the peptido-
glycan layer of dividing bacteria, since disruption of
chains did not decrease the viability of bacteria. Conse-
quently, DIP1281 function seems to be limited to the
outer protein layer of C. diphtheriae, which is not
 Publications
Ott et al. BMC Microbiology 2010, 10:2 Page 5 of 9
http://www.biomedcentral.com/1471-2180/10/2

Figure 3 Analysis of surface proteins. Surface proteins were isolated from C. diphtheriae wild-type and mutant strains and subjected to SDS-
PAGE (A), Western blotting (B), and 2-D PAGE (C). For SDS-PAGE 25 μg of protein prepared from strains ISS3319 (lane 2), Lilo1 (lane 3), ISS4060
(lane 4), and Lilo2 (lane 5) were applied per lane on a 10% polyacrylamide gel and silver-stained after electrophoresis. Molecular weight of
marker proteins (lane 1, from top to bottom): 250, 130, 95, 72, 55, 36, 28, 17, 11 kDa. Western blotting was carried out after SDS-PAGE using a
polyclonal antiserum directed against C. diphtheriae DSM44123 surface proteins. For 2-D PAGE surface protein preparations were separated
according to their isoelectric point and molecular mass using a pH range of 3-10 for isoelectric focussing and 12.5% polyacrylamide gels for SDS-
PAGE. Gels were stained with Coomassie Brilliant Blue. Molecular weight of marker proteins (from top to bottom): 150, 120, 100, 85, 70, 60, 50,
40, 30, 25, 20, 15 kDa.

uniformly organized in a surface layer lattice, but com-
prises more than 50 different proteins [16]. If the other
NlpC/P60 family members in C. diphtheriae besides
DIP1281, namely DIP0640, DIP1621, and DIP1622 [18]
have similar functions in cell surface layer organization
is unknown and has to be investigated in future projects.
Tsuge and co-workers reported cell separation defects
in Corynebacterium glutamicum R, when the DIP1281
homolog cgR_1596 and another member of the NlpC/
P60 protein family cgR_2070 were mutated [22]. Also in
this study, cell separation was not impaired in respect to
separation of peptidoglycan and mycolic acid layers of
daughter cells, but mainly restricted to the surface pro-
tein layer of the bacteria. However, using transmission
electron microscopy of thin sections of cells, in this
study also formation of multiple septa within single bac-
teria was observed in response to cgR_1596 mutations.
Furthermore, growth of mutant strains was examined.
In contrast to the situation in C. diphtheriae, where we
found an unaltered growth rate and a strongly increased

29

Ott et al. BMC Microbiology 2010, 10:2
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Figure 4 Ultrastructural analysis of the cell surface of C. diphtheriae strains. (A) ISS3319, (B) Lilo1; red boxes in the low magnification
images on the left hand side mark three areas shown with a higher magnification on the right hand side (upper row: topography/height, lower
row: phase). Colour scale bars at the right hand side give height and phase magnitudes.
biomass formation caused by lack of DIP1281, in
C. glutamicum R mutation of cgR_1596 led to a slightly
decreased growth rate and unaltered final optical density
of the culture. The exact function of the NlpC/P60 pro-
tein family members in C. glutamicum was also not
unravelled until now.
In respect to adhesion and internalization of
C. diphtheriae to epithelial cells, the results obtained in
this study suggest that DIP1281 is crucial for localiza-
tion and function of adhesion and invasion factors and
consequently, structural alterations caused by lack
DIP1281 prevent adhesion of corresponding mutants to
host cells and invasion into these cells.
Conclusions
C. diphtheriae protein DIP1281 was, as its homologs
Ce1659, Cg1735, and JK0967 in Corynebacterium effi-
ciens, Corynebacterium glutamicum, and Corynebacter-
ium jeikeium, previously annotated as hypothetical
invasion-associated protein. Generation and analyses of
mutant strains indicate that DIP1281 is predominantly
involved in the organization of the outer surface protein
layer of C. diphtheriae rather than in the separation of
the peptidoglycan cell wall of dividing bacteria. The
adhesion- and invasion-negative phenotype of corre-
sponding mutant strains is an effect of rearrangements
30
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of the outer surface of bacteria. Specific interaction part-
ners for DIP1281 and its homologs in other corynebac-
teria are unknown and might be the focus of further
studies to unravel the specific functions and targets of
these proteins on a molecular level.
Methods
Bacterial strains and growth
Strains used in this study are listed in Table 2. Escheri-
chia coli DH5aMCR was grown in Luria Bertani (LB)
medium at 37°C, C. diphtheriae in Heart Infusion (HI)
broth at 37°C. If appropriate, kanamycin was added
(30 μg/ml for E. coli; 50 μg/ml for C. diphtheriae).
Preparation of C. diphtheriae protein extracts
To prepare surface proteins, bacteria were grown in
20 ml HI broth (with kanamycin added for the mutant
strains) for approximately six hours and used to inocu-
late 250 ml HI broth for overnight growth. Bacteria
were harvested by centrifugation at 5,000 × g for 20
min, washed twice with pre-cooled (4°C) 50 mM Tris-
HCl buffer (pH 7.2), resuspended in 50 mM Tris-HCl
(pH 7.2) containing 2% 3-[(3-choamidopropyl)-dimethy-
lammonio] propanesulfonate (CHAPS) and incubated on
ice overnight, followed by centrifugation at 3,500 × g
and 4°C for 30 min to separate the cell surface proteins.
After filtration of the protein solution using 0.45 μm
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Table 2 Bacterial strains and eukaryotic cells used in this 20 mM DTT, 2% CHAPS, a trace of bromophenol blue,
study. and 0.5% Pharmalyte 3-10). IEF was performed with
Strains Description Reference commercially available IPG strips (18 cm, pH 3-10)
C. diphtheriae and the Ettan IPGphor II (GE Healthcare, Munich,
DSM44123 non-toxigenic isolate, type DSMZ Germany). The following voltage profile was used for
strain (Braunschweig) IEF: 1 h, 0 V; 12 h, 30 V; 2 h, 60 V; 1 h, 500 V; 1 h,
ISS3319 C. diphtheriae var. mitis, non- [9] 1000 V followed by a linear increase to 8000 V. The
toxigenic isolate final phase of 8000 V was terminated after 90,000 Vh.
ISS4060 C. diphtheriae var. gravis, non- [9] The IPG strips were equilibrated for 30 min each in 5
toxigenic isolate
ml of solution C (6 M urea, 50 mM Tris-HCl (pH 6.8),
Lilo1 ISS3319 DIP1281:: This study
pK18mob'DIP1281''
30% glycerol, 2% SDS, 1% DTT) and in 5 ml of solution
Lilo2 ISS4060 DIP1281:: This study
D (6 M urea, 50 mM Tris-HCl (pH 6.8), 30% glycerol,
pK18mob'DIP1281'' 2% SDS, 4% iodacetamide). The isolated proteins were
E. coli separated in 12.5% acrylamide/bis-acrylamide gels
DH5aMCR endA1 supE44 thi-1 l- recA1 [28] (37.5:1) with an Ettan Dalt II system (GE Healthcare,
gyrA96 relA1 deoR ∆(lacZYA- Munich, Germany) applying approximately 15 mA per
argF) U196 !80∆lacZ ∆M15 gel. To visualize the separated proteins, gels were
mcrA ∆(mmr hsdRMSmcrBC)
stained in Coomassie staining solution (5% methanol,
Cell lines
42.5% ethanol, 10% acetic acid, 0.25% Serva-G250), and
Detroit562 human hypopharyngeal [29]
carcinoma cells
destained with 10% acetic acid.
Immuno-fluorescence
For immuno-fluorescence staining a rabbit antiserum
pore-size filters (SARSTEDT, Nümbrecht, Germany), directed against the C. diphtheriae surface proteome
further preparation of the surface proteins by phenolic was used as primary antibody. As secondary antibody
acid extraction and methanol precipitation followed a Alexa-Fluor 488 (green) goat anti-rabbit IgGs were
protocol described by Watt and co-workers [23]. The applied. All antibodies were diluted in blocking solution
precipitated proteins were harvested by centrifugation at (2% goat serum, 2% BSA). Bacterial cells were dried on
3,500 × g and 4°C for 30 min. The pellet was washed coverslips (37°C), fixed with 3% PFA (10 min at room
twice with 3 ml of 70% ethanol (-20°C) and once with temperature) and finally washed thrice with 1 × PBS.
3 ml of acetone (-20°C). Finally, the protein pellet was Bacterial cells were incubated in staining solution for at
dried on ice and solubilised in 450 μl of dehydration least 1 h at room temperature and washed thrice with
buffer (8 M urea, 20 mM DTT, 2% CHAPS). Protein PBS between staining steps. Coverslips were mounted
concentrations were determined spectrophotometrically on glass slides using Fluoroprep (Biomerieux, Craponne,
using a NanoDrop spectrophotometer (peqLab, Erlan- France). Imaging was done on an AxioVert 200 M
gen, Germany). Surface proteins prepared from strain inverted optical microscope (Carl Zeiss Micromaging
DSM44123 were used for the immunization of rabbits GmbH, Jena, Germany). For additional image processing
to generate C. diphtheriae surface protein-specific anti- Photoshop CS2 (Adobe, Munich, Germany) was used.
sera (Eurogentec, Liege, Belgium). LIVE/DEAD staining
SDS-PAGE, silver staining, and Western blot analysis Overnight cultures grown in 20 ml HI broth plus kana-
Proteins of the cell surface fraction of wild-type and mycin added for the mutant strains were washed once
mutant strains were separated using Tricine-buffered in 20 ml 1 × PBS and resuspended in 10 ml 1 × PBS.
10% SDS gels as described [24]. After SDS-PAGE pro- To distinguish live and dead bacteria the LIVE/DEAD
tein bands were visualized by silver staining [25]. For Baclight Bacterial Viability Kit for microscopy (Invitro-
Western blotting, the SDS gel-separated proteins were gen, Eugene, OR) was used according to the supplier’s
transferred onto a polyvinylidene difluoride membrane protocol. Imaging was done on an AxioVert 200 M
by electroblotting (PVDF, Roth, Karlsruhe, Germany) inverse microscope (Carl Zeiss Micromaging GmbH,
and incubated with C. diphtheriae surface protein-speci- Jena, Germany).
fic antisera generated in rabbits. Antibody binding was Atomic force microscopy (AFM)
visualized by using goat anti-rabbit IgG coupled to alka- Overnight cultures grown in 20 ml HI broth plus kana-
line phosphatase and the BCIP/NBT alkaline phospha- mycin added for the mutant strains were washed five
tase substrate (Sigma-Aldrich, Darmstadt, Germany). times in 20 ml ice cold distilled water and finally resus-
2-D-PAGE of C. diphtheriae surface proteins pended in 10 ml ice cold distilled water. 5 μl of each
2-D polyacryalmide gels were loaded with 300 μg of sample were fixed on a glass slide by drying using com-
proteins dissolved in 450 μl of solution B (8 M urea, pressed air. An AFM instrument (MFP-3D, Asylum

31

Ott et al. BMC Microbiology 2010, 10:2
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Research, Santa Barbara, CA) with standard silicon can-
tilever probes (NCH-W, Nanosensors, Neuchatel, Swit-
zerland) was used under ambient laboratory conditions
and operated in tapping mode. AFM topography and
phase images were recorded simultaneously.
Adhesion assays
D562 cells were seeded in 24 well plates (Greiner bio-
one Cellstar, Frickenhausen, Germany) at a density of
2 × 105 cells per well 48 h prior to infection. Bacteria
were inoculated to an OD600 of 0.1 from overnight cul-
tures and grown in HI broth for 3.5 h. Subsequently,
the bacteria were harvested by centrifugation and
adjusted to an OD600 of 0.2. A master mix of the inocu-
lum was prepared in DMEM (Dulbecco’s modified
Eagle’s medium, PAA; high glucose, 10% FCS, 2 mM
glutamine) without penicillin/streptomycin and cells
were infected for 90 min at a MOI of 200 (viable counts
experiments). The cells were washed with PBS nine
times, detached with 500 μl trypsin solution (0.12% tryp-
sin, 0.01% EDTA in PBS) per well (5 min, 37°C, 5%
CO2 , 90% humidity) and lysed with 0.025% Tween 20
for 5 min at 37°C. Serial dilutions were made in pre-
chilled 1 × PBS and plated on HI plates to determine
the number of cfu. The assay is modification of a pre-
viously described one [9].
Epithelial cell invasion model
D562 cells were seeded in 24 well plates (Greiner bio-
one Cellstar, Frickenhausen, Germany) at a density of
2 × 105 cells per well 48 h prior to infection. Overnight
cultures grown in HI were re-inoculated to an OD600 of
0.1 in fresh medium and grown aerobically for another
3.5 h. An inoculum of approximately 8 × 107 bacteria
ml -1 (MOI = 200) was prepared in DMEM without
penicillin/streptomycin and 500 μl per well were used to
infect the D562 cells. The plates were centrifuged for
5 min at 500 × g to synchronize infection and subse-
quently incubated for 90 min (37°C, 5% CO 2 , 90%
humidity). The cells were washed thrice with PBS and
500 μl of DMEM containing 100 μg ml -1 gentamicin
was applied to each well to kill remaining extracellular
bacteria. After 2 h of incubation the cell layers were
washed thrice with PBS, detached by adding 500 μl tryp-
sin solution (0.12% trypsin, 0.01% EDTA in PBS) per
well (5 min, 37°C, 5% CO2, 90% humidity), and lysed for
5 min at 37°C with 0.025% Tween 20 to liberate the
intracellular bacteria. Serial dilutions of the inoculum
and the lysates were plated on HI plates to determine
the number of colony forming units (cfu).
Construction of mutant strains
For plasmid isolation, transformation and cloning, stan-
dard techniques were used [26]. For chromosomal dis-
ruption of the C. diphtheriae DIP1281 gene an 582 bp
internal DNA fragment was amplified via PCR using
chromosomal DNA of strain ISS3319 as template and
32
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the following primers: 5’- cgc gcg ctc gcg ggc acg tca
gga agc tg - 3’; 5’- cgc gcg ccc ggg cga atc caa ttt tat taa
aa - 3’. Using the AvaI and XmaI sites introduced in via
the PCR primers (shown in bold) the DNA fragment
was ligated to AvaI/XmaI-restricted and dephosphory-
lated pK18 mob DNA [27]. The resulting plasmid pK18
mobDIP1281’ was amplified in E. coli DH5aMCR. One
microgram of unmethylated plasmid isolated from this
E. coli strain was used to transform C. diphtheriae using
a GenePulser II (Bio-Rad, Munich Germany). Electropo-
rated cells were added to 1 ml of HI broth containing
1% glucose and incubated for 2 h at 37°C. An appropri-
ate volume of culture was plated on medium containing
kanamycin. Since pK18 mob cannot be replicated in
C. diphtheriae, kanamycin-resistant C. diphtheriae car-
ried the vector integrated via recombination in the
chromosomal DIP1281 gene and were designated Lilo1
(resulting from the strain ISS3319) and Lilo2 (resulting
from the strain ISS4060).
Acknowledgements
The authors wish to thank C. v. Hunolstein (Istituto Superiore di Sanita’,
Rome) for providing strain ISS3319 and ISS4060, A. Völzke (Erlangen) for
preparation of surface proteins for antibody generation and the Deutsche
Forschungsgemeinschaft for financial support in frame of SFB 796 (projects
B5 and Z).
Author details
1
Friedrich-Alexander-Universität Erlangen-Nürnberg, Lehrstuhl für
Mikrobiologie, Staudtstr. 5, 91058 Erlangen, Germany. 2Mikrobiologisches
Institut des Universitätsklinikums Erlangen, Wasserturmstr. 3-5, 91054,
Erlangen, Germany. 3Friedrich-Alexander-Universität Erlangen-Nürnberg,
Lehrstuhl für Angewandte Physik, Staudtstr. 5, 91058 Erlangen, Germany.
4
Robert-Koch-Institut, Burgstr. 37, 38855 Wernigerode, Germany.
5
Arbeitsgruppe Mikrobiologie, Universität Osnabrück, Barbarastr. 11, 49076
Osnabrück, Germany.
Authors’ contributions
LO carried out growth mutagenesis experiments, invasion assays,
fluorescence microscopy, protein preparation and analysis, MHö carried out
adhesion experiments, RGG and MHe supported LO and MHö in respect to
cell culture, adhesion and invasion analysis and fluorescence microscopy,
AFM experiments were carried out in cooperation with JR and TES, AB
supervised the experiments of LO and MHö and was responsible for the
draft and final version of the manuscript. All authors read and approved the
final manuscript.
Received: 23 July 2009
Accepted: 5 January 2010 Published: 5 January 2010
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3.2 Strain-specific differences in pili formation and the interaction of

Corynebacterium diphtheriae with host cells (Ott et al, 2010b)

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RESEARCH ARTICLE Open Access

Strain-specific differences in pili formation and

the interaction of Corynebacterium diphtheriae
with host cells
Lisa Ott1, Martina Höller1, Johannes Rheinlaender2, Tilman E Schäffer2, Michael Hensel3,4, Andreas Burkovski1*

Abstract
Background: Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin
production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we
investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili
in different isolates.
Results: Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly
coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in
macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number
and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation
between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-
specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic
detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562
cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use
epithelial cells as an environmental niche supplying protection against antibodies and macrophages.
Conclusions: The results obtained suggest that it is necessary to investigate various isolates on a molecular level
to understand and to predict the colonization process of different C. diphtheriae strains.

Background able to gain access to deeper tissues and to persist inside

Corynebacterium diphtheriae is the causative agent of
diphtheria, a toxaemic localized infection of the respira-
tory tract. While this disease is well-controlled by vacci-
nation against the diphtheria toxin in e. g. Western
Europe [1-3], it is still a severe health problem in less
developed countries. Furthermore, C. diphtheriae is not
only the aetiological agent of diphtheria, but can cause
other infections as well. Non-toxigenic strains have been
increasingly documented [4-6] and found to be the
cause of invasive diseases such as endocarditis, bacterae-
mia, pneumonia, osteomyelitis, spleen abscesses, and
septic arthritis [7,8]. As indicated by these systemic
infections, C. diphtheriae is not only able to attach to
host epithelial cells of larynx and pharynx, but must be
* Correspondence: aburkov@biologie.uni-erlangen.de
1
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-
Nürnberg, Nürnberg, Germany
Full list of author information is available at the end of the article
tissues or cells.
A possible clue for the background of persistence of
C. diphtheriae came from investigations of adherence
and invasion of toxigenic and non-toxigenic strains by
different groups. Using a combination of gentamicin
protection assays and thin-section electron microscopy,
Hirata and co-workers [9] showed that toxigenic
C. diphtheriae are not only able to adhere to laryngeal
HEp-2 cells, but also enter these cells and survive after
internalization. Similar observations were made for non-
toxigenic strains [10] showing that also pharyngeal
Detroit 562 cells can be invaded by C. diphtheriae and
that viable intracellular bacteria can be detected up to
48 h after infection.
While host cell receptors and invasion-associated pro-
teins of the pathogen are still unknown, bacterial adhe-
sion factors have been recently at least partially
characterized on the molecular level. C. diphtheriae

© 2010 Ott et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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strain NCTC13129 is able to assemble three distinct
types of pili on its surface [11,12]. Mutant analyses
showed that the SpaA-type pilus is sufficient for adhe-
sion of this strain to pharynx cells, shaft proteins are
not crucial for pathogen-host interaction, and adherence
to pharyngeal cells is greatly diminished when minor
pili proteins SpaB and SpaC are lacking [13]. The results
obtained in other studies indicated the existence of addi-
tional proteins besides pili subunits involved in adhesion
to larynx, pharynx, and lung epithelial cells, since a total
loss of attachment to pharyngeal cells due to mutagen-
esis of pili- and sortase-encoding genes could not be
observed and attachment to lung or larynx cells was less
affected by the mutations. This is in line with a number
of studies suggesting the multifactorial mechanism of
adhesion (reviewed in [14]). Furthermore, Hirata and
co-workers [7,15] described three distinct patterns of
adherence to HEp-2 cells, an aggregative, a localized,
and a diffuse form, an observation that hints also to the
existence of several adhesion factors and different recep-
tors on the host cell surface. The involvement of differ-
ent C. diphtheriae proteins to adherence to distinct cell
types is further supported by work on adhesion to
human erythrocytes, showing that non-fimbrial surface
proteins 67p and 72p, which were up to now only char-
acterized by their apparent mass, are involved in this
process [16]. Interestingly, besides strain-specific differ-
ences in adherences (see references cited above), also
growth-dependent effects were observed. In a study
using two toxigenic C. diphtheriae strains and erythro-
cytes as well as HEp-2 cells, de Oliveira Moreira and
co-workers [17] showed an effect of iron supply on
hemagglutination and lectin binding properties of the
microorganisms.
In this study, we present a characterization of different
non-toxigenic C. diphtheriae and a toxin-producing
strain with respect to adhesion to and internalization
into epithelial cells. Analyses reveal significant strain-
specific differences in host colonization and macromole-
cular surface structures of the studied strains, while
neither of the strains evoked rapid cell damage under
the conditions tested.
Results
Adhesion of C. diphtheriae to epithelial cells, invasion of
host cells and intracellular survival
In this study, adhesion of six non-toxigenic strains and
one toxin-producing C. diphtheriae to Detroit562 cells
was analyzed (Fig. 1). In these experiments tox+ strain
DSM43989 showed the lowest adhesion rate with 0.34 ±
0.05%. While in general higher than the tox+ strain, the
non-toxigenic strains differed significantly in their adhe-
sion rate, varying between 0.69 ± 0.12% for strain
DSM43988 and 7.34 ± 2.33% for strain ISS4749 (Fig. 1).
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Once attached to the surface of an epithelial cell,
C. diphtheriae might invade the host cell and persist
within the cell. In order to investigate this process for
the different strains studied here, gentamicin protection
assays were carried out. For this purpose, cells were
incubated for 1.5 h with bacteria, gentamicin was added
to kill remaining extracellular C. diphtheriae and survi-
val of intracellular bacteria was analyzed after different
times of incubation (Fig. 2). When invasion into D562
cells was analyzed for the six non-toxigenic strains and
the toxigenic C. diphtheriae strain after 2 h, tox+ strain
DSM43989 showed the lowest internalization rate with
0.014 ± 0.007%. As in the adhesion assay, the non-toxi-
genic strains showed in general a higher rate compared
to the toxin-producer strain and again rates differed sig-
nificantly between the non-toxigenic strains, varying
between 0.018 ± 0.006% for strain ISS4749 and 0.060 ±
0.027 for strain ISS4060 (Fig. 2A). The comparison of
strains in respect to adhesion and internalization rates
suggested that although a high adhesion seems to favour
internalization, adhesion and invasion are not strictly
coupled processes. Plating and counting of internalized
cells after 8.5 and 18.5 h revealed decreasing numbers
of colony forming units (Fig. 2B-C). Even after 18.5 h,
no strain was completely eliminated from the cells and
survival of bacteria ranged from 0.002 ± 0.001% of the
inoculums for DSM43989 to 0.005 ± 0.001% for
ISS4060.
In addition to the gentamicin protection assay, which
gives quantitative data, immune-fluorescence micro-
scopy was applied as an independent method to investi-
gate host cell interaction of C. diphtheriae strains. This
method has the advantage of allowing direct visualiza-
tion, although only on a qualitative level. Using an anti-
serum directed against C. diphtheriae surface proteins
and antibody staining before and after permeabilization
of the host cell, internalized C. diphtheriae were
detected (Fig. 3). Interestingly, V-shaped C. diphtheriae
dimers within the cells were observed. These V-forms
are the result of the Corynebacterium-specific snapping
division and indicate growing bacteria. Together with a
tendency towards formation of clusters of cells (Fig. 3C
and 3F), this observation suggests that bacteria replicate
within the host cells and growth and elimination
described above (Fig. 2A-C) are parallel processes.
Influence of C. diphtheriae on the transepithelial
resistance of cell monolayers
Some pathogens, such as Salmonella enteric serovar
Typhimurium (S. Typhimurium), can cause severe
damage on cell membranes and due to the resulting loss
of cell integrity, the transepithelial resistance of mono-
layers is dramatically reduced (for example see [18]). In
this study, we used S. Typhimurium NCTC12023 as a
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Figure 1 Adhesion of C. diphtheriae strains to D562 cell layers. D562 cells were infected with different C. diphtheriae strains. Besides
DSM43989, which is tox+, the isolates are non-toxigenic. The cells were washed with PBS, detached with trypsin solution, lysed with Tween 20,
and the number of colony forming units (cfus) was determined. Adhesion is expressed as percentage of the inoculum, showing means and
standard deviations of ten independent measurements (biological replicates) with 3 samples each (technical replicates). All strains, except
ISS4746 and ISS4749, show statistically significant differences in adhesion rates (students TTEST values below 0.04).

positive control (Fig. 4A) and tested the influence of dif-
ferent C. diphtheriae strains on transepithelial resistance
(Fig. 4B). Infection of Detroit562 monolayers with
S. Typhimurium caused a dramatic break-down of trans-
epithelial resistance within 1.5 h while all tested
C. diphtheriae strains including tox+ strain DSM43989
had no effect on transepithelial resistance within a time
span of three hours. Since the incubation conditions
used in this assay are similar to that applied for the
adhesion and invasion assays, this observation indicates
that a detrimental effect of toxin production can be
excluded as a reason for the low adhesion and internali-
zation rates of strain DSM43989.
Overnight incubation of D562 cells with C. diphther-
iae was tested as well. In this case, the Dulbecco’s modi-
fied Eagle’s medium had to be exchanged after 3 h with
fresh medium to remove not adhered bacteria in order
to avoid that the pH of the medium dropped due to the
bacterial metabolism leading to secondary detrimental
effects. In contrast to short term incubation and to the
non-toxigenic strains, long term measurement (Fig. 4B,
overnight time point) of transepithelial resistance of cell
monolayers infected with DSM43989 showed a signifi-
cant effect, which might be caused by toxin production.
Ultrastructural analysis of C. diphtheriae strains
Since we suspected that the differences in adhesion
might be the result of different surface structures, we
started an ultrastructure analysis of selected C. diphther-
iae. For this purpose, non-toxigenic strains as well as
tox+ strain DSM43989 were analyzed by atomic force
microscopy (Fig. 5A). With this technique, which allows
imaging surfaces topography at high resolution, signifi-
cant different macromolecular surface structures were
found between the different investigated C. diphtheriae
strains. While for ISS4060 and DSM43988 pili were not
detectable at all, ISS3319 and DSM44123 revealed short,
spike-like pili, ISS4746, ISS4749 and DSM43989 showed
long, hair-like protrusions (Fig. 5A). Also the number of
pili (counted from at least six specimens of each strain)
differed significantly (5B). Interestingly, adhesion and
pili formation were not coupled, since ISS3319, which
revealed spike-like pile and ISS4060, lacking these,
showed comparable adhesion rates, while ISS4746 and
ISS4749 had different numbers of long hair-like pili but
showed identical adhesion rates. Also no correlation
between invasion and pili formation was found. Since
strain-specific differences in pili formation have not
been observed before, the background for this

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Figure 2 Invasion of epithelial cells by C. diphtheriae strains. D562
cells were infected with different C. diphtheriae strains (DSM43989 tox+,
all others are non-toxigenic), washed, and incubated 2.0 (A), 8.5 (B) and
18.5 (C) hours with 100 μg ml-1 gentamicin. Subsequently, cells were
washed, detached with trypsin solution, lysed with Tween 20, and the
number of intracellular cfus was determined. Invasion is shown as
percentage of the inoculum internalized (means and standard
deviations of three independent biological replicates with three
samples each (technical replicates). Statistically relevant differences
between the strains (based on students TTEST values below 0.05) are
indicated by letters above columns.
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phenomenon was investigated in more detail in subse-
quent experiments.
Strain-specific expression of pili subunits
To analyze the molecular basis of strain-specific differ-
ences in pili formation, RNA hybridization experiments
were carried out to study the mRNA levels of the C.
diphtheriae spa genes. These genes are organized in
three different clusters together with the corresponding
sortase-encoding genes in the sequenced strain
NCTC13129 [13,19]. The first cluster comprises the
genes spaA, spaB, and spaC, which are most likely orga-
nized as an operon; the second cluster is formed by
spaD and a putative spaE-spaF operon, and a third clus-
ter comprises the spaG, spaH, and spaI gene, which are
most likely independently transcribed. Strain-specific
differences were detected, when probes for the detection
of all genes of cluster I and III were applied in RNA
hybridization experiments (Fig. 6A).
Strongest hybridization signals with spaA, spaB, and
spaC probes were detected with RNA isolated from
strains ISS4746 and ISS4749, slightly lower signal inten-
sities were observed with strain DSM43989, while only
faint signals were obtained for cluster I mRNA for the
other investigated strains. Strong transcription of spaG,
spaH, and spaI were again detected in strains ISS4746
and ISS4749, while other strains did not express cluster
III genes deduced from RNA hybridization experiments.
The data are in accordance with the AFM experiments
presented in Fig. 5, which show formation of a high
number of extended pili for strains ISS4746 and
ISS4749, followed by DSM43989; however, hybridization
signals may differ not only due to mRNA abundance,
but also due to sequence alteration.
To elucidate whether the missing transcripts in var-
ious strains are the result of regulatory processes or
have genetic reasons, PCR experiments were carried
out, which showed that missing transcripts are corre-
lated to lacking PCR products making regulatory
effects unlikely (Fig. 6B). Furthermore, reproducible
strain-specific differences in sizes of the PCR products
were observed for spaA and in band intensities for
spaB fragments, suggesting that also sequence devia-
tions exist besides strain-specific differences in the spa
gene repertoire. Interestingly, we were unable to gener-
ate any probes for genes of cluster II, comprising spaD,
spaE, and spaF, when chromosomal DNA of the used
ISS and DSM strains were used as template, while all
genes could be amplified from NCTC13129 DNA (Fig.
7). Deduced from these PCR experiments, these genes
seem to be absent in the investigated C. diphtheriae
strains. As an additional approach, we tested
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Figure 3 Detection of intracellular C. diphtheriae in Detroit562 cells by immune-fluorescence microscopy. D562 cells were seeded on
coverslips 48 h prior to infection and infected with C. diphtheriae (DSM43989 tox+, all others are non-toxigenic) for 4 h with at a MOI of 200 as
described earlier [26]. Antibodies directed against the surface proteome of C. diphtheriae were used as primary, Alexa Fluor 488 goat anti-rabbit
IgGs and Alexa-Fluor 568 goat anti-rabbit IgGs as secondary antibodies (A, D: intact D562, B, E: permeabilized D562, C, F: overlay with blue
F-actin stain Phalloidin-Alexa-Fluor 647, A-C: ISS3319, D-F: ISS4060. Green stain in panels A and D indicate extracellular bacteria. Dark red stain in
panels B and E indicates internalized C. diphtheriae, while adherent bacteria appear in light red. In the overlay (C, F) extracellular C. diphtheriae
appear orange, while internalized bacteria are stained dark red. Scale bars: 20 μm.

Figure 4 Transepithelial resistance of polarized D562 monolayers grown on transwells. (A) Control experiments of cells, which were
incubated without bacteria (open circles) and S. enterica serovar Typhimurium (open squares). (B) Incubation with C. diphtheriae strains
DSM43989 (tox+, open stars), ISS4749 (inverted closed triangles), ISS4746 (closed triangles), ISS4060 closed circles, ISS3319 (closed square),
DSM43988 (closed hexagons), and DSM44123 (closed diamonds). Experiments were carried out independently at least thrice and typical results
are shown.

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Figure 5 Ultrastructural analysis of the cell surface of C. diphtheriae strains. (A) Bacteria were fixed on glass slides by drying using
compressed air. Atomic force microscopy was carried out under ambient laboratory conditions and operated in tapping mode. Scale bars: 500
nm. (B) AFM images were analyzed in respect to pili number per bacterium. For each strain pili of at least six bacteria were counted; error bars
indicate deviations from mean values.

expression of SpaD in the different strains by Western
blot experiments. Cell extracts of strains ISS3319,
ISS4040, ISS4746, ISS4749, DSM43988, DSM43989,
and DSM44123 as well as purified SpaD protein as a
positive control were separated by SDS-PAGE and sub-
jected to Western blotting. SpaD-specific antiserum
reacted exclusively with the SpaD control, while no
signal was detectable in the investigated cell extracts
(data not shown).
To address the hypothesis that pili expression patterns
might change, when bacteria were in exposed to host
cells, Green fluorescent protein (GFP) fluorescence of C.
diphtheriae transformed with plasmids carrying spa
gene upstream DNA and a promoter-less gfpuv gene

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was determined without and after 1.5 h of host cell con-
tact. However, analysis of 80 to 140 bacteria for GFP
fluorescence before and after host cell contact revealed
no significant differences (data not shown).
Figure 6 Strain-specific distribution and expression of pili-
encoding genes. (A) Levels of spa gene transcripts in different
C. diphtheriae strains. Total RNA was isolated from the indicated
C. diphtheriae strains and hybridized with probes monitoring 16SrRNA
for control as well as spa gene transcription. (B) PCR detection of spa
genes. Chromosomal DNA of the indicated C. diphtheriae strains was
used as template for PCR using specific oligonucleotide pairs for the
spa genes indicated at the right side of the figure.
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Discussion
In this study, different non-toxigenic C. diphtheriae and
a toxin-producing strain were characterized in respect
to adhesion to and invasion of epithelial cells. All strains
were able to attach to host cells and immuno-fluores-
cence microscopy revealed internalization and growth of
C. diphtheriae within epithelial cells. We could show
that adhesion and invasion are not strictly coupled, indi-
cating that different proteins and mechanisms play a
role in these processes. Despite the fact that the number
of internalized bacteria decreased over time for all inves-
tigated strains, a considerable number of bacteria sur-
vived prolonged internalization for more than 18 h.
Furthermore, V-shaped division forms as well as forma-
tion of microcolonies were observed by fluorescence
microscopy, suggesting that the epithelial cells might
support growth of C. diphtheriae.
While proteins responsible for invasion and intracellu-
lar persistence are completely unknown for C. diphther-
iae, for the sequenced strain NCTC13129 the influence
of pili subunits on adhesion was characterized recently.
It was shown that the minor pili subunits SpaB and
SpaC are crucial for adhesion of strain NCTC13129 to
epithelial cells [13], while pili length is influenced by the
major pili subunits SpaA, SpaD, and SpaH, which form
the shaft of the structure [11,12,19]. The strains investi-
gated here showed significant variations in adhesion
rates and compared to the tox + strain, for all studied
non-toxigenic strains higher adhesion rates were
observed; however, adhesion and pili formation were
not strictly coupled processes. Strain-specific differences
of appearance and numbers of pili-like structures on the
surface of C. diphtheriae strains were shown by ultra-
structural analyses via atomic force microscopy. Addi-
tionally, RNA hybridization and Western blotting
experiments revealed distinct differences in the expres-
sion patterns of pili subunits for the investigated strains.
To our knowledge, this is the first time that isolate-spe-
cific differences in pili formation were characterized.
Mandlik and co-workers [13] showed that type III pili
Figure 7 PCR detection of spa genes in C. diphtheriae strain
NCTC 13129. Chromosomal DNA of C. diphtheriae strain NCTC
13129 was used as template for PCR using specific oligonucleotide
pairs for the indicated spa genes. In all cases, DNA fragments of the
expected size were amplified.

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length of strain NCTC13129 depends on spaH expres-
sion and can be manipulated by deletion or overexpres-
sion of spaH. These results are supplemented here by
showing that this is a phenomenon which occurs also as
natural variation in different C. diphtheriae wild type
isolates. Strains ISS4746 and ISS4749 showed the most
extended pili structures, an observation which is corre-
lated with high expression of spaA and spaH in these
strains, while medium-length pili of DSM43989 are cor-
related with lack of spaH expression. As mentioned
above, it was shown by mutant analyses of strain
NCTC13129 that expression of spaB and spaC is crucial
for adhesion to D562 cells [13]. Natural variations of the
spaB and spaC expression patterns observed here indi-
cate that this correlation is not as strict as suggested,
since strain ISS4060 shows only low spaB and no spaC
expression but a high adhesion rate, indicating that
other factors are important for adhesion as well and
expression of these might differ in various isolates.
The lack of any PCR product for spaD, spaE, and spaF
and the absence of a SpaD signal in Western blotting
experiments suggest that these genes are absent in the
investigated strains. All pili-encoding genes of
C. diphtheriae are located on pathogenicity islands
[20,21]. Based on the genome sequence of strain
NCTC13129, C. diphtheriae possesses 13 of these geno-
mic islands [20,22] and pili cluster II is located on geno-
mic island CDGI-2, which has a size of 17.5 kb and is
located directly adjacent to 36.5 kb pathogenicity island
CDGI-1, the tox+ corynephage [20]. Data of PCR experi-
ments (not shown) indicate that the pili-encoding genes
located on CDGI-2 are missing in all investigated ISS
and DSM strains and consequently the genetic reper-
toire of C. diphtheriae isolates is rather variable. This
observation is in agreement with a recent genome sur-
vey of C. diphtheriae C7(-) and PW8 strains [23] indi-
cating that 11 of the 13 putative pathogenicity islands of
the sequenced reference strain NCTC13129 are absent
in the C7(-) strain.
The importance of bacterial appendices and surface
proteins for host cell contact were also shown recently
for a non-fimbrial protein, DIP1281, previously anno-
tated as invasion-associated protein. This protein is a
virulence factor involved in cell surface organization,
adhesion and internalization in epithelial cells. Corre-
sponding mutant strains lack the ability to adhere to
host cells or invade these [24].
Conclusions
The results obtained in this study show that adhesion
and invasion are not necessarily coupled processes.
Adhesion rates are not strictly correlated with pili for-
mation and in summary the pili repertoire of the inves-
tigated strains is highly variable. As shown by genome
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comparisons [23] it is necessary to investigate various
isolates on a molecular level to understand and to pre-
dict the colonization process of different C. diphtheriae
strains.
Methods
Bacterial strains and growth
Strains used in this study are listed in Table 1.
C. diphtheriae strains were grown in Heart Infusion
(HI) broth or on Columbia agar with sheep blood
(Oxoid, Wesel, Germany) at 37°C. S. Typhimurium and
Escherichia coli DH5aMCR were grown in Luria Broth
(LB) [25] at 37°C. If appropriate, kanamycin was added
(30 μg ml-1 for E. coli; 50 μg ml-1 for C. diphtheriae).
Transformation of competent C. diphtheriae
For preparation of electrocompetent cells, 10 ml of an
overnight culture of C. diphtheriae were inoculated in
200 ml of Brain Heart Infusion (BHI) containing 2% gly-
cine and 15% sucrose, at 37°C in an orbital shaker until
an OD600 nm of 0.5 was reached. After storing the cells
on ice for 15 min, bacteria were harvested by centrifuga-
tion (4,000 × g, 4°C), washed thrice with 15% glycerol,
and resuspended in 1 ml of 15% glycerol. 100 μl aliquots
of the competent cells were frozen in liquid nitrogen
and stored at -80°C.
For transformation the aliquots were thawed on ice.
Plasmid DNA used for transformation was extracted
from E. coli strain DH5aMCR, which is unable to
methylate DNA. One microgram of plasmid DNA was
used to transform C. diphtheriae cells using a GenePul-
ser II apparatus (Bio-Rad, Munich, Germany) and 200
Ω, 2.5 kV, 25 μF. Electroporated cells were added to 1
ml of HI broth containing 1% glucose, incubated for 2 h
at 37°C, and plated on medium containing 10 μg ml-1
kanamycin. Subsequently, transformants were cultivated
in the presence of 50 μg ml-1 kanamycin.
Construction of GFP reporter plasmids and reporter gene
assay
For construction of reporter plasmids, promoter regions
of the pili- encoding spaA, spaG, spaH, and spaI genes
were amplified from chromosomal DNA by PCR (for
oligonucleotides used see Table 2). The PCR products
were inserted into plasmid pEPR1, using the NsiI and
BamHI restriction site upstream of the promoterless
gfpuv gene. Plasmids constructed (Table 3) were used to
transform electrocompetent C. diphtheriae and resulting
plasmid-carrying strains were applied to analyze spa
promoter activity before and in response to host cell
contact. For this purpose, Detroit562 cells were seeded
on coverslips 48 h prior to infection. Adhesion assays
with Detroit562 cells were carried out as described
below (4 μl of the inoculi dried on coverslips were used
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as no contact control). Subsequently, the infected cells
were fixed with 3% para-formaldehyde (PFA) in PBS (10
min, room temperature), coverslips were mounted on
glass slides using Fluoroprep (Biomerieux, Craponne,
France), and imaging was done on a LSM700 confocal
laser microscope (Carl Zeiss Micromaging GmbH, Jena,
Germany). GFP fluorescence emission of C. diphtheriae
without and with 1.5 h of host cell contact was deter-
mined for ISS4746 and the different reporter plasmids
(Table 3) by analyzing between 80 and 140 bacteria for
each condition using the open source ImageJ program
package (http://rsbweb.nih.gov/ij/index.html).
RNA preparation, hybridization analyses
Total C. diphtheriae RNA was prepared from 20 ml cul-
tures using the NucleoSpin RNA II Kit (Macherey
Nagel, Düren, Germany). For the generation of antisense
probes, internal DNA fragments of the corresponding
genes were amplified by PCR (oligonucleotide primers
for the different probes are listed in Table 2). The
reverse primers encoded the promoter region for T7
polymerase, which allowed in vitro transcription of
probes using T7 polymerase and subsequent labelling
with DIG RNA-labeling mix (Roche, Mannheim, Ger-
many). RNA (2.5 μg per time point) was spotted onto
nylon membranes using a Schleicher & Schuell (Dassel,
Germany) Minifold I Dot Blotter. Hybridization of
digoxigenin-labelled RNA probes was detected with X-
ray films (Hyperfilm MP; GE Healthcare, Munich, Ger-
many) using alkaline phosphatase-conjugated anti-digox-
igenin Fab fragments and CSPD as light-emitting
substrate as recommended by the supplier (Roche, Man-
nheim, Germany).
SDS-PAGE and Western blotting
Cell extracts of C. diphtheriae strains were separated by
Tricine-buffered 9.5% SDS gels as described [26], pro-
teins were transferred onto a polyvinylidene difluoride
membrane (PVDF, Immobilon-P, pore size 0.45 μm,
Millipore, Bedford, MA, USA) by semi-dry electroblot-
ting, and immunodetection of SpaD was performed with
antibodies directed against this pili subunit produced in
rabbits [13]. Antibody binding was visualised by using
appropriate anti-antibodies coupled to alkaline phospha-
tase (Sigma-Aldrich, Taufkirchen, Germany) and the
BCIP/NBT alkaline phosphatase substrate (Sigma-
Aldrich, Taufkirchen, Germany).
Atomic force microscopy (AFM)
Overnight cultures grown in 20 ml HI broth were
washed five times in 20 ml ice cold distilled water and
finally resuspended in 10 ml ice cold distilled water

Table 1 Bacterial strains and eukaryotic cell lines used in this study
Strains Description Reference
C. diphtheriae
DSM43988 non-toxigenic, isolated from throat culture DSMZ, Braunschweig,
Germany
DSM43989 tox+, unknown source DSMZ, Braunschweig,
Germany
DSM44123 non-toxigenic isolate, type-strain, unknown source DSMZ, Braunschweig,
Germany
ISS3319 C. diphtheriae var. mitis, non-toxigenic, isolated from patients affected by [1]
pharyngitis/tonsilitis
ISS4060 C. diphtheriae var. gravis, non-toxigenic, isolated from patients affected by [1]
pharyngitis/tonsilitis
ISS4746 C. diphtheriae var. gravis, non-toxigenic, isolated from patients affected by [21]
pharyngitis/tonsilitis
ISS4749 C. diphtheriae var. gravis, non-toxigenic, isolated from patients affected by [21]
pharyngitis/tonsilitis
NCTC13129 C. diphtheriae var. gravis, non-toxigenic, isolated from pharyngeal membrane, [2]
patient with clinical diphtheria
E. coli
DH5aMCR endA1 supE44 thi-1 l- recA1 gyrA96 relA1 deoR ∆(lacZYA-argF) U196 j80∆lacZ [9]
∆M15mcrA ∆(mmr hsdRMSmcrBC)
Salmonella enterica serovar Typhimurium
(S. Typhimurium)
NCTC12023 wild type identical to ATCC14028 NCTC, Colindale, UK
Cell lines
Detroit562 human hypopharyngeal carcinoma cells [20]

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Table 2 Oligonucleotides used in this study

Designation Sequence (5’®3’ direction) Application
DIP0235-as ctt ggt tgc cgg agc agc ctc ctt PCR spaD
DIP0235-s ccg aaa cca aga ccg aga aga ccg tca ag PCR spaD
DIP0237-as gca cac cag tca gcg cca agt cgc PCR spaE
DIP0237-s cgc gac tac gga acc gac acg ctg a PCR spaE
DIP0238-as gaa gtt gaa agg tcg gcc act aca gca a PCR spaF
DIP0238-s aaa ggg cta cta cat caa cat tcc aga cac PCR spaF
DIP2010asT7 cgc gta ata cga ctc act ata ggg tcc atc acg agg aac gac aac ggt ttt aga spaC probe
DIP2010-s cgc tac tcc tat ggg caa gca cct act gat att c spaC probe
DIP2011asT7 cgc gta ata cga ctc act ata ggg tgg tgg cga tgg cca gca gtc cga spaB probe
DIP2011-s tgc agc att cgc cga cga cca acc spaB probe
SondeDIP2013-asT7 ggg ccc taa tac gac tca cta tag gga gga ctg gag tgt tgc gcc g spaA probe
SondeDIP2013-s ggc gtc gaa aat caa gct gg spaA probe
DIP2223asT7 cgc gta ata cga ctc act ata ggg atc ggt aac ttc ctt acg gaa ctt ctc tgg cag spaI probe
DIP2223-s ttg ccc gcg gga act atc gac gga spaI probe
SondeDIP2226-asT7 ggg ccc taa tac gac tca cta tag ggc cca gcc cct gcg acg tc spaH probe
SondeDIP2226-s gga ggg ctg gga ggc agt ca spaH probe
DIP2227asT7 cgc gta ata cga ctc act ata ggg tcg acc ttg gac cag tgg acc tta gcg spaG probe
DIP2227-s ccg gac aga aga ttg ctg ccg agg ca spaG probe
PromDIP2223-as cgc ggg atc cag tag ggc gtc ctt tca gga construction reporter plasmid for spaI
PromDIP2223-s cgc gat gca tgt gac gcc attt tat gta cgc construction reporter plasmid for spaI
PromDIP2226-as cgc ggg atc cag ggt gtt ttc ctt tca gga construction reporter plasmid for spaH
PromDIP2226-s cgc gat gca tcg tca aag tta cgg ccg acc construction reporter plasmid for spaH
PromDIP2227-as cgc ggg atc cag tga aaa cac ctt cta ggg construction reporter plasmid for spaG
PromDIP2227-s cgc gat gca ttg aac cgg aat cat ttc tta construction reporter plasmid for spaG
PromDIP2013-neu-as cgc ggg atc ccc ctc aac tta ttt att tgg caa aaa g construction reporter plasmid for spaA
PromDIP2013-neu-s cgc gat gca tat tgg tga gac tac ttc ctt aaa gct ggt construction reporter plasmid for spaA

(centrifugation steps: 10 min 4,500 × g; resuspension by
vortexing). 5 μl of each sample were fixed on a glass
slide by drying using compressed air. An AFM instru-
ment (MFP-3D, Asylum Research, Santa Barbara, CA)
with standard silicon cantilever probes (NCH-W, Nano-
sensors, Neuchatel, Switzerland) was used under ambi-
ent laboratory conditions and operated in tapping mode
[26].
Measurement of transepithelial resistance
D562 cells were seeded in transwells (6.5 mm, 0.4 μm,
polyester membrane, 24 well plate, Corning Costar) at a
density of 5 × 10 4 cells per well and cultivated in
DMEM (Dulbecco’s modified Eagle’s medium, PAA;
high glucose, 10% FCS, 2 mM glutamine) for 14 days
until they build a transepithelial resistance of at least
1600 Ω·cm-2. Bacteria were subcultured (OD600 of 0.1
from overnight cultures) in 20 ml HI broth for 3.5 h.
The pellet was resuspended in 500 μl 1 × PBS. 50 μl of
the suspension were used for infection. Measurements
of transepithelial resistance of D562 cells during the
infection with C. diphtheriae were carried out with a
volt-ohm-meter (EVOM2, World Precision Instruments,
Berlin, Germany) every 30 min. After 3 h the

Table 3 Plasmids used in this study

Plasmid Description Reference/Source
pEPR1_spaApromoter promoter sequence (500 bp) of C. diphtheriae spaA gene This study
pEPR1_spaGpromoter promoter sequence (500 bp) of C. diphtheriae spaG gene This study
pEPR1_spaHpromoter promoter sequence (500 bp) of C. diphtheriae spaH gene This study
pEPR1_spaIpromoter promoter sequence (500 bp) of C. diphtheriae spaI gene This study
pEPR1 gfpuv, KmR, rep, per, T1, T2 [27]

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supernatant of infected D562 cells was removed and the
cells were incubated in fresh DMEM overnight to avoid
detrimental effects of excessive bacterial growth.
Adhesion assays
D562 cells were seeded in 24 well plates (bio-one Cell-
star, Greiner, Frickenhausen, Germany) at a density of 2
× 10 5 cells per well 48 h prior to infection. Bacteria
were subcultured (OD600 of 0.1 from overnight cultures)
in HI broth for 3.5 h and adjusted to an OD600 of 0.2. A
master mix of the inoculum was prepared in DMEM
without penicillin/streptomycin at a MOI of 200 (viable
counts experiments). The plates were centrifuged for 5
min at 500 × g to synchronize infection and subse-
quently incubated for 1.5 h. The cells were washed with
PBS nine times, detached with 500 μl trypsin solution
(0.12% trypsin, 0.01% EDTA in PBS) per well (5 min,
37°C, 5% CO 2 , 90% humidity) and lysed with 0.025%
Tween 20 for 5 min at 37°C. Serial dilutions were made
in pre-chilled 1 × PBS and plated on blood agar plates
to determine the number of colony forming units (cfu).
From this, the percentage of invasive bacteria was calcu-
lated [24].
Epithelial cell invasion model
D562 cells were seeded in 24 well plates (bio-one Cell-
star, Greiner, Frickenhausen, Germany) at a density of 2
× 105 cells per well 48 h prior to infection. Overnight
cultures of C. diphtheriae grown in HI were re-inocu-
lated to an OD 600 of 0.1 in fresh medium and grown
aerobically for another 3.5 h. An inoculum of approxi-
mately 1.6 × 10 8 bacteria ml -1 (MOI = 200) was pre-
pared in DMEM without penicillin/streptomycin and
500 μl per well were used to infect the D562 cells. The
plates were centrifuged for 5 min at 500 × g to synchro-
nize infection and subsequently incubated for 1.5 h (37°
C, 5% CO2, 90% humidity). The cells were washed thrice
with PBS and subsequently 500 μl of DMEM containing
100 μg ml-1 gentamicin were applied to each well to kill
remaining extracellular bacteria. After 2, 8.5 and 18.5 h
of incubation the cell layers were washed thrice with
PBS, detached by adding 500 μl trypsin solution (0.12%
trypsin, 0.01% EDTA in PBS) per well (5 min, 37°C, 5%
CO2, 90% humidity) and lysed for 5 min at 37°C with
0.025% Tween 20 to liberate the intracellular bacteria.
Serial dilutions of the inoculum and the lysates were
plated on blood agar plates to determine the number of
colony forming units (cfu).
Immuno-fluorescence
For immuno-fluorescence staining an antibody against
the C. diphtheriae surface proteome was used, which
was raised in rabbits. For antibody generation, surface
proteins were prepared as described [24]. As secondary
45
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antibodies Alexa-Fluor 488 (green) goat anti-rabbit IgGs
and Alexa-Fluor 568 (red) goat anti-rabbit IgGs were
used. Phalloidin Alexa-Fluor 647 was used for staining
the cytoskeleton of D562 cells. All antibodies were
diluted in blocking solution (2% goat serum, 2% BSA in
PBS). Cell lines were seeded on round coverslips in 24
well plates 48 h prior to infection and fixed after the
respective assay with 3% PFA in PBS (10 min at room
temperature). For immuno-fluorescence staining the
preparations were washed thrice with 1 × PBS and incu-
bated with primary antibodies for at least 1 h at room
temperature, washed thrice with PBS again, and subse-
quently incubated with Alexa-Fluor 488 (green) goat
anti-rabbit for 45 min. After permeabilization with 0.1%
Triton X-100 (5 min room temperature) and three
washing steps with PBS, staining with Alexa-Fluor 568
(red) goat anti-rabbit was carried out as described
above. F-actin was stained in parallel with Phalloidin-
Alexa-Fluor 647 (blue). Coverslips were mounted on
glass slides using Fluoroprep (Biomerieux, Craponne,
France). Imaging was done on an AxioVert 200 M
inverted optical microscope (Carl Zeiss Micromaging
GmbH, Jena, Germany).
Acknowledgements
The authors wish to thank C. von Hunolstein (Istituto Superiore di Sanità,
Rome) for providing strain ISS3319, ISS4060, ISS4746, and ISS4749, as well as
H. Ton-That (University of Texas Health Science Center, Houston, TX) for
SpaD antibodies, SpaD protein, and chromosomal DNA of strain NCTC13129.
The help of R. G. Gerlach (Mikrobiologisches Institut des Universitätsklinikums
Erlangen) to establish the epithelial resistance assay is gratefully
acknowledged. This study was financially supported by the Deutsche
Forschungsgemeinschaft for in frame of SFB 796 (projects B5, C1, and Z).
Author details
1
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-
Nürnberg, Nürnberg, Germany. 2Lehrstuhl für Angewandte Physik, Friedrich-
Alexander-Universität Erlangen-Nürnberg, Nürnberg, Germany.
3
Mikrobiologisches Institut des Universitätsklinikums Erlangen, Erlangen,
Germany. 4Abteilung Mikrobiologie, Universität Osnabrück, Osnabrück,
Germany.
Authors’ contributions
LO carried out invasion assays, fluorescence microscopy, TER measurements,
and RNA and protein analyses, MHö carried out adhesion and invasion
analysis, AFM experiments were carried out in cooperation with JR and TES,
MHe supported LO and MHö in respect to cell culture, AB supervised the
experiments of LO and MHö and was responsible for the draft and final
version of the manuscript. All authors read and approved the final
manuscript.
Received: 11 August 2010 Accepted: 13 October 2010
Published: 13 October 2010
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46
 Publications

3.3 The complete genome sequence of Corynebacterium

pseudotuberculosis FRC41 isolated from a 12-year-old girl with
necrotizing lymphadenitis reveals insights into gene-regulatory
networks contributing to virulence (Trost et al, 2010b)

47
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RESEARCH ARTICLE Open Access

The complete genome sequence of

Corynebacterium pseudotuberculosis FRC41
isolated from a 12-year-old girl with necrotizing
lymphadenitis reveals insights into gene-
regulatory networks contributing to virulence
Eva Trost1,2, Lisa Ott3, Jessica Schneider1,2,4, Jasmin Schröder1, Sebastian Jaenicke4, Alexander Goesmann4,
Peter Husemann5, Jens Stoye5, Fernanda Alves Dorella6, Flavia Souza Rocha6, Siomar de Castro Soares6,
Vívian D’Afonseca6, Anderson Miyoshi6, Jeronimo Ruiz7, Artur Silva8, Vasco Azevedo6, Andreas Burkovski3,
Nicole Guiso9, Olivier F Join-Lambert10, Samer Kayal11, Andreas Tauch1*

Abstract
Background: Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely
infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as
C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing
lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human
isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally
annotated.
Results: Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with
a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc
homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-
regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41.
Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can
probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery
of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are
encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases,
neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved
in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a
key role in controlling the expression of several genes contributing to virulence.
Conclusion: The functional data deduced from the genome sequencing and the extended knowledge of virulence
factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting
its survival under unfavorable environmental conditions encountered in the mammalian host.

* Correspondence: tauch@cebitec.uni-bielefeld.de
1
Institut für Genomforschung und Systembiologie, Centrum für
Biotechnologie, Universität Bielefeld, Universitätsstraße 27, D-33615 Bielefeld,
Germany
Full list of author information is available at the end of the article

© 2010 Trost et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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Background
Corynebacterium pseudotuberculosis is generally regarded
as an important animal pathogen and the etiological agent
of a disease that is commonly called caseous
lymphadenitis [1,2]. This bacterium is predominantly iso-
lated from sheep and goats (biovar ovis), but has been
recognized also in other animals, including horses and cat-
tle (biovar equi) [1]. The importance of caseous lymphade-
nitis varies greatly around the world, but this disease is
found in all major sheep and goat production areas [2]. C.
pseudotuberculosis is a significant cause of morbidity in
sheep and goats, and caseous lymphadenitis in these ani-
mals resulted in economic losses, for instance in wool,
milk and meat production [2,3]. C. pseudotuberculosis is a
facultative intracellular pathogen that is able to survive
and grow in macrophages, thus escaping the immune
response of the host [1,4]. A close phylogenetic relation-
ship between C. pseudotuberculosis and Corynebacterium
ulcerans was proposed as both species are unique among
the corynebacteria in producing phospholipase D. The
sphingomyelin-degrading enzyme is regarded as the major
virulence factor for C. pseudotuberculosis [5,6]. This exo-
toxin promotes the hydrolysis of ester bonds in sphingo-
myelin in mammalian cell membranes and contributes to
the spread of the bacterium from the initial site of infec-
tion to secondary sites within the host.
Although infections due to C. pseudotuberculosis are
predominantly observed in sheep and goats, infections
due to this pathogen also occur in humans [7,8]. The
number of human infections is rare, but it might be
underestimated as corynebacteria are often considered
as skin contaminants in clinical specimens [9]. Published
cases of human infections by C. pseudotuberculosis
usually presented as suppurative lymphadenitis [7,8],
with the exception of one case of eosinophilic pneumo-
nia [10]. Most patients revealed a classical risk exposure
of close contact with animals, in particular with sheep.
C. pseudotuberculosis infects humans via superficial
wounds, forming abscesses in the regional draining
lymph nodes after an incubation period ranging from
weeks to months. In most of the published cases, anti-
biotic treatment alone was unsuccessful and the antimi-
crobial therapy was therefore supplemented by surgical
interventions [7,8]. The general problem in achieving an
effective treatment of C. pseudotuberculosis infections in
humans and animals is probably related to the faculta-
tive intracellular lifestyle of this bacterium, as it can sur-
vive and multiply in macrophages [4]. The cell death
and subsequent release of the pathogen lead to necrotic
lesions and the formation of thick collagen capsules that
cannot be penetrated by antibiotics [11].
In the present study, we characterize the genome of
C. pseudotuberculosis FRC41 that was isolated from the
inguinal lymph node of a 12-year-old French girl with
49
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necrotizing lymphadenitis [12]. This patient had no
underlying disease or predisposing conditions. The diag-
nosis of necrotizing lymphadenitis was supported by 16
S rDNA gene sequencing, a taxonomic classification of
the bacterial isolate and Western blot assays revealing
the presence of phospholipase D in the patient’s serum.
The patient relapsed many times despite a surgical drai-
nage and an adapted antimicrobial treatment, although
C. pseudotuberculosis FRC41 turned out to be highly
susceptible to a wide range of antibiotics in vitro. How-
ever, the patient recovered after a broad spectrum intra-
venous antimicrobial therapy with imipenem-cilastatin,
rifampin and ofloxacin for four months, followed by an
oral therapy with rifampin and ofloxacin for six months
[12]. Here, we report the functional annotation of the
complete genome sequence of C. pseudotuberculosis
FRC41, the detection of potential virulence factors and
the deduced gene-regulatory networks controlling their
expression.
Results and discussion
Pyrosequencing and annotation of the C.
pseudotuberculosis FRC41 genome
The DNA sequence of the C. pseudotuberculosis FRC41
chromosome was determined by a whole-genome shotgun
approach using pyrosequencing. A quarter of a sequencing
run with the Genome Sequencer FLX Instrument yielded
286,938 reads and 94,447,635 bases that were assembled
into ten large contigs (≥ 500 bases) and one small contig
(313 bases), indicating a very low number of repetitive
sequences in the C. pseudotuberculosis FRC41 genome. A
search for repetitive DNA elements in the complete gen-
ome sequence revealed the absence of insertion sequences
in C. pseudotuberculosis FRC41, whereas the small contig
was present in three tandem copies in the assembled chro-
mosome (data not shown). The remaining gaps were
closed by a PCR strategy that was supported by the related
reference contig arrangement tool r2cat [13], using the
Corynebacterium diphtheriae NCTC 13129 genome
sequence as a reference [14]. The final assembly of the
DNA sequences yielded a circular chromosome with a size
of 2,337,913 bp and a mean G+C content of 52.2% (Figure
1A). Considering the final size of the C. pseudotuberculosis
FRC41 chromosome, a 40-fold coverage was initially
obtained by pyrosequencing.
The annotation of the C. pseudotuberculosis FRC41
genome sequence was performed with the GenDB soft-
ware system [15] and resulted in the detection of 2,110
protein-coding regions. Furthermore, 49 tRNA genes
were predicted by the tRNAscan-SE program [16] and
four rrn operons were detected on the leading strands
of the chromosome (Figure 1B). A plot of the calculated
G/C skew [(G-C)/(G+C)] indicated a bi-directional repli-
cation mechanism of the C. pseudotuberculosis
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Figure 1 Annotation and analysis of the C. pseudotuberculosis FRC41 genome sequence. (A), Plot of the annotated C. pseudotuberculosis
FRC41 chromosome. The circles represent from the outside: circle 1, DNA base position [kb]; circle 2, protein-coding regions transcribed
clockwise; circle 3, protein-coding regions transcribed anticlockwise; circle 4, G/C skew plotted using a 10-kb window; circle 5, G+C content
plotted using a 10-kb window. The protein-coding regions are coloured according to their functional classification into the Clusters of
Orthologous Groups of proteins [111]. (B), Architecture imparting sequences in the C. pseudotuberculosis FRC41 chromosome. The distribution of
the octamers G(A/T/C)GGGGGA and (T/C)GGGGGAG on the leading and lagging strands is shown. The origin of replication (oriC) is marked. The
deduced dif locus is located at around 1.1 Mbp of the chromosomal map. The 28-bp sequence of the predicted dif region is shown. The
location of the four rRNA operons (rrnA-rrnD) on the leading strands is indicated. (C), Synteny between the chromosomes of C.
pseudotuberculosis FRC41 and C. diphtheriae NCTC 13129. The X-Y plot is composed of dots forming syntenic regions between both
chromosomes. The dots represent predicted C. pseudotuberculosis FRC41 proteins having an orthologue in the genome of C. diphtheriae NCTC
13129 with co-ordinates corresponding to the position of the respective coding region in each genome sequence and indicated in kb.
Orthologous proteins were detected by reciprocal best BLASTP matches.

chromosome (Figure 1A). According to the presence
and distribution of six conserved DnaA boxes, the oriC
is located downstream of the dnaA coding region [17].
The G/C skew and the biased distribution of architec-
ture imparting sequences (AIMS) on the leading and
lagging strands indicated the presence of a dif region
[18] at the expected position of 180° from oriC, dividing
the chromosome of C. pseudotuberculosis FRC41 into
two replichores of similar size (Figure 1B). Synteny ana-
lysis by reciprocal best matches with BLASTP [19]
revealed a highly conserved order of orthologous genes
between the chromosomes of C. pseudotuberculosis
FRC41 and C. diphtheriae NCTC 13129 (Figure 1C),
which is consistent with the close phylogenetic relation-
ship of both species [1] and the observation that genetic
rearrangements are rare in the genomes of species
belonging to the main lineage of the genus Corynebac-
terium [20,21]. The calculated reciprocal best BLASTP
hits [19] were used also to compare the predicted
proteome of C. pseudotuberculosis FRC41 with the com-
plete set of proteins encoded in the genome of
C. diphtheriae NCTC 13129 [14]. This comparative con-
tent analysis at the proteome level revealed that 1610
proteins (76.3%) of C. pseudotuberculosis FRC41 share a
homologue in the genome of C. diphtheriae NCTC
13129 (data not shown). The characteristic features of
C. pseudotuberculosis FRC41 are apparently based on a
distinct gene set, defining its lifestyle and pathogenicity,
such as the pld gene encoding phospholipase D [22]. In
the following sections, we describe a collection of rele-
vant genes contributing to the lifestyle and pathogenicity
of C. pseudotuberculosis FRC41 and deduce their inte-
gration into a transcriptional gene-regulatory network.
The transcriptional regulatory repertoire of C.
pseudotuberculosis FRC41
The repertoire of candidate transcription regulators
encoded in the C. pseudotuberculosis FRC41 genome

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was deduced from the functional genome annotation
taking into account the comprehensive knowledge of
the reconstructed gene-regulatory network of Coryne-
bacterium glutamicum ATCC 13032 [23]. A collection
of 83 genes encoding DNA-binding transcription regula-
tors, sigma factors and response regulators of two-
component systems can be regarded as the minimal reg-
ulatory repertoire of C. pseudotuberculosis FRC41
(Figure 2). This set of candidate transcription regulators
represents 3.9% of the predicted protein-coding genes of
the C. pseudotuberculosis FRC41 genome. This value is
in agreement with previous observations that less than
10% of the total number of predicted proteins are asso-
ciated with transcriptional regulatory processes in
bacteria [24] and is in the range known from other
pathogenic corynebacteria, such as C. diphtheriae and
Corynebacterium jeikeium [25]. The collection of poten-
tial transcription regulators was grouped into regulatory
protein families according to their amino acid sequence
similarities and domain organizations [26]. This bioin-
formatic classification assigned the candidate transcrip-
tion regulators to 31 regulatory protein families, with
one regulator (cpfrc_01413) remaining unclassified. The
regulatory protein families detected in C. pseudotubercu-
losis FRC41 vary significantly in their number of
Figure 2 Classification of the predicted transcriptional regulatory repertoire of C. pseudotuberculosis FRC41 into regulatory protein
families. The number of candidate transcription regulators assigned to a regulatory protein family is shown.
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representatives (Figure 2). The largest family of DNA-
binding transcription regulators is TetR with 11 mem-
bers, followed by GntR with 5 proteins. The TetR family
of transcription regulators is widely distributed among
bacterial species [27] and is also the most prevalent
group of regulatory proteins in other corynebacteria
[25]. It is noteworthy that the detected collection of
transcription regulators includes only 23 out of the 24
proteins that hitherto constituted the core of DNA-
binding transcription regulators in corynebacteria, as C.
pseudotuberculosis FRC41 lacks a gene for an orthologue
of the conserved TetR regulator MfsR (Cg0454) [23,25].
The knowledge of the transcriptional regulatory reper-
toire of C. pseudotuberculosis FRC41 was combined
with regulons contributing to virulence by bioinformatic
motif searches for DNA-binding sites of prominent reg-
ulatory proteins.
Iron regulation in physiology and virulence of C.
pseudotuberculosis
For most bacteria, iron is essential as a cofactor for
proteins involved in important cellular functions, such
as DNA biosynthesis and respiration [28]. Thus, iron
acquisition is a vital function for bacterial survival. As
iron limitation is a common strategy by which a
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mammalian host suppresses bacterial growth, iron has
a decisive role in infectious diseases. On the one hand,
pathogenic bacteria have to compete for iron in the
host so that they can multiply and establish a success-
ful infection. On the other hand, they must regulate
iron metabolism to prevent excess iron that can initi-
ate the generation of toxic oxygen radicals from nor-
mal products of metabolism by Fenton chemistry.
Bacteria have solved the problem of iron acquisition
and homeostasis by encoding a variety of high-affinity
uptake systems that are tightly regulated at the tran-
scriptional level [28]. The fagCBA-fagD genes of
C. pseudotuberculosis, for instance, encode an iron
uptake system that is regulated by iron in vitro [29]. A
fag mutant showed a reduced virulence in a goat
model of caseous lymphadenitis when compared with a
wild-type control, although no defect in iron utilization
by the mutant strain was determined [29]. As the
expression of the fagABCD genes contributed to the
virulence of C. pseudotuberculosis from animal sources,
the complete genome sequence of FRC41 was screened
for the presence of these genes, additional iron uptake
systems and the responsible transcription regulator
(Figure 3).

Figure 3 Regulons involved in iron regulation of C. pseudotuberculosis FRC41. The DtxR and RipA regulons controlling iron homeostasis
and the respective gene-regulatory interactions were deduced from a genome-scale network transfer approach [114] and the combined use of
hidden Markov models and position weight matrices [74]. The assignment of the transcription regulators into the regulatory protein families of
C. pseudotuberculosis FRC41 is indicated. Predicted DNA-binding sites are listed by sequence and are shown as black boxes, regulated target
genes are shown as arrows and coloured as follows: grey, regulatory gene; yellow, gene involved in iron uptake and iron storage; orange, gene
involved in central metabolism; red, gene contributing to the oxidative stress response. The highly conserved coregulation of the hmuO gene by
DtxR and the response regulators HrrA and ChrA in corynebacteria is indicated [23]. The 19-bp consensus sequence of the DtxR-binding site of
C. pseudotuberculosis FRC41 is shown as DNA sequence logo. The 16 predicted DNA-binding sites of DtxR were used as input data for the
WebLogo tool [115].

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The dtxR gene of C. pseudotuberculosis FRC41
encodes a homologue of the diphtheria toxin repressor
DtxR that is activated by iron and controls a complex
gene-regulatory network involved in iron homeostasis in
corynebacteria [30,31]. The detection of a putative
DtxR-binding site in the fagA-fagD intergenic region
supports the previously observed differential expression
pattern of the fagABC operon in vitro [29]. We com-
bined the functional annotation of the C. pseudotubercu-
losis FRC41 genome sequence with a bioinformatic
motif search for DtxR-binding sites using a hidden Mar-
kov model and a position weight matrix with input data
from the DtxR regulon of C. glutamicum [31]. In this
way, additional genes were assigned to the DtxR regulon
of C. pseudotuberculosis FRC41, including several gene
clusters involved in the utilization of various host com-
pounds as iron sources (Figure 3). One gene region
revealed similarity to the hemin utilization system Hmu-
TUV from C. diphtheriae [32]. The corresponding
hemin binding protein HtaA is probably associated with
the cell envelope and involved in the utilization of heme
iron [33]. Two gene clusters assigned to the DtxR regu-
lon of C. pseudotuberculosis FRC41 include htaA-like
genes (htaD and htaF) that are associated with genes
encoding membrane proteins (htaE and htaG), suggest-
ing a role of these clusters in the acquisition of iron
from the host (Figure 3). The hmuO gene of the DtxR
regulon encodes heme oxygenase that releases iron from
the protoporphyrin ring of heme and facilitates the
acquisition of iron from heme and hemoglobin [34].
Because of the potential toxicity of both, iron and heme,
the expression of hmuO in C. diphtheriae is under com-
plex control, comprising the iron-responsive repressor
DtxR and the heme-dependent activators ChrA and
HrrA that are part of the two-component signal trans-
duction systems ChrA-ChrS and HrrA-HrrS [35,36]. A
BLAST search across the C. pseudotuberculosis FRC41
genome revealed two response regulators that share
similarity with ChrA and HrrA. As the hrrA gene of C.
pseudotuberculosis FRC41 is part of the DtxR regulon, a
complex hierarchical control of hmuO gene expression
might be established in this bacterium (Figure 3).
Iron acquisition can moreover involve the synthesis
and secretion of high-affinity iron chelators, termed
siderophores, which are synthesized by nonribosomal
peptide synthetases or by biosynthesis pathways inde-
pendent of these multimodular enzymes [37,38]. The
genome of C. pseudotuberculosis FRC41 contains two
DtxR-regulated gene clusters that are probably asso-
ciated with pathways for siderophore biosynthesis inde-
pendent of nonribosomal peptide synthetases (Figure 3).
The ciu locus comprises the ciuABCD (ABC-type trans-
porter), ciuE (siderophore biosynthesis-related protein)
and ciuF (putative efflux protein) genes. The predicted
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product of the ciuE gene is similar to aerobactin bio-
synthesis enzymes [39]. The expression of the ciuA gene
encoding the lipoprotein receptor of the ABC transport
system has been detected in vivo by a reporter transpo-
son system [40]. The ciu gene region detected in the
genome of C. pseudotuberculosis FRC41 is similar to the
ciu gene locus from C. diphtheriae NCTC 13129, with
the exception that it lacks the ciuG gene, encoding a
protein of unknown function [41].
The second DtxR-regulated gene cluster related to
siderophore biosynthesis and excretion includes four
genes probably constituting the biosynthesis pathway
(ogs, ocd, odc and tsb), a gene encoding an efflux pro-
tein (mdtK) and four genes encoding an ABC-type
transporter (stsABCD) (Figure 3). Ornithine cyclodea-
minase (ocd) and ornithine decarboxylase (odc) as well
as monooxygenase (ogs) and synthetase (tsb) functions
encoded in this gene cluster are components of widely
distributed routes for siderophore biosynthesis [39].
Additional DtxR-binding sites were detected in front of
ftn (ferritin) and fas (fatty acid synthase) and the
sdhCAB (succinate dehydrogenase) operon that are
also part of the DtxR regulon in C. glutamicum
[31,42]. Ferritins act primarily in iron storage and are
thus central to the natural regulation of iron in the
bacterial cell [28].
Furthermore, the ripA gene encoding a DNA-binding
transcription regulator of the AraC protein family was
assigned to the DtxR regulon of C. pseudotuberculosis
FRC41 (Figure 3). The expression of the orthologous
ripA gene from C. glutamicum is also controlled directly
by DtxR [42]. Under iron limitation, the RipA protein
acts as a repressor of genes coding for iron proteins in
C. glutamicum [42]. Candidate RipA-binding sites were
detected by bioinformatic pattern searches in the gen-
ome sequence of C. pseudotuberculosis FRC41 in front
of the acn (aconitase) gene and upstream of the sdhCAB
(succinate dehydrogenase) and cydABDC operons (cyto-
chrome bd-type menaquinol oxidase and ABC-type
transporter), thereby linking the availability of iron with
the expression of genes in the citrate cycle and the
respiratory energy metabolism of C. pseudotuberculosis.
Due to its high oxygen affinity, the cytochrome bd oxi-
dase is used in many bacteria under microaerobic
growth conditions [43]. Additional RipA-binding sites
were detected in front of the katA (catalase) gene and in
the dps-fpg1 (DNA protection during starvation protein,
formamidopyrimidine-DNA glycosylase) intergenic
region, integrating protective functions into the RipA
regulon (Figure 3). Dps-like proteins effectively protect
DNA against oxidizing agents by nonspecific DNA-bind-
ing and physical sequestration that limits DNA accessi-
bility to detrimental factors [44]. They also act as
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oxidation of ferrous iron to ferric iron by H2O2, which
prevents the formation of hydroxyl radicals by the Fen-
ton reaction [45]. The lsr2 gene of C. pseudotuberculosis
FRC41 encodes the multifunctional histone-like protein
and transcription regulator Lsr2 (Figure 2) that shares a
number of physical properties with Dps-like proteins
and the ability to bind DNA sequences with little speci-
ficity [46]. Like Dps, the Lsr2 protein may physically
protect corynebacterial DNA against reactive oxygen
intermediates [47]. Formamidopyrimidine-DNA glycosy-
lase is a primary participant in the repair of DNA
lesions caused by oxidative damage [48]. A second gene
(fpg2) encoding formamidopyrimidine-DNA glycosylase
was detected in the genome of C. pseudotuberculosis
FRC41, but the upstream region of fpg2 apparently lacks
a RipA-binding site. Catalase is another important pro-
tective component in the bacterial oxidative stress
response which is involved in the detoxification of H2O2
[49]. Hence, the gene composition of the DtxR-RipA
gene-regulatory network revealed the interdependence
of iron metabolism and oxidative stress response and
the regulatory connection between distinct physiological
functionalities of the corynebacterial cell, including an
important role in virulence of C. pseudotuberculosis.
Manganese and zinc regulation in physiology and
virulence of C. pseudotuberculosis
Peroxynitrite and other reactive nitrogen and oxygen
intermediates are produced by macrophages as part of
their antimicrobial response [50]. Consequently, many
pathogenic bacteria have evolved protection mechanisms
against these reactive nitrogen and oxygen intermediates
that have potent antimicrobial activity [51]. Four genes
encoding protective enzymes probably involved in corre-
sponding detoxification reactions were identified in the
genome sequence of C. pseudotuberculosis FRC41,
including alkyl hydroperoxide reductase (ahpCD), man-
ganese-dependent superoxide dismutase (sodA) and cop-
per, zinc-dependent superoxide dismutase (sodC). A
bioinformatic pattern search with actinobacterial OxyR-
binding sites as input data revealed that these genes are
most likely under transcriptional control by OxyR (Fig-
ure 4A). The oxyR gene of C. pseudotuberculosis FRC41
is linked to the ahpCD genes and its gene product may
act as a repressor of gene expression [52]. AhpC is a
member of a large family of peroxidases that contribute
to the antioxidant defense in bacteria [53]. The AhpC
protein directly reduces peroxides and is in turn
reduced by AhpD [54]. The mycobacterial AhpC protein
also catalyzes the rapid conversion of peroxynitrite to
nitrite to avoid the formation of deleterious nitrogen
dioxide and hydroxyl radicals [55]. Superoxide dismu-
tase converts superoxide anions into molecular oxygen
and H 2 O2 , the latter being broken in turn to H2 O by
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the enzymatic activity of catalase [56]. Superoxide dis-
mutases were classified into three evolutionarily distinct
families according to the type of metal cofactors. Most
bacteria possess either a manganese-dependent (Mn-
SOD) or an iron-dependent (Fe-SOD) superoxide
dismutase in their cytoplasm, while secreted copper,
zinc-dependent superoxide dismutases (Cu,Zn-SODs)
have been detected in pathogenic or endosymbiontic
bacteria [57]. The genome of C. pseudotuberculosis
FRC41 encodes two types of superoxide dismutases, a
cytoplasmic Mn-SOD (SodA) and a secreted Cu,Zn-
SOD (SodC) that is characterized by a lipobox motif
and may be anchored in the cell membrane [58]. The
extracellular location of SodC suggests that it may pro-
tect the surface of C. pseudotuberculosis cells against
superoxide generated externally by the mammalian host
cells. Likewise, the mycobacterial SodC protein contri-
butes to the resistance of Mycobacterium tuberculosis
against oxidative burst products generated by activated
macrophages [59,60]. The protective activity of Cu,Zn-
SODs has been associated with virulence in many bac-
teria, such as Neisseria meningitidis and Haemophilus
ducreyi [61,62]. However, further experimental work is
necessary to elucidate which protective enzyme contri-
butes to the virulence of C. pseudotuberculosis.
As manganese and zinc ions are apparently involved as
cofactors in the oxidative stress response of pathogens, the
corresponding regulons involved in metal ion uptake were
identified in the genome sequence of C. pseudotuberculosis
FRC41 (Figure 4B). The uptake of manganese in C. pseu-
dotuberculosis FRC41 is mediated by a typical manganese
ABC-type transport system (mntABCD) that is negatively
controlled at the transcriptional level by the metalloregula-
tor MntR (Figure 4B). The manganese ABC transporter is
composed of: a lipoprotein receptor (MntA), anchored to
the cell membrane and functioning as an extracellular
cation-binding protein; a cytoplasmic ATP-binding protein
(MntB); and two integral membrane proteins (MntC and
MntD) that mediate the cation flux [63]. A homologous
gene cluster and its manganese-dependent transcriptional
control by the metalloregulator MntR have been examined
in the genome of C. diphtheriae [64]. The uptake of zinc
ions in C. pseudotuberculosis FRC41 is probably mediated
by two ABC transport systems (znuB1C1A1 and znu-
B2A2C2), as both gene regions are specified by the pre-
sence of Zur-binding sites (Figure 4C). The Zur protein is
a metalloregulator of the ferric uptake regulator (FUR)
family of DNA-binding transcription regulators [65,66].
The znu ABC transporter genes are key components of
actinobacterial Zur regulons and their expression is gener-
ally repressed by Zur in a zinc-dependent manner [66].
Zinc resistance might be facilitated in C. pseudotuberculo-
sis FRC41 by the ArsR-type transcription regulator Znr
that probably controls the expression of the czcE gene
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Figure 4 Regulons involved in the oxidative stress response and in zinc and manganese regulation of C. pseudotuberculosis FRC41. (A),
The OxyR regulon controlling the oxidative stress response. (B), The MntR operon controlling the uptake of manganese. (C), The Zur regulon
controlling zinc metabolism. The assignment of the transcription regulators into the regulatory protein families of C. pseudotuberculosis FRC41 is
indicated. The regulatory interactions were deduced from a genome-scale network transfer approach [114] and the combined use of a hidden
Markov model and a position weight matrix [74]. Predicted DNA-binding sites are shown as black boxes and indicated by DNA sequence. The
regulated target genes are shown as arrows and coloured as follows: grey, regulatory gene; red, gene contributing to the oxidative stress
response; dark blue, gene involved in maganese uptake; light blue, gene involved in zinc metabolism.

encoding a cobalt/zinc/cadmium efflux system [23,67,68].
The yciC gene coding for a putative P-loop GTPase of the
COG0523 protein family is also part of the Zur regulon in
C. pseudotuberculosis FRC41 (Figure 4C). The YciC pro-
tein may function as a metallochaperone/insertase to
enable the in vivo assembly of zinc-containing proteins
under environmental conditions of zinc deficiency [69].
Moreover, the genome-wide motif search for Zur-
binding sites in C. pseudotuberculosis FRC41 detected
three genes (cpfrc_00168, cpfrc_00313, cpfrc_02059)
encoding proteins with a carboxy-terminal sorting
(LPxTG) signal that is generally used by Gram-positive
bacteria to anchor proteins to the cell wall [70]. The
Cpfrc_00168 protein contains two CnaB-like domains
that may be involved in the positioning of a ligand bind-
ing domain away from the corynebacterial cell surface
and is encoded adjacent to a putative sialoprotein-bind-
ing protein. The secreted proteins Cpfrc_00313 and
Cpfrc_02059 contain actinobacterial surface-anchored
protein domains for their covalent attachment to the
cell wall [56]. The Cpfrc_00313 protein is encoded next
to the components of a transporter and may act
together with the Cpfrc_00314 protein as a substrate
receptor for this system. Hence, the reconstruction of
regulons participating in metal ion uptake of C. pseudo-
tuberculosis FRC41 led to the detection of genes that
may fulfill novel functions in sensing the presence of
zinc in the environment.
Genes encoding adhesive pili in C. pseudotuberculosis
FRC41
The complete set of predicted protein-coding regions of
C. pseudotuberculosis FCR41 was subsequently screened
for the presence of further proteins containing a typical
sorting signal. This approach revealed ten additional
proteins without any conserved domain organization
[56] that were annotated as hypothetical proteins with
LPxTG motif and, more interestingly, six proteins show-
ing similarity to subunits of adhesive pili from
C. diphtheriae NCTC 13129 (Figure 5). The correspond-
ing coding regions are organized in two gene clusters
that include sortase genes involved in the process of
pilus polymerization [71,72]. The housekeeping sortase
gene (cpfrc_02014; srtD) of C. pseudotuberculosis
FCR41, necessary for the cell wall anchoring of pilin
monomers and pilus polymers [73], is located elsewhere
in the chromosome. The adhesive pili of C. pseudotuber-
culosis FCR41 consist of major pilin subunits (SpaA,
SpaD), minor pilin subunits (SpaB, SpaE) and tip pro-
teins (SpaC, SpaF) that are characterized by conserved
amino acid motifs (Figure 5). The function of the
hypothetical proteins SpaX and SpaY encoded in the

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Figure 5 Gene regions encoding adhesive pili of C. pseudotuberculosis FRC41. The gene clusters involved in the synthesis of adhesive (Spa-
like) pili of C. pseudotuberculosis FRC41 are shown. The gene clusters encode sortases required for the assembly of the pilus (blue), major pilins
(red), minor pilins (yellow), pilus tip proteins (green), and proteins of unknown function (grey). The detected sorting (LPxTG) signals are indicated.
Specifically marked in the major pilin proteins are the characteristic pilin boxes (blue) and E-boxes (white). The predicted binding of the
transcription regulator GlxR in the spaA-srtB intergenic region is shown.
spa gene regions of C. pseudotuberculosis FCR41 is cur-
rently unknown. According to alkaline phosphatase
(phoZ) gene fusions generated with a reporter transpo-
son system in C. pseudotuberculosis, at least the SpaABC
pilus is expressed in this species [1]. The spaABC gene
cluster contains a putative DNA-binding site for the
cAMP-sensing transcription regulator GlxR in the spaA-
srtB intergenic region (Figure 5), thereby connecting the
expression of pilus genes with a global gene-regulatory
network in C. pseudotuberculosis [74,75]. The adhesive
pili of C. diphtheriae NCTC 13129 are covalently
anchored to the cell wall and can mediate the initial
adhesion to host tissues and other bacterial cells [76].
The adherence of C. diphtheriae to pharyngeal epithelial
cells is mediated also by the minor pilin of the adhesive
pilus, as this pilin subunit is covalently anchored to the
cell wall and can provide tight contact between the bac-
terial cell and the host tissue in the absence of a pilus
shaft [77]. Considering a similar functioning of the pre-
dicted SpaABC and SpaDEF pilus proteins from C. pseu-
dotuberculosis, either a complete pilus structure or the
minor pilins SpaB and SpaE can probably make the
initial contact with host cell receptors to enable addi-
tional ligand-receptor interactions and to facilitate the
efficient delivery of virulence factors and intracellular
invasion.
Candidate virulence factors of C. pseudotuberculosis
FRC41 and their integration into a gene-regulatory
network
The observation that adhesive pili promote the adher-
ence of C. diphtheriae to host tissue suggests that the
SpaABC and SpaDEF pili from C. pseudotuberculosis
FRC41 can be regarded as potential virulence factors
[77]. To extend the view on proteins contributing to the
pathogenicity of C. pseudotuberculosis FRC41, the
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genome sequence was screened for further candidate
genes encoding virulence factors (Table 1). The major
virulence factor of C. pseudotuberculosis is the sphingo-
myelin-degrading phospholipase D that facilitates the
persistence and spread of the bacterium within the host
[5,6]. The expression of the pld gene is regulated by
multiple environmental stimuli, including heat, and
plays a role in the reduction of macrophage viability fol-
lowing infection [78,79]. The secreted corynebacterial
protease CP40 was detected previously as a protective
antigen of C. pseudotuberculosis and shown to be of the
serine protease type, although BLAST searches revealed
homology to endoglycosidases [80]. The extracellular
CP40 enzyme may contribute to the virulence of
C. pseudotuberculosis by its proteolytic activity, but the
enzymatic activity of CP40 was not detectable in culture
supernatants [80]. However, vaccination of sheep with
this antigen resulted in protection against infection with
C. pseudotuberculosis, probably by affecting directly the
function of the CP40 protein and indirectly the growth
of the pathogen [11]. The genome sequence of C. pseu-
dotuberculosis FRC41 revealed three additional genes
encoding secreted proteases, including two subtilisin-
like serine proteases and one trypsin-like serine protease
(Table 1). Extracellular proteases may exhibit a wide
range of pathogenic potentials when interacting with the
defense mechanisms and tissue components of the host.
Redundant enzymatic systems are moreover suitable to
promote the survival of pathogens under unfavorable
environmental conditions encountered in the infected
host [81]. The genome sequence of C. pseudotuberculo-
sis FRC41 also encodes a secreted protein of the SGNH-
hydrolase subfamily (Table 1). SGNH-hydrolases are a
diverse family of lipases and esterases which are known
to act as virulence factors in other bacteria, such as
Streptomyces scabies, the causal agent of the potato scab
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Table 1 Candidate determinants contributing to virulence of C. pseudotuberculosis FRC41

Identifier Gene Predicted protein function
cpfrc_00029 pld phospholipase D (sphingomyelin-degrading enzyme)
cpfrc_01895 cpp corynebacterial protease CP40 (serine protease)
cpfrc_00397 - secreted subtilisin-like serine protease
cpfrc_01634 - secreted subtilisin-like serine protease
cpfrc_00562 - secreted trypsin-like serine protease
cpfrc_00536 - secreted SGNH-hydrolase
cpfrc_00386 nanH neuraminidase H (sialidase)
cpfrc_01079 rpfI resuscitation-promoting factor interacting protein (D,L-endopeptidase)
cpfrc_00594 rpfA resuscitation-promoting factor A (muralytic enzyme)
cpfrc_00679 rpfB resuscitation-promoting factor B (muralytic enzyme)
cpfrc_00128 nor nitric oxide reductase
cpfrc_00565 nrpS1 nonribosomal peptide synthetase 1
cpfrc_01801 nrpS2 nonribosomal peptide synthetase 2
cpfrc_00492 dtsR1 acetyl-CoA carboxylase b-subunit involved in fatty acid synthesis
cpfrc_00491 dtsR2 acyl-CoA carboxylase b-subunit involved in mycolic acid synthesis
cpfrc_01953 accD3 acyl-CoA carboxylase b-subunit involved in mycolic acid synthesis

disease [56]. Further experimental studies are required
to elucidate whether the expression of the secreted
enzymes promotes the virulence of C. pseudotuberculo-
sis FRC41.
Another candidate virulence factor of C. pseudotuber-
culosis FRC41 is the extracellular neuraminidase NanH
(Table 1). Neuraminidases, or sialidases, belong to a
class of glycosyl hydrolases that catalyze the removal of
terminal sialic acid residues from a variety of glycocon-
jugates and can contribute to the recognition of sialic
acids exposed on host cell surfaces [82,83]. The homolo-
gous counterpart of NanH was recently characterized in
C. diphtheriae KCTC3075 and shown to be a protein
containing neuraminidase and trans-sialidase activities
[84]. Trans-sialidases located on the bacterial cell sur-
face can be used for the decoration of sugar moiety
acceptors with sialic acid to enable the invasion of hosts
under certain conditions. The trans-sialidase activity is
of importance for many pathogenic bacteria and the
corresponding proteins are therefore considered poten-
tial virulence factors [82]. Iron limitation reduced the
number of sialic acid residues on the surface of C.
diphtheriae cells and their adhesive properties, indicat-
ing that the expression of dissimilar virulence determi-
nants is coordinately controlled by a gene-regulatory
system [85]. By bioinformatic pattern searches, a GlxR-
binding site was detected in the upstream region of the
nanH gene of C. pseudotuberculosis FRC41, suggesting
that the cAMP-sensing transcription regulator GlxR
might be involved in the control of this virulence factor
gene (Figure 6).
Likewise, a DNA-binding site for GlxR was detected in
front of the rpfI gene encoding an invasion-associated
protein that is involved in cell surface organization and
adhesion of corynebacteria [86]. The homologue of RpfI
in M. tuberculosis (named RipA) revealed endopeptidase
activity and interacts with the resuscitation-promoting
factor RpfB, representing a peptidoglycan glycosidase
[86,87]. Two genes (rpfA and rpfB) encoding resuscita-
tion-promoting factors are present in the genome of
C. pseudotuberculosis FRC41 (Table 1). Important roles
in pathogenesis for peptidoglycan hydrolytic enzymes
have been proposed [88] and an analogous system com-
bining the activities of a muramidase and an endopepti-
dase contributed to the virulence of Listeria
monocytogenes [89]. As previously demonstrated in
C. glutamicum [74,90], the expression of rpfI, rpfA and
rpfB in C. pseudotuberculosis FRC41 is probably under
complex control by three regulatory proteins, GlxR,
RamB and RamA (Figure 6).
Another potential virulence factor of C. pseudotuber-
culosis FRC41 is represented by the nor gene encoding
nitric oxide reductase (Table 1). This enzyme is gener-
ally involved in the detoxification of nitric oxide and
consequently necessary for the long-term persistence of
pathogens in macrophages [91]. The toxic properties of
nitric oxide are used by the host immune system to kill
or slow down the growth of pathogenic bacteria [51].
Interestingly, the expression of the nor gene was not
induced upon the infection of macrophages by animal
C. pseudotuberculosis [4]. As the expression of nor is
typically activated by a transcription regulator in
response to the presence of nitric oxide [92], the regula-
tory pattern of nor transcription and its contribution to
the protection against nitric oxide remains unclear.
The previous search for macrophage-induced genes of
animal C. pseudotuberculosis by means of a cloned pro-
moter library provided two gene tags showing significant

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Figure 6 Regulatory interactions involved in the control of potential virulence factors of C. pseudotuberculosis FRC41. Transcription
regulators controlling the expression of candidate virulence factors are shown. The regulatory interactions were deduced from a genome-scale
network transfer approach [114] and the combined use of a hidden Markov model and a position weight matrix [74]. The assignment of the
transcription regulators into the regulatory protein families of C. pseudotuberculosis FRC41 is indicated. Predicted DNA-binding sites of RamA and
RamB are shown as white boxes and predicted DNA-binding sites of GlxR as black boxes. Regulated target genes are shown as arrows and
coloured as follows: grey, regulatory gene; yellow, gene involved in iron storage; orange, gene of central metabolism or fatty acid/mycolic acid
biosynthesis; violet, gene involved in the resuscitation process; light green, gene involved in the assembly of adhesive pilus; dark green, other
potential virulence factor gene. The 16-bp consensus sequence of the GlxR-binding site of C. pseudotuberculosis FRC41 is shown as DNA
sequence logo. The 17 predicted DNA-binding sites of GlxR were used as input data for the WebLogo tool [115].

58

Trost et al. BMC Genomics 2010, 11:728
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induction rates in macrophages [4]. The nucleotide
sequence of the respective gene tags revealed similarity
to nonribosomal peptide synthetases (44-fold induction)
and to the b-subunit of acyl-CoA carboxylases (24-fold
induction), respectively. The genome sequence of C.
pseudotuberculosis FRC41 encodes two nonribosomal
peptide synthetases, NrpS1 and NrpS2 (Table 1). These
modular enzymes are used by microorganisms to partici-
pate in the synthesis of many secondary metabolites,
including for instance siderophores and antibiotics [93].
As both nrpS genes were not assigned to the iron-respon-
sive DtxR regulon of C. pseudotuberculosis FRC41 and
siderophore biosynthesis is carried out by alternative
pathways independent of nonribosomal peptide synthe-
tases, a physiological role in iron metabolism of the two
proteins cannot be deduced from the current data. How-
ever, the strong upregulation of gene expression in
macrophages points toward a protective or toxic function
during the infection of at least one nonribosomal peptide
synthetase [4]. A role in virulence of a secondary metabo-
lite produced by a nonribosomal peptide synthetase has
been demonstrated in Streptomyces acidiscabies. This
phytopathogen produces thaxtomin A which is necessary
for the infection of potato tubers [94].
Three genes coding for b-subunits of acyl-CoA car-
boxylases are present in the genome of C. pseudotuber-
culosis FRC41 (Table 1). These genes are located in
highly conserved regions of corynebacterial genomes
and are essential for either fatty acid synthesis (dtsR1)
or mycolic acid synthesis (dtsR2 and accD3) [95]. The
acetyl-CoA carboxylase of C. glutamicum consists of the
biotinylated a-subunit AccBC, the b-subunit AccD1
(DtsR1) and the small AccE protein. The acyl-CoA car-
boxylase involved in mycolic acid synthesis of C. gluta-
micum consists of the two b-subunits AccD2 and
AccD3 (DtsR2 and AccD3) in addition to AccBC and
AccE [95,96]. The expression of the respective genes in
C. pseudotuberculosis FRC41 is probably controlled by
the global transcription regulator GlxR (Figure 6), pro-
viding hints that this regulatory protein has a key func-
tion in connecting both the central metabolism and the
expression of candidate virulence factors in this patho-
genic bacterium. Mycolic acids, which are long-chain a-
alkyl-b-hydroxy fatty acids, are important components
of the corynebacterial cell wall [97] and probably asso-
ciated with the pathogenicity of C. pseudotuberculosis
[98]. They can provide a thick layer at the outer surface
of the cell that protects the bacterium from antibiotics
and the host’s immune system [99]. Moreover, the non-
covalently bound trehalose dimycolate is a well-estab-
lished immunostimulatory compound with toxic
properties [97]. Variations in the amount of mycolic
acids and differences in other cell surface properties
provide a basis for explaining the aggregation capacity
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of C. pseudotuberculosis in suspension, the strong aggre-
gation within macrophages, the formation of thick
capsules after the release of the pathogen from macro-
phages [11], and the occurrence of virulent and attenu-
ated strains [98].
Conclusions
The complete genome sequence of C. pseudotuberculosis
FRC41 of human origin provides detailed insights into
the gene repertoire contributing to the virulence of this
bacterium that was isolated from a rare case of necrotiz-
ing lymphadenitis [12]. By combining in silico data
obtained from the genome annotation with previous
experimental knowledge, occasional observations on
genes that affect the virulence of C. pseudotuberculosis
were integrated into a global view on the pathogenicity
of this species. The reconstruction of the DtxR regulon
for instance provides a comprehensive set of genes
involved in the acquisition of iron and extends the
initial observation that a fagB mutant was not impaired
in iron uptake [29]. A systematic mutational characteri-
zation of the newly detected iron transporters and side-
rophore biosynthesis gene clusters may help to dissect
the contribution of each system to the virulence of
C. pseudotuberculosis FRC41. It is very likely that the
ciu siderophore biosynthesis and transport system may
complement the uptake of iron by the FagABCD trans-
porter under certain environmental conditions, as the
ciuA gene was shown earlier to be expressed in C. pseu-
dotuberculosis [1]. Likewise, a reporter transposon sys-
tem indicated that a gene coding for a fimbrial subunit
is expressed during growth of C. pseudotuberculosis in
standard medium [40]. As the genome sequence of
C. pseudotuberculosis FRC41 revealed two gene clusters
encoding adhesive pili, it is interesting to examine how
the respective pilin monomers or pilus polymers contri-
bute to the adherence of C. pseudotuberculosis to host
tissue and how their expression is controlled. We
detected a DNA-binding site for the cAMP-sensing
transcription regulator GlxR in the spaA-srtB intergenic
region, whereas the iron-responsive regulator DtxR was
proposed to control the assembly of pilin subunits in
C. diphtheriae NCTC 13129 [100]. Another previous
study provided two DNA sequence tags for macro-
phage-induced genes [4] that were linked to the nonri-
bosomal peptide synthetase genes nrpS1 and nrpS2 and
to the acyl-CoA carboxylase subunit genes dtsR1, dtsR2
and accD3 by means of the genome sequence of
C. pseudotuberculosis FRC41. The detection of the latter
genes is consistent with the observation that the cell
surface of C. pseudotuberculosis is an important factor
contributing to virulence [98]. Future work should com-
pare the knowledge deduced from the genome sequence
of C. pseudotuberculosis FRC41 with the genetic

Trost et al. BMC Genomics 2010, 11:728
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information generated from other human isolates or ani-
mal pathogens of the different biovars [101], or even
extend the genome comparison to the pangenomic level.
This global comparative approach with a larger set of
sequenced genomes may provide comprehensive insights
into the distinctive features of each biovar or strain. As
the currently available commercial vaccines are unable
to fully protect susceptible animals [102,103], the
extended knowledge of potential virulence factors and
novel antigens of C. pseudotuberculosis might be helpful
for the design of more effective vaccines and molecular
diagnostics to control caseous lymphadenitis in sheep
and goats to reduce thereby the occupational risk expo-
sure for humans.
Methods
Bacterial strain and growth conditions
C. pseudotuberculosis FRC41 was isolated from the ingu-
inal lymph node of a 12-year-old French girl with necro-
tizing lymphadenitis [12]. The taxonomic identification
of this clinical isolate was recently confirmed using mul-
tiplex PCR and the nucleotide sequence of the pld gene
(N. Guiso, unpublished data). C. pseudotuberculosis
FRC41 was routinely grown in brain-heart-infusion (BHI)
broth or on Columbia agar with sheep blood at 37°C.
Preparation of chromosomal DNA for genome sequencing
The preparation of chromosomal DNA from C. pseudo-
tuberculosis FRC41 was performed as follows: 50-ml ali-
quots of cultures grown for 48-72 h were centrifuged at
4°C and 2,000 × g for 20 min. The cell pellets were
resuspended in 0.6 ml Tris/NaCl buffer [10 mM Tris
(pH 7.0), 10 mM EDTA (pH 8.0), 300 mM NaCl] and
transferred to VK01 Precellys lysing tubes. The cells
were lysed by means of a Precellys 24-Dual Tissue
Homogenizer, using two cycles of 6,500 rpm for 15 sec
with an interval of 30 sec. The chromosomal DNA was
purified by phenol/chloroform/isoamyl alcohol (25:24:1)
extraction and precipitated with ethanol. The DNA con-
centration was determined with a Tecan Infinite 200
Microplate Reader.
Sequencing of the C. pseudotuberculosis FRC41 genome
A total of 5 μg of purified chromosomal DNA was used
for constructing a single-stranded template DNA library.
The DNA concentration of the library was measured by
using the Agilent RNA 6000 Nano Kit. The preparation
of the single-stranded template DNA library and DNA
sequencing were performed according to manufacturer’s
protocols (Roche Applied Science). The sequencing of
C. pseudotuberculosis DNA was carried out with the
Genome Sequencer FLX Instrument and Titanium
chemistry (Roche Applied Science). The sequence data
were assembled with the GS de novo Assembler
60
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Software (Version 2.3). According to the 454 Newbler
Metrics file, 286,938 reads representing 94,447,635 bases
were assembled. Using the default cutoff of 500 bases
for the size classification of the contigs, ten large contigs
(≥ 500 bases) and one small contig (313 bases) were
obtained to give a total size of 2,319,243 bases. The gap
closure process was supported by the related reference
contig arrangement tool r2cat, using the C. diphtheriae
NCTC 13129 genome sequence as a reference [13]. The
remaining gaps in the genome sequence were closed by
PCR with Phusion hot start high-fidelity DNA polymer-
ase (Finnzymes) and genomic template DNA. All pri-
mers used in this study were synthesized by Metabion.
The PCR assays were carried out with a TProfessional
PCR thermocycler (Biometra) according to standard
protocols (Finnzymes). The amplified DNA fragments
linking the individual contigs were sequenced by IIT
Biotech. All DNA sequences were uploaded into the
Consed program [104] to generate the complete genome
sequence of C. pseudotuberculosis FRC41.
Bioinformatic analysis of the complete genome sequence
The assembled sequence of C. pseudotuberculosis FRC41
was uploaded into the bacterial genome annotation sys-
tem GenDB [15]. The annotation of the complete gen-
ome sequence was performed as described previously
[105], followed by manual curation. Analyses of the pre-
dicted gene content and the metabolic properties of
C. pseudotuberculosis FRC41 were accomplished by the
computer programs EDGAR [19] and CARMEN [106].
The synteny between the genomes of C. pseudotubercu-
losis FRC41 and C. diphtheriae NCTC 13129 was calcu-
lated by the EDGAR software [19]. The origin of
chromosomal DNA replication was predicted with the
Ori-Finder tool [17]. The genome sequence of C. pseu-
dotuberculosis FRC41 has been deposited in the Gen-
Bank database with accession number CP002097.
Bioinformatic analysis of the regulatory repertoire
The detection of the transcriptional regulatory reper-
toire of C. pseudotuberculosis FRC41 was performed by
a combined bioinformatic approach using several tools
and programs [26]. Proteins containing putative DNA-
binding domains were detected by means of the HMM
library and genome assignments server Superfamily ver-
sion 1.75 [107]. To identify among the set of potential
DNA-binding proteins those representing transcription
regulators, hidden Markov model (HMM) profiles of
regulatory protein families were downloaded from the
Pfam database version 24.0 [108] and used for searches
against the predicted C. pseudotuberculosis FRC41 pro-
teins by applying the HMMsearch module of the profile
hidden Markov model software HMMER [109]. More-
over, the helix-turn-helix (HTH) recognition tool [110]

Trost et al. BMC Genomics 2010, 11:728
http://www.biomedcentral.com/1471-2164/11/728
integrated in the GenDB platform was applied to scan
the putative DNA-binding transcription regulators for
the presence of HTH motifs. The classification of
C. pseudotuberculosis FRC41 proteins into Clusters of
Orthologous Groups of proteins [111] during genome
annotation provided further indications on the role of
the predicted proteins in transcriptional regulation. The
genome-wide search was extended by using data on
known transcription regulators from C. glutamicum
[23]. A validation step was included by performing
BLASTP [112] searches against the NCBI protein data-
base and evaluating results generated with the Con-
served Domain Search program [56]. During the final
step of data analysis, the DNA-binding transcription
regulators of C. pseudotuberculosis FRC41 were grouped
into regulatory protein families [23,26].
The detection of DNA-binding sites in the genome
sequence of C. pseudotuberculosis FRC41 followed a
combined workflow, using both position weight matrices
and hidden Markov models [74]. The programs PoSSuM-
search [113] and HMMsearch [109] were applied to scan
the complete genome sequence of C. pseudotuberculosis
FRC41. As HMMsearch does not support searching in
both directions of a double strand, scanning of the
reverse complementary DNA sequence was implemen-
ted. PoSSuMsearch was configured for lazy probability
evaluation [113]. The comprehensive set of validated
DNA-binding sites sequences from C. glutamicum was
downloaded from the reference database CoryneRegNet
[114] and used as input for PoSSuMsearch and
HMMsearch. The respective gene-regulatory network
transfer between C. glutamicum and other corynebacteria
on the genome-scale was described previously [114]. E-
value cut-offs were used as described for the genome-
wide pattern recognition approach in C. glutamicum
[75]. Sequence logos of the detected DNA-binding sites
were generated with WebLogo version 2.8.2 [115].
Acknowledgements
ET and JSchn acknowledge the receipt of scholarships which are embedded
into the Cluster Industrial Biotechnology CLIB2021 and financed by Bielefeld
University and the Ministry of Innovation, Science, Research and Technology
of North Rhine-Westphalia. LO and AB were supported by the Deutsche
Forschungsgemeinschaft in frame of SFB 796 (project B5). The valuable
contribution of the CeBiTec sequencing team (A. Albersmeier, K. Brinkrolf, S.
Schatschneider, R. Szczepanowski, P. Viehoever, A. Winkler) to this genome
project is gratefullly acknowledged. The authors thank the sequencing team
from IIT Biotech for the help during the gap closure process.
Author details
1
Institut für Genomforschung und Systembiologie, Centrum für
Biotechnologie, Universität Bielefeld, Universitätsstraße 27, D-33615 Bielefeld,
Germany. 2CLIB Graduate Cluster Industrial Biotechnology, Centrum für
Biotechnologie, Universität Bielefeld, Universitätsstraße 27, D-33615 Bielefeld,
Germany. 3Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität
Erlangen-Nürnberg, Staudtstraße 5, D-91058 Erlangen, Germany.
4
Bioinformatics Resource Facility, Centrum für Biotechnologie, Universität
Bielefeld, Universitätsstraße 27, D-33615 Bielefeld, Germany. 5AG
61
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Page 14 of 17
Genominformatik, Technische Fakultät, Universität Bielefeld, Universitätsstraße
25, D-33615 Bielefeld, Germany. 6Laboratório de Genética Celular e
Molecular, Departamento de Biologia Geral, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Pampulha,
Belo Horizonte, MG, Brazil. 7Cellular and Molecular Parasitology Laboratory,
Rene Rachou Research Center, Oswaldo Cruz Foundation (FIOCRUZ), Belo
Horizonte, MG, Brazil. 8Instituto de Ciências Biológicas, Universidade Federal
do Pará, Rua Augusto Corrêa, 01 - Guamá, Belém, PA, Brazil. 9Institut Pasteur,
Unité de Prévention et Thérapies Moléculaires des Maladies Humaines,
National Centre of Reference of Toxigenic Corynebacteria, 25 rue du Dr.
Roux, 75724 Paris Cedex 15, France. 10Medical Faculty, University Paris
Descartes, Hospital Necker Enfants Malades, Department of Microbiology,
147 rue de Sèvres, 75015 Paris, France. 11Medical Faculty, University Rennes
1, Hospital Pontchaillou, Department of Microbiology, 2 rue Henri le Guilloux,
35000 Rennes, France.
Authors’ contributions
ET sequenced and annotated the FRC41 genome and prepared the
manuscript. JSchn implemented and maintained the GenDB project for
annotation. JSchr participated in the gene-regulatory network analysis. SJ
and AG provided bioinformatic support. PH and JSt participated in the gap
closure process. FAD and FSR purified the genomic DNA. LO, SCS, VD, AM,
JR and AS participated in data evaluation. NG performed the taxonomic
classification of FRC41. OFJL and SK discovered FRC41 and provided the
isolate for the sequencing project. VA, AB and AT supervised the project. All
authors read and approved the final version of the manuscript.
Received: 8 September 2010 Accepted: 30 December 2010
Published: 30 December 2010
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doi:10.1186/1471-2164-11-728
Cite this article as: Trost et al.: The complete genome sequence of
Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old
girl with necrotizing lymphadenitis reveals insights into gene-regulatory
networks contributing to virulence. BMC Genomics 2010 11:728.

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64
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3.4 Impact of improved potassium accumulation on pH homeostasis,

membrane potential adjustment and survival of Corynebacterium
glutamicum (Ochrombel et al, 2011)

65
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Biochimica et Biophysica Acta 1807 (2011) 444–450

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a b i o

Impact of improved potassium accumulation on pH homeostasis, membrane

potential adjustment and survival of Corynebacterium glutamicum
Ines Ochrombel a, Lisa Ott b, Reinhard Krämer a, Andreas Burkovski b, Kay Marin a,⁎
a
Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany
b
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Metal ion uptake is crucial for all living cells and an essential part of cellular bioenergetic homeostasis. In this
Received 22 July 2010 study the uptake and the impact of the most abundant internal cation, potassium, were investigated in
Received in revised form 26 January 2011 Actinobacteria, a group of high G + C Gram-positives with a number of prominent biotechnologically and
Accepted 28 January 2011
medically important members. Genome analyses revealed a variety of different potassium uptake systems in
Available online 2 February 2011
this monophyletic group ranging from potassium channels common in virtually all Actinobacteria to different
Keywords:
active carriers that were present predominantly in pathogenic members able to cope with various stress
Potassium transport conditions. By applying Corynebacterium glutamicum as model system we provide experimental evidence that
Potassium channel under optimal conditions a potassium channel is sufficient in bacteria for the maintenance of internal pH and
Corynebacterium membrane potential ensuring survival of cells under stress conditions. Under potassium limitation, however,
pH homeostasis viability of C. glutamicum was increased under acidic stress or during desiccation when a functional KtrAB
Membrane potential adjustment potassium transporter from the pathogen Corynebacterium jeikeium was heterologously expressed. We
provide experimental evidence that the KtrAB mediated enhanced potassium accumulation improved
maintenance of internal pH and membrane potential. The results indicate that the occurrence of active
potassium transport systems correlates with an improved potassium-dependent bioenergetic homeostasis
and survival of bacterial cells under stress conditions.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction third type of uptake system is the secondary Kup-type transporter

Ion homeostasis is an essential part of life. Since the cell membrane
is impermeable for ions, transport systems are mandatory for ion
uptake and extrusion and carrier proteins regulate the internal ion
content. The alkali metal ion potassium plays an outstanding role in
this respect [1]. With concentrations between 0.1 and 0.6 M
potassium is the most abundant cation in bacteria, while in general
only traces are available in the environment (0.1–10 mM). It is
involved in membrane potential adjustment and required for the
activity of the respiratory chain [2]. Potassium acts as second
messenger for stress signalling and as regulatory element for
transcription control as well as counter ion for glutamate and other
compounds during osmotic stress response [3]. The impact of
potassium for bacterial life is indicated by the diversity of transport
systems present for this particular ion. In many enteric bacteria
primary active P-type ATPases of the Kdp-type are present in addition
to secondary carriers of the Trk-type. Ktr-type systems are related to
Trk transporters and are present in many Gram-positive bacteria. A
⁎ Corresponding author at: University of Cologne, Institute of Biochemistry,
Zuelpicher Str. 47, 50674 Cologne, Germany. Tel.: +49 221 470 6476; fax: + 49 221
470 5091.
E-mail address: kay.marin@uni-koeln.de (K. Marin).
that is present in many Gram-positive and Gram-negative bacteria. All
secondary active carriers are supposed to use the membrane potential
for uptake of potassium [3]. Additionally, several ligand-gated channel
proteins are involved in potassium uptake [4,5].
The distribution of potassium transport systems in bacteria was
proposed to be correlated with the potassium availability in their
natural habitat [6]. Bacteria like Escherichia coli or Bacillus subtilis
harbor different types of potassium carriers and maintain high
internal potassium levels under both stress and standard cultivation
conditions [7,8], while Corynebacterium glutamicum, Klebsiella pneu-
moniae and Bacillus stearothermophilus do not require potassium
under optimal growth conditions and can be propagated in its virtual
absence [1,2]. However, under acidic stress C. glutamicum accumulates
high cytoplasmic potassium concentrations as well. A Kup-type
transporter and a potassium channel encoding gene were found in
the C. glutamicum genome, respectively. Interestingly, no activity was
observed for the secondary active potassium carrier Kup and the
potassium channel CglK was found to represent the main uptake
system under standard conditions [2]. CglK represents the first
example of a bacterial potassium channel for which the activity was
proven in its natural membrane environment. Additionally, the
analysis of CglK in C. glutamicum unravelled for the first time the
physiological function of a bacterial potassium channel, namely, the

0005-2728/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbabio.2011.01.008

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I. Ochrombel et al. / Biochimica et Biophysica Acta 1807 (2011) 444–450 445

decease of the membrane potential and the maintenance of a neutral
internal pH under conditions of acidic stress. The CglK channel is
sufficient for potassium accumulation in presence of high potassium
concentrations or at conditions of low potassium demand. However, if
the potassium availability is limited or the requirement for potassium
is increased the sole presence of a potassium channel might restrict
growth and consequently active carriers might be required [6].
Taxonomically, C. glutamicum belongs to the Actinobacteria that are
widespread in nature and live as soil bacteria, saprophytes, members
of the microflora of humans or animal and plant pathogens. Medically
important members are Mycobacterium tuberculosis, Nocardia farci-
nica, Propionibacterium acnes, Corynebacterium diphtheriae and Cory-
nebacterium jeikeium, while C. glutamicum, Corynebacterium efficiens,
Bifidobacterium longum as well as Streptomyces coelicolor are impor-
tant in biotechnological processes [9].
In this study we analyzed the monophyletic group of Actinobac-
teria regarding the equipment with potassium transport systems. We
show that all pathogenic strains, supposed to be exposed to
challenging environmental limitations and stress conditions, harbor
active potassium transport systems in addition to potassium channels.
We addressed the impact of the mode of potassium transport by
active carriers and/or channels on growth and survival from a
bioenergetic point of view. Improvement of potassium transport of
C. glutamicum harboring a functional potassium channel by additional
presence of the transporter KtrAB from C. jeikeium led to an increased
potassium content and was beneficial for the maintenance of
membrane potential and internal pH accompanied by improved
growth and survival of C. glutamicum under stress conditions.
2. Materials and methods
gas-permeable membrane in a volume of 200 μl shaken at 1200 rpm.
Plates were prepared by the addition of 15 g l− 1 agar to the medium. For
all experiments precultivation was performed as described [2] and
experiments were performed with cultures entered the exponential
growth phase. Whereas BHI contains 10 mM potassium, MMI contains
37 mM potassium. The pH was adjusted by appropriate buffer
substances (250 mM) as described [11]. If necessary, the medium was
supplemented with kanamycin (25 μg ml− 1) and for induction of
expression with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).
Growth was followed by measuring the OD600. Standard molecular
cloning techniques were applied using E. coli DH5αMCR cells grown in
Luria-Bertani (LB) medium at 37 °C.
For amplification of the ktr locus of C. diphtheriae ISS3319 or C.
jeikeium K411, primers listed in Table 1 were applied. The genomic
fragments, harboring both ktrB and ktrA as well as 500 bp upstream of
ktrB, including the putative promoter region and the ribosome
binding site (Fig. S1), were cloned into the plasmid pDRIVE (Qiagen,
Hilden, Germany) and the sequence of the resulting vectors
pDRIVE_ktrBA_Cd and pDRIVE_ktrBA_Cj was confirmed (GATC, Kon-
stanz, Germany). The PCR primers were designed in order to
introduce a Strep-tag coding sequence at the 3′ end of the ktrA
gene. Subsequently, the ktrBA fragments were cloned into the vector
pEKEX2 mediating the IPTG inducible expression of genes under the
control of the lac promoter [12], resulting in plasmids pEKEX2_ktr-
BA_Cd and pEKEX2_ktrBA_Cj. After electroporation of the C. glutami-
cum cells, the presence of the plasmids was proven by cultivation on
kanamycin-containing plates, as well as by PCR. The resulting strains
are listed in Table 1. Additionally, expression of the Strep-tagged KtrA
protein was proven by Western blot analysis as described previously
[2] using a Strep tag antibody (Sigma).

2.1. Bacterial strains, growth conditions and construction of mutants
C. glutamicum strain ATCC 13032 served as wild type and was grown
either in brain heart infusion (BHI) medium (Becton-Dickenson,
Heidelberg, Germany) or in minimal medium MMI [10] at 30 °C in
Erlenmeyer flasks shaken at 130 rpm or in microtiter plates sealed with a
2.2. Measurement of external and internal potassium concentration as
well as bioenergetic parameters
Monitoring of both external and internal potassium concentration
was performed by flame photometry (ELEX 6361; Eppendorf,
Germany) as described previously [2]. In short, after precultivation

Table 1
Strains, plasmids and primers used in this study.

Strain or plasmid Related genotype or descriptiona Reference

E. coli
DH5αMCR endA1 supE44 recA1 gyr96 relA1 deoR U169 ϕ80ΔlacZΔM15 mcrA Δ Grant et al. [22]
(mrr-hsdRMS-mcrBC)
C. glutamicum strains
ATCC 13032 Wild type Abe et al. [23]
Δkup Deletion of kup (cg0187) in ATCC 13032 Follmann et al. [2]
ΔcglK Deletion of cglK (cg0887) in ATCC 13032 Follmann et al. [2]
ΔcglKΔkup Deletion of kup and cglK in ATCC 13032 Follmann et al. [2]
Cg_p ATCC 13032 harboring plasmid pEKEX2 This study
Cg_pktrBA ATCC 13032 harboring plasmid pEKEX2_ktrBA_Cj This study
Cg_pktrBA ATCC 13032 harboring plasmid pEKEX2_ktrBA_Cd This study
ΔcglKΔkup_pktrBA_Cj ΔkupΔcglK harboring plasmid pEKEX2_ktrBA_Cj This study
Plasmids
pDRIVE A-T cloning vector (KmR, ApR) Qiagen, Hilden, Germany
pDRIVE_cglK_Cd pDRIVE harboring an internal fragment of gene DIP0724 of C. diphtheriae This study
pDRIVE_ktrB_Cd pDRIVE harboring an internal fragment of gene DIP1931 of C. diphtheriae This study
pDRIVE_ktrBA_Cj pDRIVE harboring the ktrBA genomic locus of C. jeikeium This study
pDRIVE_ktrBA_Cd pDRIVE harboring the ktrBA genomic locus of C. diphtheriae This study
pEKEX2 E. coli–C. glutamicum shuttle expression vector (KmR) Eikmanns et al. [12]
pEKEX2_ktrBA_Cj pEKEX2 harboring the ktrBA genomic locus of C. jeikeium This study
pEKEX2_ktrBA_Cd pEKEX2 harboring the ktrBA genomic locus of C. diphtheriae This study
Primerb
Pre500_ktrB_Cjeik_5` 5′-GCCTGCAGGGAGACTCAGCCCGTGCTGCGTTTGC-3′ (PstI) This study
ktrA_CStrep_Cjeik_3′ 5′-GCGAATTCGCCTATTTTTCGAACTGCGGGTGGCTCCAGGAATCCGCAAACTTATCCAGGT-3′ (EcoRI) This study
Pre500_ktrB_Cdiph_5′ 5′-GCCTGCAGGAAACTCAGGCGGTGTTGGCGTTGCTGC-3′ (XhoI) This study
ktrA_CStrep_Cdiph_3′ 5′-CGAATTCGCCTATTTTTCGAACTGCGGGTGGCTCCA-ACCAATGATCAGCCTTTCTACAGG-3′ (EcoRI) This study
a
Abbreviations: ApR, ampicillin resistance; KmR, kanamycin resistance.
b
Letters in bold indicate the recognition site for the restriction enzyme given in parentheses, the stop codon is underlined and the Strep tag-coding sequence is shown in italic
letters.

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cells were washed three times with MMI medium without potassium channel Kch was applied as query beside the potassium channel KcsA
and then inoculated in MMI medium containing 1 mM KCl at a defined of Streptomyces lividans. Sequences highly similar to the known
pH and an OD600 of 2 in 100 ml of medium. External potassium potassium transport systems were employed for a cluster analysis
concentration was measured after removal of cells by centrifugation including the sequence of the E. coli sodium transporter NhaA as
in the supernatant. For detection of internal potassium concentra- outgroup. As seen in Fig. 1, with either KcsA or CglK all strains harbor
tions, cells of 2 ml culture were collected by centrifugation and the at least one potassium channel protein, while in N. farcinica and S.
pellet was washed twice with potassium-free MMI. Cell disruption coelicolor both types of potassium channels are present. In Gram-
was carried out by ultrasonication 80% (Sonorex Digital DK 512 P;
Badelin, Berlin) at 85 °C in 1.7 ml H2O for 20 min. After centrifugation
the potassium concentration in the supernatant was measured.
The cytoplasmic volume was determined by the distribution of 3H-
labeled H2O (0.55 mCi l− 1) and 14C-labeled inulin (0.14 mCi l− 1)
between the cell pellet and the supernatant to be 1.8 μl mg− 1 cell dry
matter (CDM) as described previously [2]. Processing of samples for
rapid separation of extra- and intracellular fluids was performed by
using silicone oil centrifugation with perchloric acid in the bottom
layer [2]. The membrane potential was determined by measuring the
distribution of tetraphenylphosphonium (TPP) (final concentration
5 μM, specific radioactivity 0.995 Ci mol− 1) as described previously
[2]. Internal pH was determined by measuring the distribution of 14C-
labeled benzoic acid (final concentration 15 μM, specific radioactivity
3.12 Ci mol− 1) as described [2].
Rates of oxygen consumption by C. glutamicum cells were measured
by a Clark-type electrode (Oxygraph; Hansatech, Reutlingen, Germany)
at 30 °C in a total volume of 1 ml minimal medium, pH 6 (250 mM MES)
with cells harvested at the exponential phase and resuspended at an
OD600 of 0.3 to 0.5 in MMI medium. Steady-state oxygen consumption
rates were determined in absence or presence of potassium (50 mM).

2.3. Determination of pH and desiccation tolerance of C. glutamicum

For the drop assays, cells were depleted of potassium by

precultivation in minimal medium without potassium supplementa-
tion. Subsequently, cultures were diluted in minimal medium without
potassium to the OD values indicated. Four microliters of each cell
suspension was dropped onto minimal medium agar plates with a pH
of 7.5 and 7.0 (250 mM MOPS) as well as pH 6.5 and 6.0 (250 mM
MES) and the indicated potassium concentrations. After 48 h of
incubation, results were documented by photography. Desiccation
tolerance was analyzed with cells depleted of potassium diluted in
minimal medium with 5 mM potassium to an OD600 of 1. In a well of a
microtiter plate 100 μl of each cell suspension was exposed to
desiccation by shaking at 1200 rpm at 30 °C. After 24 and 48 h dried
cells were resuspended in 100 μl NaPi -puffer, pH 7.4 and dilutions
were made as indicated. Subsequently, 4 μl of each cell suspension
was dropped onto BHI agar plates containing 100 mM KCl. After 24 h
of incubation, results were documented by photography.

3. Results

3.1. Occurrence of different potassium transport systems in Actinobacteria

Addressing the question whether the existence of active potassium

transport is correlated with a particular bacterial life style, a
bioinformatic analysis of (putative) potassium transport systems in
Actinobacteria including the non-pathogenic species C. efficiens and C.
glutamicum strain R and the pathogens Corynebacterium aurimucosum,
C. diphtheriae, C. jeikeium, Corynebacterium urealyticum and Coryne-
bacterium kroppenstedtii was performed. Outside the genus Coryne-
bacterium, the closely related species M. tuberculosis, Mycobacterium
abscessus, N. farcinica, P. acnes and Clavibacter michiganensis were
included, which are prominent human or plant pathogens as well as
the non-pathogenic and biotechnologically important strains B.
longum, Streptomyces avermitilis and S. coelicolor [9]. The sequence
of (subunits of) the known E. coli potassium transport systems Trk,
Kup and Kdp, the potassium exporter KefB as well as the potassium
Fig. 1. Occurrence of different types of potassium transporters in Actinobacteria.
Proteins similar to known potassium transporter subunits in E. coli (Ecoli, Kch, KefB,
TrkH, KdpA) or S. coelicolor (Scoel, KcsA) were identified by genomicBlast searches
against the genomes of Corynebacterium aurimucosum (Cauri), Corynebacterium
diphtheriae biotype gravis strain NCTC13129 (Cdiph), C. efficiens strain YS-314 (Ceffi),
Corynebacterium glutamicum strain ATCC 13032 (Cglut), Corynebacterium glutamicum
strain R (CglutR), Corynebacterium jeikeium K411 (Cjeik), C. urealyticum DSM 7109
(Curea), C. kroppenstedtii DSM 44385 (Ckrop), Mycobacterium tuberculosis strain H37RV
(Mtube), M. abscessus ATCC 19977 (Mabsc), Nocardia farcinica strain IFM 10152 (Nfarc),
Propionibacterium acnes strain KPA171202 (Pacne), S. avermitilis strain MA-4680
(Saver), S. coelicolor strain A3(2) M145 (Scoel), B. longum strain NCC2705 (Blong) and
C. michiganensis subsp. michiganensis NCPPB 382 (Cmich). The sequences were grouped
according to a cluster analysis performed by using ClustalX and visualised by TreeView
[24,25]. The sequence of the E. coli sodium transporter NhaA was used as an outgroup.
The (putative) subunit composition and the mode of transport for each subgroup are
indicated on the right.

68
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I. Ochrombel et al. / Biochimica et Biophysica Acta 1807 (2011) 444–450 447

positive bacteria homologs of the E. coli Trk transporter, designated
Ktr in this group, and the Kdp-type uptake system are present almost
exclusively in pathogenic species, whereas the Kup-type transporter
is found in non-pathogenic strains predominantly. The KefB carrier is
mostly present in pathogenic strains. In conclusion, non-pathogenic
strains harbor potassium channels and Kup-type potassium uptake
systems whereas pathogenic strains are equipped with Ktr- and Trk-
type potassium uptake systems. Because pathogenic strains are
exposed to numerous stress conditions like acidic pH and/or low
potassium availability the presence of appropriate potassium trans-
port systems might be beneficial for survival of pathogenic Actino-
bacteria [13,14].
We hypothesized that the exclusive presence of a potassium channel
may represent a bottleneck for the maintenance of bioenergetic
parameters that can be overcome by an additional active transport
system. To test this hypothesis experimentally C. glutamicum was chosen
as model system because mutants lacking particular potassium transpor-
ters are available and a variety of methods is established to address the
impact of potassium transport on bioenergetic parameters and survival.
Our strategy was to improve potassium accumulation in C. glutamicum by
expression of ktrAB genes from C. diphtheriae or C. jeikeium. Since no
expression of genes derived from C. diphtheriae in C. glutamicum was
detected (data not shown) we focused on the impact of KtrAB from
C. jeikeium on C. glutamicum.
3.2. Expression of functional C. jeikeium ktrBA in C. glutamicum
or harboring the ktrBA expression plasmid. Comparable rates of
potassium accumulation and comparable internal potassium levels
were observed in both strains within 8 h (data not shown, Fig. 2).
Obviously, the contribution of the KtrAB transport system to
potassium uptake in C. glutamicum is not significant in comparison
to potassium uptake via the channel CglK under these conditions.
Consequently, we performed experiments under conditions of an
increased demand for potassium, namely at neutral and acidic
medium pH. In comparison to the results obtained at pH 8.5 the
internal potassium concentration in both C. glutamicum strains was
maintained at higher levels at pH 7.5 and 6.5 after 8 h of incubation.
Whereas at pH 8.5 the internal potassium concentration was
comparable in both cell types, at pH 7.5 and 6.5 the internal
potassium concentration in the recombinant strain was 170 mM
and 120 mM higher than in wild type cells, respectively (Fig. 2). These
findings underline the increased potassium requirement of C.
glutamicum under neutral and acidic conditions and demonstrate
that potassium accumulation in C. glutamicum is increased by the
heterologous uptake system KtrAB of C. jeikeium under acidic stress
conditions.
The known KtrAB transport systems require sodium ions for
activity but it is unknown whether sodium activates the transporter
A

The Ktr transport system of C. jeikeium putatively consists of two

subunits which are encoded by the genes ktrB (jk0347) and ktrA (jk0348).
For the KtrB subunit eight transmembrane domains, a molecular weight of
49 kDa is predicted, whereas the KtrA subunit is a soluble 23 kDa protein
harboring a putative NAD-binding domain. The presence of the KtrAB
system was demonstrated by detection of the C-terminal Strep-tag of the
KtrA protein in cell extracts of Cg_pktrBA. Upon addition of IPTG to cell
cultures and protein extraction a protein band of approx. 27 kDa was
detected, corresponding to the Strep-tagged version of KtrA (Fig. S1).
Functionality of the KtrAB transport system was addressed by
expression of ktrBA genes in C. glutamicum wild type cells and cells of
the mutant lacking both potassium transporters (CglK and Kup).
Under standard laboratory conditions C. glutamicum does not require
addition of potassium and mutants lacking potassium transporters
grow indistinguishable from wild type cells at alkaline pH [2].
However, potassium accumulation was shown to be crucial for B
survival under acidic conditions. Cells were inoculated at pH 7.5 on
plates containing different potassium concentrations. While in the
presence of 50 mM potassium all strains were able to grow to a
comparable extent, at 5 mM potassium growth of the strain lacking all
potassium uptake systems was impaired. This strain did not grow at
all if no potassium was added (Fig. 5A). Growth of wild type cells was
slightly decreased in presence of low potassium concentrations, but
mutants harboring the ktrBA genes were not affected upon addition of
5 mM or if potassium was lacking in comparison to plates containing
50 mM potassium (Fig. 5A). Moreover, at very low potassium
concentrations the presence of ktrBA genes resulted in slightly better
growth in comparison to wild type cells (Fig. 5A). From these
observations it was concluded that loss of potassium uptake systems
in C. glutamicum can be complemented by the expression of the ktrBA
operon from C. jeikeium indicating that the KtrAB transporter is
Fig. 2. Impact of ktrBA expression on potassium uptake and accumulation in C.
functional in C. glutamicum.
glutamicum and sodium dependence of KtrAB function. (A) Cells of the wild type
harboring a control plasmid (white bars) or the ktrBA expression vector (strain
3.3. Impact of ktrBA expression on potassium uptake and accumulation Cg_pktrBA, black bars) were inoculated in minimal medium of pH 8.5, 7.5 and 6.5 in
presence of 1 mM potassium. The internal potassium content was measured by flame
As a basis for further investigations, potassium uptake and the photometry and the values after 8 h of incubation are shown. (B) Cells of the strains
ΔcglKΔkup and ΔcglKΔkup_pktrBA were grown in liquid media at a pH level of 6.5 at the
cytoplasmic potassium concentration in C. glutamicum cells were indicated potassium concentrations in absence (white bars) or presence (black bars) of
analyzed by flame photometry [2]. Under optimal growth conditions sodium (no addition or addition of 100 mM NaCl), respectively, and growth rates were
(pH 8.5), external potassium was taken up by wild type cells lacking estimated.

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448 I. Ochrombel et al. / Biochimica et Biophysica Acta 1807 (2011) 444–450
or KtrAB functions as a K+/Na+ symporter [15]. To test the
dependence of the KtrAB transporter of C. jeikeium on sodium, growth
experiments in absence or presence of NaCl under potassium limiting
conditions using the ΔcglKΔkup mutant at low pH were performed.
Whereas cells of the ΔcglKΔkup mutant were not affected by addition
of NaCl (data not shown), expression of ktrBA caused significantly
increased growth rates of the mutant at low potassium concentrations
in the presence of NaCl only (Fig. 2B). At high potassium concentra-
tions no difference of ΔcglKΔkup cells harboring or lacking KtrAB was
found (Fig. 2B). The data indicate that the demand for potassium of C.
glutamicum can be accomplished by KtrAB in a sodium-dependent
manner pointing to the important role of sodium ions for the KtrAB
function.
3.4. Impact of ktrBA expression on bioenergetic parameters
To investigate the impact of the KtrAB-dependent potassium
transport on the maintenance of bioenergetic homeostasis, mem-
brane potential (ΔΨ), internal pH and proton motive force (pmf)
were measured in cells of the wild type and the ΔcglKΔkup mutant as
well as the corresponding strains harboring the KtrAB transporter
under acidic stress conditions. Cells were grown in the absence of
potassium in minimal medium at pH 7.5 for 3 h in order to deplete the
internal potassium concentration, and subsequently the external pH
was shifted to 6.0. Simultaneously, bioenergetic parameters were
A in the ΔcglKΔkup mutant resulted in higher rates of oxygen
Fig. 3. Impact of ktrBA expression on bioenergetic parameters in C. glutamicum. Cells of
the wild type (WT) and the mutant lacking the cglK and kup gene (ΔcglKΔkup) as well
as both strains harboring the ktrBA expression vector (Cg_pktrBA, ΔcglKΔkup_pktrBA)
were exposed to a pH shift in minimal medium from pH 7.5 to pH 6 without addition of
potassium (A) or in presence of 50 mM potassium (B). Membrane potential (white
bars) and pH gradient (grey bars) were measured and the corresponding pmf (black
bars) was calculated. All values are presented as potential and are given in mV. On the
right site the corresponding internal pH is indicated.
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measured in absence of potassium or immediately after addition of
50 mM potassium. In C. glutamicum cells lacking the potassium
transport systems CglK and Kup, a collapse of the pH gradient was
observed indicated by low internal pH values of 6.3 and 6.1
irrespective of the potassium concentration (Fig. 3). Consequently,
high values for the membrane potential were determined in both
strains (184 and 183 mV). In contrast, wild type cells maintained a
more neutral internal pH of 6.7 and a lower membrane potential
(143 mV) in the presence of potassium. In its absence, however, the
internal pH dropped to 6.2 and the membrane potential was found to
be increased to 183 mV (Fig. 3). Expression of ktrBA enabled the
ΔcglKΔkup mutant and, interestingly, even wild type cells to maintain
more neutral internal pH values (6.8 and 6.9) and lower values of the
membrane potential (143 and 126 mV), respectively, than the
parental strains (Fig. 3). The improved pH homeostasis was even
more pronounced at low potassium concentrations resulting in
0.7 units higher internal pH values in ktrBA expression strains
compared to the parental strains. For all strains comparable pmf
values were observed (Fig. 3).
In order to investigate further consequences of the KtrAB-
dependent improvement of pH homeostasis the activity of the
respiratory chain was examined in the same experimental set up.
Respiratory activity in wild type cells without addition of potassium
was 38 nmol O 2 min− 1 ml− 1 OD − 1
600 and increased by 50% in
presence of 5 or 50 mM potassium (Fig. 4). For the ΔcglKΔkup
mutant the respiratory activity was significantly lower than in wild
type cells (25 nmol O2 min− 1 ml− 1 OD− 1
600) and did not increase
upon addition of potassium (Fig. 4). In contrast, expression of ktrBA
consumption in comparison to the parental strain. In the wild type
background, however, expression of the ktrBA genes did not
increase oxygen consumption irrespective of the potassium
concentration.
3.5. Impact of ktrBA expression on survival and growth of C. glutamicum
under stress conditions
After proving the functionality of C. jeikeium KtrAB in C.
glutamicum and quantifying the beneficial impact on bioenergetic
homeostasis, we investigated whether growth and survival of C.
Fig. 4. Impact of ktrBA expression on the activity of the respiratory chain in C.
glutamicum. Cells of the wild type (grey bars) and the mutant lacking the cglK and kup
gene (white bars) as well as both strains harboring the ktrBA expression vector
(hatched bars) were inoculated in minimal medium pH 6 in presence of the indicated
potassium concentration. Activity of the respiratory chain was measured by
determining the oxygen consumption rate.
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I. Ochrombel et al. / Biochimica et Biophysica Acta 1807 (2011) 444–450 449

glutamicum are affected in a KtrAB-dependent manner under stress 4. Discussion

conditions. C. jeikeium is part of the human skin flora and exposed to
low pH values (around pH 5.5) and desiccation in combination with
low potassium concentrations (approx. 10 mM) during colonization
of this habitat. Therefore, the KtrAB-dependent pH and desiccation
tolerance of C. glutamicum were analyzed as an example. The
increased tolerance of C. glutamicum cells expressing the ktrBA
genes towards neutral pH was already described and interestingly
even the pH tolerance of wild type cells was increased in a KtrAB-
dependent manner (Fig. 5A). At pH 6.5 even at high potassium
concentrations growth of wild type cells harboring the control vector
without potassium transporter genes was strongly impaired
(Fig. 5C). In contrast, cells harboring the KtrAB transporter grew
even at low potassium concentrations down to 1 mM. At pH 6 growth
of both strains was strongly impaired (Fig. 5C). In comparison, upon
ectopic overexpression of the channel encoding gene cglK growth of
C. glutamicum was not improved (Fig. S2). The results demonstrate
that the KtrAB transport system of C. jeikeium confers a higher
resistance towards potassium limitation under acidic stress condi-
tions to C. glutamicum.
To test the correlation between potassium transport and survival of
dehydration, wild type and the ΔcglKΔkup mutant harboring the control
vector pEKEX2 or the ktrBA expression plasmid were exposed to
desiccation in the presence of 5 mM potassium. After 24 and 48 h survival
of cells was determined by re-suspending the cells and transferring 5 μl of
this suspension onto BHI agar plates. The mutant ΔcglKΔkup, lacking all
potassium transport systems, was strongly impaired in survival in
comparison to wild type cells (Fig. 5B). Expression of the ktrBA genes
did complement the sensitivity of the ΔcglKΔkup strain and improved
survival of wild type cells, respectively (Fig. 5B). In conclusion, under these
conditions the presence of potassium and appropriate potassium uptake
systems are crucial for long-term survival and resistance of C. glutamicum
towards desiccation stress.
4.1. Potassium channels are sufficient for stress-dependent potassium
uptake
The requirement for potassium under stress conditions was
observed for bacteria as well as eukaryotic organisms [3]. Potassium
accumulation as an immediate response upon an osmotic upshift is
observed in many bacteria like E. coli, B. subtilis or cyanobacteria and
was shown to be essential for survival [1,7,16]. During desiccation
bacterial cells are exposed to physiological challenges comparable to
osmotic stress situations [17]. In this study, we showed that a CglK
mutant of C. glutamicum was more sensitive towards acidic conditions
and desiccation than wild type cells indicating that potassium uptake
is required under these conditions. Also in Helicobacter pylori the
potassium channel HpKchA acts as sole potassium uptake system and
it was demonstrated that the presence of this channel is required for
the persistence of H. pylori in the gastric environment [6]. Conse-
quently, supply of cells with potassium seems to be required during
various stress conditions and a potassium channel is sufficient to meet
the demand.
4.2. Potassium uptake represents a bottleneck for bioenergetic homeostasis
The sole presence of a potassium channel seems not to be
sufficient, however, under potassium limitation as indicated by the
significantly improved survival of C. glutamicum strains expressing
ktrBA. A plausible reason for this limitation is the sole driving force for
the CglK-dependent potassium accumulation, the membrane poten-
tial, which was determined in C. glutamicum wild type cells as 143 mV
minimum at low pH. This facilitates a 400-fold internal accumulation
of potassium according to the Nernst equation. Since the internal
potassium concentration in C. glutamicum at acidic pH was around

Fig. 5. Impact of C. jeikeium KtrAB on growth and survival of C. glutamicum during potassium limitation and/or acidic stress as well as desiccation. Cells of the wild type (WT), the
ΔcglKΔkup mutant and the strains Cg_pktrBA and ΔcglKΔkup_pktrBA, harboring the plasmid pEKEX2_ktrBA, were grown on agar plates of pH 7.5 (A) supplemented with 50, 5 mM or
no potassium or exposed to desiccation (B) for 0, 24 and 48 h and survival was determined by the drop assay. The potassium-dependent acidic stress resistance of wild type and
Cg_ktrBA cells was addressed (C) by growth on agar plates of pH 7.0, 6.5 and 6.0 supplemented with 50, 10, 5, 1 mM or no potassium. The OD600 values indicate the cell density of
cultures applied for the assay by transfer of 4 μl onto the plate (A, C) or exposed to desiccation (B). Upon incubation for 24 h documentation by photography was performed.

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450 I. Ochrombel et al. / Biochimica et Biophysica Acta 1807 (2011) 444–450
600 mM an external potassium concentration of at least 1.5 mM is
required. If the potassium availability is lower, lower levels of internal
potassium can be maintained causing the collapse of internal pH
homeostasis under acidic stress conditions. Overexpression of the
homologous potassium channel encoding gene cglK did not lead to
improved survival under acidic conditions and did not cause an
improved maintenance of bioenergetic parameters [2]. The potassium
potential at the cytoplasmic membrane might be adjusted faster upon
overexpression of cglK because more channels mediate a higher
potassium flux, but at thermodynamical equilibrium the same
internal potassium concentration will be achieved. Consequently,
survival of cells depends on the availability of potassium and efficient
transport systems are required for growth and survival [6]. This
conclusion is in agreement with the exclusive occurrence of Ktr- and
Trk-type potassium transport systems in representatives of the
Actinobacteria that are supposed to be challenged by such stress
conditions.
The presence of the C. jeikeium potassium transporter KtrAB could
complement the loss of homologous potassium transport systems in
C. glutamicum and, moreover, was found to be beneficial even for
C. glutamicum wild type cells. Cells harboring the KtrAB transporter can
accumulate higher levels of internal potassium which causes an improved
homeostasis of the internal pH and the membrane potential in particular
under conditions of potassium limitation. The internal pH in ktrBA
expressing cells was kept 0.7 units higher upon application of acidic stress.
A decrease of the internal pH by 0.7 units under the same conditions
caused the reduction of growth rate by 50% in wild type cells [11]. This fact
seems to be the basic advantage of ktrBA expressing cells over wild type
cells. In agreement with the more neutral internal pH during acidic stress
the membrane potential was found to be not increased in C. glutamicum
cells harboring the KtrAB system and the respiratory activity can be
increased in a KtrAB-dependent manner.
4.4. The presence of active potassium transport systems improves
survival under stress conditions
We showed that the maintenance of bioenergetic parameters in C.
glutamicum upon expression of the ktrBA genes of C. jeikeium was
improved and this fact was correlated to the significantly enhanced
growth at acidic pH and survival during desiccation stress. The
increased survival of C. glutamicum cells harboring KtrAB was more
pronounced under potassium limitation and in the presence of
sodium, which is in agreement with the properties of KtrAB
transporters. Interestingly, C. jeikeium is exposed to both types of
stress conditions during colonization of the human skin. The pH on
dry skin is approx. 5.5 or can drop to 5.0 in sweat and low potassium
(5 mM) and high sodium concentrations (80 mM) on skin or in sweat
can be found [18]. This is in agreement with the hypothesis that the
KtrAB system is of relevance for acclimatization of Actinobacteria
towards low pH and desiccation stress. In agreement, desiccation
tolerance was concluded for C. diphtheriae which was isolated after
9 weeks of desiccation in silica gel-dried swabs. It was also shown for
M. tuberculosis that can remain viable in dust for up to 120 days and
upon storage under vegetable oils for approx. 2 years [19,20]. The
comparison of potassium transport systems in these strains revealed
that in contrast to C. glutamicum mycobacteria harbor genes encoding
the high-affinity potassium ATPase Kdp and C. diphtheriae carries a
KtrAB transport system in addition to a CglK-type channel. The
correlation between desiccation tolerance and effective potassium
uptake is underlined by the presence of genes encoding the KtrAB and
Kdp carrier in the genome of the highly desiccation-tolerant
Deinococcus radiodurans R1 [21].
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Supplementary materials related to this article can be found online
at doi:10.1016/j.bbabio.2011.01.008.
Acknowledgements
We would like to thank the Bundesministerium für Bildung und
Forschung and the Deutsche Forschungsgemeinschaft for financial
support (BMBF SysMap program (0313704) and SFB796, B5).
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3.5 Evaluation of invertebrate infection models for pathogenic

corynebacteria (Ott et al, 2012a)

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RESEARCH ARTICLE

Evaluation of invertebrate infection models for pathogenic

corynebacteria
Lisa Ott1, Ashleigh McKenzie2, Maria Teresa Baltazar2, Sabine Britting1,2, Andrea Bischof1,2,
Andreas Burkovski1 & Paul A. Hoskisson2
1
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; and 2Strathclyde Institute of Pharmacy and
Biomedical Sciences, University of Strathclyde, Glasgow, UK

Correspondence: Andreas Burkovski, Abstract

Lehrstuhl für Mikrobiologie, Friedrich-
IMMUNOLOGY & MEDICAL MICROBIOLOGY

Alexander-Universität Erlangen-Nürnberg,
Staudtstr. 5, 91058 Erlangen,
Germany. Tel.: +49 9131 85 28086;
Fax: +49 9131 85 28082; e-mail:
aburkov@biologie.uni-erlangen.de
Received 7 October 2011; revised 8 March
2012; accepted 9 March 2012.
DOI: 10.1111/j.1574-695X.2012.00963.x
Editor: Nicholas Carbonetti
Keywords
Acanthamoeba; Caenorhabtitis
elegans; Corynebacterium diphtheriae;
Corynebacterium pseudotuberculosis;
Corynebacterium ulcerans; Galleria
mellonella.
For several pathogenic bacteria, model systems for host–pathogen interactions
were developed, which provide the possibility of quick and cost-effective high
throughput screening of mutant bacteria for genes involved in pathogenesis. A
number of different model systems, including amoeba, nematodes, insects, and
fish, have been introduced, and it was observed that different bacteria respond
in different ways to putative surrogate hosts, and distinct model systems might
be more or less suitable for a certain pathogen. The aim of this study was to
develop a suitable invertebrate model for the human and animal pathogens
Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, and Coryne-
bacterium ulcerans. The results obtained in this study indicate that Acanth-
amoeba polyphaga is not optimal as surrogate host, while both Caenorhabtitis
elegans and Galleria larvae seem to offer tractable models for rapid assessment
of virulence between strains. Caenorhabtitis elegans gives more differentiated
results and might be the best model system for pathogenic corynebacteria,
given the tractability of bacteria and the range of mutant nematodes available
to investigate the host response in combination with bacterial virulence. Never-
theless, Galleria will also be useful in respect to innate immune responses to
pathogens because insects offer a more complex cell-based innate immune sys-
tem compared with the simple innate immune system of C. elegans.

best-investigated pathogenic corynebacterium; however,

Introduction
The genus Corynebacterium belongs to the class of
Actinobacteria (high G+C gram-positive bacteria) and
comprises a collection of morphologically similar, irregu-
lar-shaped, or club-shaped microorganisms (Ventura
et al., 2007; Zhi et al., 2009). To date, more than 80
species were taxonomically classified into the genus
Corynebacterium including industrially important bacteria,
commensals of animals and humans as well as pathogens.
A well-known member of the pathogenic species is Cory-
nebacterium diphtheriae, which is also the type species of
the whole genus.
Corynebacterium diphtheriae is the etiological agent of
diphtheria, a localized toxaemic infection of respiratory
tract and skin that can be fatal (Hadfield et al., 2000).
Owing to its medical importance, C. diphtheriae is the
even for this species, only a few virulence factors have
been characterized. Beside the diphtheria toxin, these
include mainly pili and other adhesion factors (for
reviews, see Collier, 2001; Rogers et al., 2011). Already
these data show a high degree of variation in different
isolates, for example, in respect to pili formation (Ott
et al., 2010). Molecular data on infection are even scarcer
for two closely related species, Corynebacterium ulcerans
and Corynebacterium pseudotuberculosis, which are separate
species within a distinct cluster of the genus Corynebacte-
rium but share the ability to harbor diphtheria toxin-
encoding lysogenic corynephage (Riegel et al., 1995).
Corynebacterium ulcerans has been detected as a
commensal in domestic and wild animals, which may
serve as reservoirs for zoonotic infections. During the last
decade, the frequency and severity of human infections

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2 L. Ott et al.
associated with C. ulcerans appear to be increasing in var-
ious countries. Respiratory diphtheria-like illnesses caused
by toxigenic C. ulcerans strains are increasingly reported
from various industrialized countries (Tiwari et al., 2008)
and have become more common than C. diphtheriae
infections in the United Kingdom (Wagner et al., 2010).
Infections of humans with toxigenic C. ulcerans can be
fatal in unvaccinated patients. They usually occur in
adults who have consumed raw milk or had close contact
with domestic animals (Bostock et al., 1984; Hart, 1984;
Wagner et al., 2010). Beside respiratory diphtheria-like
illnesses, C. ulcerans can also cause extrapharyngeal infec-
tions in humans, including severe pulmonary infections
(Dessau et al., 1995; Nureki et al., 2007; Cosson & Sold-
ati, 2008).
Corynebacterium pseudotuberculosis is the etiological
agent of caseous lymphadenitis, which is prevalent in
sheep and goat herds throughout the world (Dorella
et al., 2006; Baird & Fontaine, 2007). Infections due to
C. pseudotuberculosis are rare in humans, but this patho-
gen is occasionally recovered from cases of suppurative
lymphadenitis in patients with a classical risk exposure of
close contact with sheep. Corynebacterium pseudotubercu-
losis is a facultative intracellular pathogen that is able to
survive and grow in macrophages, thus escaping the
immune response of the host (McKean et al., 2005;
Dorella et al., 2006).
As mentioned earlier, the three described species are
not very well characterized on a molecular level, despite
the fact that genome sequences became available for
C. diphtheriae (Cerdeno-Tarraga et al., 2003), C. ulcerans
(Trost et al., 2011), and C. pseudotuberculosis (Trost
et al., 2010; Cerdeira et al., 2011a, b; Silva et al., 2011)
during the last years. This might be partly due to the lack
of a suitable, easy-to-handle infection model, which
would allow fast identification of virulence factors in
large-scale mutant pools. Besides the classical guinea-pig
model already used by Loeffler, a mouse model for septic
arthritis was successfully established for C. diphtheriae
(Puliti et al., 2006); however, nonmammalian model
systems might offer several advantages over mammals, for
example, in respect to cost effectiveness, handling, and
ethical aspects.
The aim of this study was to identify a nonmammalian
infection model for pathogenic corynebacteria with
potential for high throughput screening and testing of
mutants. A number of different model systems, including
amoeba, nematodes, insects, and fish, have been intro-
duced to study host–pathogen interactions (for recent
reviews, see Dorer & Isberg, 2006; Kurz & Ewbank, 2007,
O’Callaghan & Vergunst, 2010). Here, we present data for
three invertebrate models and their suitability as surrogate
hosts of pathogenic corynebacteria, the amoeba Acantha-
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moeba polyphaga, which is especially applied to investigate
adhesion and phagocytosis (Mattos-Guaraldi et al., 2008),
the nematode Caenorhabtitis elegans, which has been
widely used and often serves as an oral infection route
model (for review see Dorer & Isberg, 2006; Waterfield
et al., 2008; O’Callaghan & Vergunst, 2010), and larvae of
the greater wax moth (Galleria mellonella), an established
model which represents the more complex insect immune
system (Waterfield et al., 2008). Bacterial strains were
selected based on their previous characterization and the
availability of experimental data and sequence informa-
tion.
Materials and methods
Strains and growth conditions
Strains used in this study are listed in Table 1. Corynebac-
terium diphtheriae, C. ulcerans, and C. pseudotuberculosis
were grown in heart infusion (HI) broth at 37 °C, Cory-
nebacterium glutamicum was grown in brain heart infu-
sion (BHI) broth at 30 °C, and Escherichia coli OP50 was
grown in Luria broth (LB) (Sambrook et al., 1989) at
37 °C. If appropriate, kanamycin was added (25 lg mL!1
for C. glutamicum and C. pseudotuberculosis, 30 lg mL!1
for E. coli, 50 lg mL!1 for C. diphtheriae and C. ulcer-
ans). Caenorhabtitis elegans N2, used as the wild type
strain, were maintained and propagated on E. coli OP50
as described (Brenner, 1974).
Infection of Acanthamoeba polyphaga
For a amoebae-bacteria co-culture assay, A. polyphaga
cultures were grown to exponential phase (3–5 days) in
Peptone Yeast Glucose medium at 21 °C (Rowbotham,
1980). Cells were harvested by centrifugation and re-sus-
pended in phosphate-buffered saline (PBS), and live cell
titers (visualized by trypan blue exclusion) were carried
out to assess the number of amoebae for each assay (nor-
mally 2 9 105 amoebae mL!1). Corynebacteria were
grown overnight in HI medium, diluted to an OD600 nm
of 0.1 and grown to an OD600 nm of 0.6 and compared
with a standard curve of viable numbers for infection
assays at a range of multiplicity of infection (MOI). The
amoebae and bacteria were combined in a 24-well plate
at a MOI of 1 : 1 or 1 : 10. Plates were incubated at 21 °
C, with cells harvested for microscopy at the indicated
time intervals.
For a phagocytic plaque assay of amoebae killing, the
method of Froquet et al. (2009) was adapted for assaying
the interaction of A. polyphaga and Corynebacterium spe-
cies. Aliquots (0.75 mL) of HI agar in varying concentra-
tions (10–100% of the manufacturer’s recommended
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Corynebacterium infection model systems 3

Table 1. Strains, plasmids and cell lines used in this study

Strains Description Reference/Source

Corynebacterium diphtheriae
DSM 43988 Nontoxigenic, isolated from throat culture DSMZ, Braunschweig, Germany
DSM 43989 tox+, unknown source DSMZ, Braunschweig, Germany
ISS3319 C. diphtheriae var. mitis, nontoxigenic, isolated Bertuccini et al. (2004)
from patient affected by pharyngitis/tonsillitis
Tn5-46 ISS 3319 carrying a Tn5 insertion in DIP1546 Laboratory strain collection
(encoding a hypothetical protein)

Corynebacterium glutamicum
ATCC 13032 Type-strain, nonpathogenic Abe et al. (1967)

Corynebacterium pseudotuberculosis
FRC41 Isolated from the inguinal lymph node of a Join-Lambert et al. (2006)
12-year-old girl with necrotizing lymphadenitis

Corynebacterium ulcerans
809 Bronchoalveolar lavage sample from an elderly Trost et al. (2011)
woman with a fatal pulmonary infection
BR-AD22 Nasal sample of an asymptomatic female dog Trost et al. (2011)
ELHA1 Phospholipase D-deficient BR-AD22 mutant strain E. Hacker (pers. commun.)

Escherichia coli
OP50 Uracil auxotrophic E. coli B strain (Brenner, 1974)

Plasmids
pEPR1-p45gfp P45, gfpuv, KmR, rep, per, T1, T2 Knoppova et al. (2007)
pXMJ19mCherry ori colE1, oricg, ptac, mCherry, CmR M. Höller (pers. commun.)

Cell lines
Detroit 562 Human hypopharyngeal carcinoma cells Peterson et al. (1968)

concentration) were placed in each well of a 24-well plate
and dried. Diluting the media equalizes the conditions for
amoeba and bacterial growth and establishes the appro-
priate conditions for plaque formation (Froquet et al.,
2009). Corynebacterial cultures were prepared as described
earlier and 50 lL of the culture was pipetted out onto the
surface of the agar and allowed to dry. Amoebal cells were
harvested as described earlier and diluted to a concentra-
tion of 200 9 104, 20 9 104, 2 9 104 or 0.2 9 104 live
cells mL!1 with PBS. Amoebal suspension (5 lL) was pip-
etted into the centre of each well. Plates were incubated at
room temperature for 2–5 days and observed daily for signs
of plaque formation.
Infection of C. elegans
Caenorhabtitis elegans N2 were maintained on E. coli OP50
for 6–7 days until the worms become starved, indicated by
clumping behaviour (de Bono & Bargmann, 1998). The
nematodes were infected with different Corynebacterium
wild-type strains, transformed with pEPR1-p45gfp as well
as E. coli OP50 transformed with pEPR1-p45gfp. Infection
of 20 L4 stage larval worms was carried out with 200 lL of
each bacteria strain (from an overnight culture) on NGM
plates at 21 °C for 24 h. Subsequently, C. elegans was
transferred to plates with 200 lL of unlabeled E. coli OP50
for a further 24 h, to allow the gut to clear of fluorescent
organisms. Worms were assessed each day following infec-
tion, and the dead worms were counted. Transfer back to
E. coli OP50 is essential following infection as corynebacte-
ria are not a preferred prey source for C. elegans and con-
tinued feeding results in the nematodes attempting to leave
the culture plates. Nematodes were paralyzed with 0.6%
2-phenoxy-2-propanol (Sigma; Wormbook.org), mounted
onto agar pads, and photographed using a Leica DMR
fluorescence microscope.
Infection of Galleria mellonella larvae
Aliquots (20 mL) of HI broth were inoculated with bacte-
ria from an overnight culture to an OD600 nm of 0.1, and
the cultures were grown until an OD600 nm of 0.6 was
reached. Bacteria were harvested by centrifugation
(10 min, 4500 g) and resuspended in 10 mM MgSO4 to
an OD600 nm of 10 (approximately 3 9 109 CFU mL!1).
For infection, a 50-lL Hamilton syringe was used to
inject 5 lL aliquots into G. mellonella larvae via the hind-
most left proleg (Jander et al., 2000). For each biological

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4 L. Ott et al.
replicate, five larvae were infected with each strain and
incubated at 25 °C for 48 h.
Adhesion assays
Detroit 562 cells were seeded in 24-well plates (bio-one
Cellstar; Greiner, Frickenhausen, Germany) at a density
of 2 9 105 cells per well 48 h prior to infection. Bacteria
were subcultured (OD600 nm of 0.1 from overnight cul-
tures) in HI broth for 3.5 h and adjusted to an OD600 nm
of 0.2. A master mix of the inoculum was prepared in
DMEM without penicillin/streptomycin at a MOI of 200
(viable counts experiments). The plates were centrifuged
for 5 min at 500 g to synchronize infection and subse-
quently incubated for 1.5 h. The cells were washed with
PBS nine times, detached with 500 lL trypsin solution
(0.12% trypsin, 0.01% EDTA in PBS) per well (5 min,
37 °C, 5% CO2, 90% humidity), and lysed with 0.025%
Tween 20 for 5 min at 37 °C. Serial dilutions were made
in prechilled 19 PBS and plated out on blood agar plates.
After overnight incubation, the number of colony form-
ing units (CFU) was determined.
Results and discussion
The use of multiple, disparate lower eukaryotic hosts, has
been used to model bacterial pathogenesis in mammals
and is well established for some organisms (Mahajan-
Miklos et al., 2000). The application and comparison of
such models in corynebacteria to indicate strain-specific
differences has not been demonstrated before; however, it
is clear that some of these models offer utility in the
study of pathogenic corynebacteria.
Acanthamoeba polyphaga
Amoebae species are well-established model systems for a
number of pathogenic bacteria; however, some bacteria
respond in different ways to phagocytosis, and it has been
shown that some bacteria, such as Listeria monocytogenes,
do not survive ingestion by Acanthamoeba (Akya et al.,
2010). Amoebae models have been used extensively for
studies on phagocytosis of mycobacteria as surrogate
macrophages to study vacuole survival and adaptation
(Mattos-Guaraldi et al., 2008). We tested different
C. diphtheriae strains using a modified version of the
method of Froquet et al. (2009), plaques on C. diphthe-
riae lawns appeared on average after three to five days of
incubation, indicating that the corynebacteria tested were
not able to resist predation by A. polyphaga (data not
shown). This observation was further supported by co-
culture experiments and visualization by fluorescence
microscopy, which indicated digestion of the bacteria
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inside the acid vacuole of A. polyphaga (data not shown).
Although the amoebae were able to predate corynebacte-
ria, in co-culture experiments, the number of dead
amoeba increased depending on the presence of bacteria
(data not shown). The reason for this is unclear; however,
these results suggest that the A. polyphaga model was not
optimal for characterization of corynebacterial strains,
and further experiments with C. diphtheriae, C. pseudotu-
berculosis, and C. ulcerans strains were abandoned.
Caenorhabtitis elegans
Caenorhabtitis elegans has been extensively used to study
bacterial virulence and offers many advantages such as its
ease of culture, microscopic tractability, extensive genetic
resources, mutant libraries, and ease of manipulation
(Mahajan-Miklos et al., 1999). We colonized C. elegans
with a range of GFP-labeled corynebacterial strains and
subsequently transferred to plates with unlabelled E. coli
OP50 before microscopy (Fig. 1) and quantifying survival
(Fig. 2). Worms infected with different strains of coryne-
bacteria, exhibiting a range of virulence properties,
showed different characteristics in the kill curve reflecting
the differences in virulence between strains (Fig. 2), with
the clinical C. diphtheriae strain ISS 3319 resulting in
faster worm death rates than strain DSM 43988. Infecting
C. elegans with GFP-labeled E. coli or C. glutamicum
followed by transfer to unlabeled OP50 E. coli resulted in
clearance of GFP-expressing strains, suggesting that these
bacteria were not colonizing the worms pathogenically. In
the case of C. diphtheriae strain ISS 3319, foregut (buccal
cavity and pharynx) and midgut were colonized by the
bacteria, while C. diphtheriae DSM 43988 colonized solely
the midgut. Toxigenic C. diphtheriae DSM 43989 as well
as C. ulcerans 809 colonized the hindgut of the worm,
while C. ulcerans BR-AD22 and C. pseudotuberculosis
FRC41 spread all over the body. These data suggest that
different virulence properties and mechanisms used by
the wide range of strains we tested are reflected in the
localization and the killing kinetics of the experiments.
Investigation of the time required to colonize the worms
indicated that a minimum of 6 h feeding on pathogenic
corynebacteria was required for successful colonization
(data not shown). Interestingly, the colonization of the
nematodes in the foregut reflects the natural niche of
these organisms in their mammalian hosts, rather than
the disperse colonization of the gut observed in Pseudo-
monas or Serratia (Mahajan-Miklos et al., 1999; Kurz
et al., 2003). In summary, in contrast to the nonpatho-
genic C. glutamicum and E. coli, pathogenic corynebacte-
ria were able to colonize C. elegans in a strain-specific
manner and were tractable by microscopic studies. The
availability of innate immune mutant C. elegans strains
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Corynebacterium infection model systems 5

a1 a2 b1 b2

c1 c2 d1 d2

e1 e2 f1 f2

g1 g2 h1 h2

Fig. 1. Infection of Caenorhabtitis elegans with different corynebacteria. Infection of worms was carried out with 200 lL of each bacterial strain
on NGM plates at 21 °C for 24 h. Nematodes were mounted onto agar pads, paralyzed with 0.6% 2-phenoxy-2-propanol (Sigma), and
photographed using a Leica DMR fluorescent microscope (1 bright field, 2 fluorescence). In each of at least five independent experiments,
approximately 20 worms were infected. Pictures show representative results. Caenorhabtitis elegans N2 fed with (a) Escherichia coli OP50, (b)
Corynebacterium glutamicum ATCC 13032, (c) Corynebacterium diphtheriae ISS3319, (d) C. diphtheriae DSM 43988, (e) C. diphtheriae DSM
43989, (f) Corynebacterium ulcerans 809, (g) C. ulcerans BR-AD22, (h) Corynebacterium pseudotuberculosis FRC41. Tails of nematodes are
indicated by arrows.

would offer attractive possibilities for further studying
host–corynebacterium interactions.
Galleria mellonella
The greater wax moth (Galleria mellonella) model system
has also been successfully used to characterize bacterial
virulence in a range of strains, such as in Pseudomonas
aeruginosa and Burkholderia to illustrate inter-strain varia-
tion in pathogenicity (Jander et al., 2000). The larvae
show a high sensitivity towards these pathogenic bacteria
resulting in melanization in response to infection, and a
50% lethal dose (LD50) of < 10 bacteria was reported
when these were injected into the hemolymph (Jander
et al., 2000; Wand et al., 2011). In general, distinct differ-
ences in the response of the larvae were detected when
different corynebacterial strains were injected (Fig. 3).
While control larvae showed a pale whitish colour and
were highly active, injection of C. glutamicum, a non-
pathogenic Corynebacterium species of biotechnological
importance, led to the development of small melanized
spots. The melanization of larvae is associated with path-
ogen killing and phagocytosis in lepidopteran larvae
(Nappi & Christensen, 2005; Senior et al., 2011). The
effects on the larvae became more severe in response to
injection of nontoxigenic C. diphtheriae strain DSM
43988, which led to brown larvae, and toxigenic C. diph-
theriae strain DSM 43989, which caused even stronger
melanization, with all infected larvae being immobile
without external stimulation. The strongest effects on the
larvae were obtained for C. pseudotuberculosis and C. ulc-
erans strains, which exhibited high degrees of melaniza-
tion, immobility, and rapid death. These data suggest that
the C. pseudotuberculosis and C. ulcerans strains are more

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6 L. Ott et al.

Buffer control (a)

100
white, active

80
% Nematode survival

60
Infection with (b)
40 C. glutamicum
white, small black
spots in the belly,
20 active

0 (c)
Infection with
C. diphtheriae
0 20 40 60 80 100 120
ISS3319
Time (h)
white, black spots in
the belly, decreased
Fig. 2. Nematode survival following infection with Corynebacterium
activity
diphtheriae. Control – Escherichia coli OP50 (▼); C. diphtheriae
ISS3319 (▲); C. diphtheriae DSM 43988 (d); C. diphtheriae DSM Infection with (d)
43989 (&). Data are the mean of three experiments, and the error C. diphtheriae
bars represent the standard deviation of the data. DSM43988
completely brown,
decreased activity
virulent in this model, perhaps reflecting their wider host
range when compared to C. diphtheriae; a phenomenon Infection with (e)
observed in the opportunistic broad host range pathogen C. diphtheriae
P. aeruginosa (Jander et al., 2000). DSM43989
brown with black
belly, decreased
Application of model systems activity

Infection with (f)

In addition to the already obvious strain-specific differ- C. pseudotuber-
ences detectable in the C. elegans and Galleria model sys- culosis FRC41
tems, two mutant strains were tested as further proof of black, dead
principle (Fig. 4). When the C. diphtheriae wild-type ISS
3319 was compared with a corresponding transposon
mutant Tn5-46, carrying an insertion in a gene encoding a Infection with (g)
hypothetical protein (DIP1546), the mutant was drastically C. ulcerans 809
black, dead
impaired in gut colonization in the C. elegans model, while
in the Galleria system, no difference of wild type and corre-
sponding mutant was observed upon injection, indicating
that the DIP1546 protein is influencing adhesion and host
Infection with (h)
colonization. In fact, in cell culture experiments, Tn5-46
C. ulcerans
showed a decreased adhesion rate to Detroit 562 cells. BR-AD22
In contrast, the C. ulcerans phospholipase D-deficient black, dead
strain ELHA1 showed unaltered colonization behaviour
compared with the parental strain BR-AD22 in the
C. elegans system, while lack of this virulence factor causes
a less severe response in the Galleria model. In this case, Fig. 3. Infection of Galleria mellonella with different corynebacteria.
the adhesion rates of wild type and mutant were identical. A 50-lL Hamilton syringe was used to inject 5 lL aliquots of the
different bacteria into G. mellonella larvae via the hindmost left
proleg. Five larvae were infected with each strain at 25 °C for 48 h.
Conclusions Infection was carried out with (a) buffer control (10 mM MgSO4),
(b) Corynebacterium glutamicum ATCC 13032, (c) Corynebacterium
The results obtained in this study indicate that A. polyph- diphtheriae ISS3319, (d) C. diphtheriae DSM 43988, (e) C. diphtheriae
aga is not optimal as surrogate host for the study and DSM 43989, (f) Corynebacterium pseudotuberculosis FRC41, (g)
characterization of pathogenic corynebacteria. Both Corynebacterium ulcerans 809, (h) C. ulcerans BR-AD22.

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Corynebacterium infection model systems 7

Wild type Mutant Wild type Mutant

2.00

1.50

Adhesion [%]
ISS3319

1.00

0.50

0.00
ISS3319 TN5-46

80.00

Adhesion [%]
60.00
BRAD22

40.00

20.00

0.00
BRAD22 ELHA-1

Fig. 4. Application of model systems. Caenorhabtitis elegans and Galleria larvae were infected with mCherry-labelled corynebacteria (details as
described in legends to Figs 1 and 3), and adhesion rates to Detroit 562 cells were determined for the corresponding strains (experiments carried
out in at least in triplicate). Upper panel: Corynebacterium diphtheriae strain ISS 3319 and corresponding mutant Tn5-46; lower panel:
Corynebacterium ulcerans strain BR-AD22 and phospholipase D-deficient mutant ELHA1. Tails of C. elegans are indicated by arrows.

C. elegans and Galleria larvae seem to offer attractive
models for rapid assessment of virulence differences
between strains. While the Galleria larvae are very easy to
handle, C. elegans gives the more differentiated results,
has extensive genetic tools as a host, is microscopically
tractable, and might represent the best model system for
pathogenic corynebacteria, especially when factors respon-
sible for colonization are screened. Based on our data,
both of these hosts, however, will be useful given the
differences detectable in the Galleria system in response
to absence or presence of toxins (diphtheria toxin) and
other virulence factors (phospholipase D). Furthermore,
insects such as Galleria offer the possibility to study a
more complex cell-based innate immune system com-
pared with the simple innate immune system of C. ele-
gans (Mahajan-Miklos et al., 2000).
Acknowledgements
The Burkovski laboratory is supported by the Deutsche
Forschungsgemeinschaft in frame of SFB 796 (project B5).
The work in the Hoskisson laboratory is supported by
Medical Research Scotland (Grant FRG-422) and by a
Society for General Microbiology Vacation Studentship
grant to A.M. Corynebacterium ulcerans phospholipase D
mutant ELHA1 was kindly provided by E. Hacker and
plasmid pXMJ19mCherry by M. Höller (both Friedrich-
Alexander-Universität Erlangen-Nürnberg), C. elegans N2
was a gift from J. Pettitt (University of Aberdeen). The
support of R. Palmisano (Friedrich-Alexander-Universität
Erlangen-Nürnberg) in respect to fluorescence microscopy
is gratefully acknowledged.
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3.6 Contour and persistence length of Corynebacterium diphtheriae

pili by atomic force microscopy (Rheinlaender et al, 2012)

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Eur Biophys J
DOI 10.1007/s00249-012-0818-4
ORIGINAL PAPER
Contour and persistence length of Corynebacterium diphtheriae
pili by atomic force microscopy
Johannes Rheinlaender • Anna Gräbner •
Lisa Ott • Andreas Burkovski • Tilman E. Schäffer
Received: 10 January 2012 / Revised: 5 April 2012 / Accepted: 21 April 2012
! European Biophysical Societies’ Association 2012
Abstract Many bacteria are characterized by nanoscale
ultrastructures, for example S-layers, flagella, fimbriae, or
pili. The last two are especially important for attachment to
different abiotic and biotic surfaces and for host–pathogen
interactions. In this study, we investigated the geometric
and elastic properties of pili of different Corynebacterium
diphtheriae strains by atomic force microscopy (AFM). We
performed quantitative contour-length analysis of bacterial
pili and found that the visible contour length of the pili can
be described by a log-normal distribution. Our data
revealed significant strain-specific variations in the mean
visible contour length of the pili, ranging from 260 to
1,590 nm. To estimate their full contour length, which is
not directly accessible from the AFM images, we devel-
oped a simple correction model. Using this model, we
determined the mean full contour length as 510–2,060 nm.
To obtain the persistence length we used two different
methods of analysis, one based on the end-to-end distance
of the pili and one based on the bending angles of short
segments. In comparison, the bending angle analysis
Electronic supplementary material The online version of this
article (doi:10.1007/s00249-012-0818-4) contains supplementary
material, which is available to authorized users.
J. Rheinlaender ! A. Gräbner ! T. E. Schäffer
Institute of Applied Physics, University of Erlangen-Nuremberg,
Erlangen, Germany
L. Ott ! A. Burkovski
Chair for Microbiology, University of Erlangen-Nuremberg,
Erlangen, Germany
Present Address:
T. E. Schäffer (&)
Institute of Applied Physics, University of Tübingen,
Auf der Morgenstelle 10, 72076 Tübingen, Germany
e-mail: Tilman.Schaeffer@uni-tuebingen.de
84
Publications
proved to be more precise and resulted in persistence
lengths in the narrow range of 220–280 nm, with no sig-
nificant strain-specific variations. This is small compared
with some other bacterial polymers, for example type IV
pili, F-pili, or flagella.
Keywords Bacteria ! AFM ! Contour length ! Persistence
length ! Stiffness
Introduction
The first crucial steps of interaction of pathogens with their
corresponding hosts are host recognition and binding to the
host’s surface. For this purpose, many bacteria possess
fimbriae and pili, which facilitate attachment to different
abiotic and biotic surfaces.
Corynebacterium diphtheriae is the etiological agent of
diphtheria, a localized infection of the upper respiratory
tract and skin (cutaneous diphtheria), which can be fatal,
because of the potent diphtheria toxin. Adhesion of dif-
ferent C. diphtheriae strains to lung, pharyngeal, and lar-
yngeal epithelial cells and to erythrocytes has been
reported (Colombo et al. 2001; Bertuccini et al. 2004;
Hirata et al. 2002, 2004). Although it is known that
both cell-wall-linked surface proteins and fibrous protein
polymers, for example fimbriae and pili, are of major
importance in the adhesion process for many types of
Gram-positive bacteria, many aspects of the underlying
mechanisms remain unclear (Rogers et al. 2011). For
C. diphtheriae the effect of pili on recognition of and
adhesion to host cells have recently been characterized on
the genetic level. Analysis of mutants has revealed that
C. diphtheriae type strain NCTC13129 is able to assemble
pili from different pilin subunits on its surface. Pili are
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important for bacterium–host cell contact and host cell
preference (Gaspar and Ton-That 2006; Swierczynski and
Ton-That 2006; Mandlik et al. 2007), making them an
interesting focus of research to understand host–pathogen
interaction. Recently, it has been reported that the expression
of different pilin subunit proteins in C. diphtheriae affects pili
length qualitatively (Swierczynski and Ton-That 2006;
Mandlik et al. 2008; Guttilla et al. 2009). However, a
comprehensive statistical analysis of the pili, e.g. giving
the mean contour length, has not yet been reported for
C. diphtheriae. Also, the persistence length, which is an
important measure of the mechanical stiffness of filamen-
tous polymers, has not yet been reported for C. diphtheriae
pili.
Pili and other bacterial cell appendices and surface
structures are often studied by electron microscopy, which
requires staining or labeling procedures. Atomic force
microscopy (AFM) can image the topography of unstained
samples and has already been intensively applied to the
study of bacteria (Anselmetti et al. 2007; Dupres et al.
2009; Wright et al. 2010) and to bacterial pili (Touhami
et al. 2006; Miller et al. 2006; Fälker et al. 2008; Arce et al.
2009). The contour length of the pili can vary by one order
of magnitude within a single cell (Yanagawa and Honda
1976; Touhami et al. 2006). However, when imaged while
being attached to the cell body, the contour length of the
pili can be systematically underestimated.
Here we describe comprehensive characterization of pili
ultrastructure for five different C. diphtheriae strains, giv-
ing two important quantities: the mean full contour length
and the persistence length. For this purpose, we develop a
simple model for estimating the mean full contour length
from the experimentally accessible mean visible contour
length in high resolution microscopy images. To obtain
persistence length of the pili we used two different analysis
methods: one based on the end-to-end distance and one
based on the bending angles of short pili segments. Finally,
we relate the strain-specific variations in pili formation to
the different abilities for adhesion and invasion of host
cells found in previous studies and to genetic information
(Swierczynski and Ton-That 2006; Mandlik et al. 2007;
Guttilla et al. 2009; Ott et al. 2010).
Methods
Growth of bacteria and sample preparation
Corynebacterium diphtheriae overnight cultures grown in
20 ml HI broth were washed five times in 20 ml ice-cold
distilled water and were resuspended in 10 ml ice-cold
distilled water. Five microliters of each sample was fixed
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on a glass slide or freshly cleaved mica substrate by drying,
by use of compressed air.
Atomic force microscopy
The samples were imaged by AFM (MFP-3D; Asylum
Research, Santa Barbara, USA) using standard silicon
cantilever probes (NCH-W; Nanosensors, Neuchatel,
Switzerland) in tapping mode under ambient laboratory
conditions. All images are unprocessed except for line-by-
line polynomial background removal (‘‘flattening’’). All
data analysis was performed in IgorPro (Wavemetrics,
Lake Oswego, OR, USA) by use of custom-written soft-
ware and least-squares fitting routines.
Theoretical background
Persistence length
To determine the persistence length of the pili, we used
the worm-like chain (WLC) model, which describes a
semi-flexible polymer chain in thermodynamic equilib-
rium (Landau et al. 1980). The WLC model has been
applied to many filamentous biomolecules and biostruc-
tures, for example actin (Nagashima and Asakura 1980;
Yanagida et al. 1984), microtubules (Gittes et al. 1993),
DNA (Rivetti et al. 1996), bacterial flagella (Trachtenberg
and Hammel 1992), type F-pili (Silverman and Clarke
2010), and type IV pili (Skerker and Berg 2001). In this
model, the polymer is described as a homogenous elastic
rod with a circular cross-section. The energy U required
to bend a polymer segment of length DL by an angle # is
given by:
EI 2
U ð# Þ ¼ # ; ð1Þ
2DL
where E is the stiffness (Young’s modulus) and I = pr4/4
is the polymer’s cross sectional area moment of inertia,
where r is the radius of the cross section (Landau et al.
1986). In thermodynamic equilibrium, the probability p
that the segment is bent by a specific angle is given by
Boltzmann’s law:
U ð# Þ 1P
pð#Þ / e$ kB T ¼ e$2DL# :
2
ð2Þ
Consequently, the standard deviation r# of the bending
angles is given by r2# = h#2i = DL/P and the moments of
the bending angle distribution fulfill h#4i/h#2i2 = 3. The
persistence length of the polymer:
EI
P¼ ; ð3Þ
kB T
 Publications
Eur Biophys J

can be interpreted as the distance along the polymer at Experimental results

which its angular orientation becomes uncorrelated. For a
long polymer of contour length L that is confined within a Characterization of the ultrastructure of pili
plane, the WLC model predicts the mean squared end-to-
end distance as (Rivetti et al. 1996): AFM topography images of C. diphtheriae strains revealed
% $& pili on the substrate extending from the cell bodies
! 2" 2P # L
R ¼ 4PL 1 " 1 " e"2P : ð4Þ (Fig. 1a–e, arrows). Strain-specific differences in pili
L
number and morphology were apparent. For example,
Log-normal distribution whereas DSM44123 cells had only a small number of sub-
micrometer long, relatively straight pili (Fig. 1b), ISS4749
Growth processes with randomly fluctuating growth rates cells had many pili, which were much longer and at first
often have log-normal distributions (Koch 1966). Hence, sight seemed more curved (Fig. 1e). DSM43988 cells had
many quantities in biology, but also in many other fields, no pili (Fig. 1f).
for example medicine, social sciences, and economics, are To obtain quantitative, strain-specific differences among
log-normally distributed (Limpert et al. 2001). For a log- length and curvature, we performed a systematic study of
normally distributed random variable L, the variable’s the pili. First, we recorded many images of pili for the
natural logarithm x = ln(L) is normally distributed. So the different strains at high resolution. In offline analysis, we
probability density function / for x is: then manually traced the pili by selecting close-spaced
! points along their contour (Fig. 2a). Only pili that were
1 ð x " lÞ 2 clearly identifiable along their entire contour on the sub-
/l;r ð xÞ ¼ pffiffiffiffiffi exp " ; ð5Þ
2pr 2r2 strate and with a visible length of at least 100 nm were
selected for analysis. Sufficient statistical precision was
where l and r are the arithmetic mean and the standard
achieved by analyzing approximately 30 individual pili for
deviation, respectively, of the distribution. Consequently,
each strain which originated from 6 to 17 different cells.
the probability density function of L is:
To investigate the effect of the substrate on persistence
!
dx 1 ðlnðLÞ " lÞ2 length, cells prepared on both glass and mica were ana-
f ðLÞ ¼ /l;r ð xÞ ¼ pffiffiffiffiffi exp " ð6Þ lyzed. Finally, we converted the sets of points into smooth
dL 2prL 2r2
contour paths by cubic spline interpolation (Fig. 2b).
and the cumulative distribution function is: Interpolation was necessary because contour length esti-
mation of a curved structure from a discrete set of points
ZL lnðLÞ
Z can lead to significant discrepancies between actual and
0 0
F ðLÞ ¼ f ðL Þ dL ¼ /l;r ðx0 Þ dx0 determined contour length (Dorst and Smeulders 1987).
0 ( "1 ) These discrepancies can be reduced to less than 1 % by
1 lnðLÞ " l cubic spline interpolation, as was demonstrated for AFM
¼ erfc " pffiffi : ð7Þ
2 2r images of DNA molecules (Rivetti and Codeluppi 2000).
Here, ‘‘erfc’’ denotes the complementary error function. We also estimated the diameters of the pili by analyzing
For a log-normal distribution the geometric mean L! is their cross sections in the high-resolution AFM images;
they were 1–2 nm for all strains.
related to the arithmetic mean l of x by L! ¼ expðlÞ. The
standard deviation of x, r, can be transformed to the geo-
Visible contour length
metric standard deviation of L, rGSD = exp(r). For the
normally distributed x the interval l ± r ranges from
As is apparent in the AFM images (Fig. 1), the visible
l - r to l ? r and covers approximately 68.3 % of the
contour length of the pili for a given C. diphtheriae strain
data. For the log-normally distributed L the corresponding
! GSD to L! 9 rGSD. The varies. We therefore statistically analyzed the measured
interval exp(l ± r) ranges from L/r
visible contour lengths L of the imaged pili by generating a
95 % confidence interval of the mean after N measure-
pffiffiffiffi cumulative histogram for each strain (Fig. 3a, b, and Suppl.
ments, which ranges from l - t 9 r/ N to l ? t 9 Fig. S1, black curves). The log-normal distribution
pffiffiffiffi
r/ N for a normally distributed x, is transformed accord- describes the data well, as can be seen by the close match
pffiffiffiffi
!
ingly, resulting in a range from L/exp[t 9 r/ N ] to between the measured cumulative histograms and the
pffiffiffiffi
L! 9 exp[t 9 r/ N ] for a log-normally distributed L. The corresponding cumulative distribution functions, using
expression t = t0.975,N-1 denotes the 97.5th percentile of the respective L! and rGSD of the data (solid gray curves).
the Student’s t-distribution with N - 1 degrees of freedom. More formally, we confirmed this close match by a

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Fig. 1 a–f AFM topography a DSM43989 b DSM44123 c ISS3319

images of bacteria from
different C. diphtheriae strains.
Pili of different number, length,
and curvature appear as fine
lines extending from the cell
bodies (blue arrows). For
example, DSM44123 cells
(b) have only a small number of
sub-micrometer long pili,

height
whereas ISS4749 cells (e) have
many pili up to several d ISS4746 e ISS4749 f DSM43988
micrometers long. DSM43988
cells (f) do not have pili. Scan
size 4 lm 9 4 lm, height data
range 500 nm, color scale range
2 nm

Full contour length

a Topography b Contour paths

The pili in the AFM images could be identified on the

cell body
height
Fig. 2 Analysis of pili ultrastructure. a High-resolution AFM topog-
raphy image of the edge of a DSM44123 cell. Pili contours were
traced by manually selecting several points along each pilus
(markers). Only pili that were clearly identifiable along their entire
contour on the substrate and with a visible length of at least 100 nm
were selected for analysis. b A smooth contour path was generated
from selected points along each pilus by cubic spline interpolation,
enabling an accurate determination of the visible contour length L and
the visible end-to-end distance R. Scan size 500 nm 9 500 nm, color
scale range 2 nm
Kolmogorow–Smirnow test using a significance level
a = 0.05. This justifies the use of the log-normal
distribution.
The results for the mean visible contour lengths for the
different strains are plotted in Fig. 3c and are listed in
Table 1. The differences between the mean visible contour
lengths for the five strains were statistically significant as
confirmed by Student’s t tests applied to the logarithmi-
cally transformed data (Limpert and Stahel 2011; p val-
ues \ 0.01), except for the difference between DSM43989
and ISS4746 (p value = 0.025) and the difference between
DSM44123 and ISS3319 (p value = 0.083).
substrate but not on the cell bodies, owing to the relatively
large surface roughness (several nanometers) of the cell
bodies. Therefore, the full contour length of a pilus is
longer than its visible contour length by the part that lies on
the cell body (Fig. 3d). The consequence is that the mea-
sured, visible contour lengths (Fig. 3c and Table 1)
underestimate the respective full contour lengths. To esti-
mate the full contour lengths we developed a simple, one-
dimensional model using four simplifications.
1. Because C. diphtheriae cells are rod-shaped and
elongated the statistical effect of the pili at the cell
poles is small. We therefore excluded them from the
model.
2. A pilus is anchored in the cell surface at a random
position.
3. Because the persistence length is similar in size to the
width of the body of a typical cell, the pili were
modeled as straight.
4. For simplicity we assumed the pili were of equal
length for each strain and were oriented perpendicular
to the long cell axis.
We now consider the two limiting cases:
1. short pili with a full contour length smaller than the
upper perimeter B of the cell body (Fig. 4a); and
2. long pili with a full contour length larger than the
upper perimeter B of the cell body (Fig. 4b).
For the short pili, some of the pili reach the substrate and
become visible. On average their visible contour length is
half their full length: L! ! L!full =2. For long pili all pili reach
the substrate and, on average, the visible contour length

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a c
1.0 = 28

(µm)
relative frequency
1.5

mean visible contour length

0.5 = 28
ISS3319 1.0 = 29
= 256 nm
= 1.60
0.5 = 29
0.0 = 27
0.0 0.2 0.4 0.6 0.8

0.0

19

46

49
b

89

3
12

33

47

47
39
1.0

44

ISS

ISS

ISS
M4

M
relative frequency

DS

DS
d
0.5
ISS4746 AFM tip cell body
= 940 nm
= 1.31 pilus
full
0.0
0.0 0.5 1.0 1.5 2.0
visible contour length (µm)

Fig. 3 Contour length analysis. a, b Cumulative histograms of
measured visible contour lengths of pili a from strain ISS3319 and
b from strain ISS4746 (black curves) with respective geometric means
L! and geometric standard deviations rGSD. The corresponding log-
normal cumulative distribution functions (Eq. 7) (solid gray curves)
match the experimental data well. Also shown are the corresponding
probability density functions (Eq. 6) (not normalized) (dashed gray
curves). The cumulative histograms for the remaining strains are
shown in Suppl. Fig. S1. c Strain-specific visible contour lengths
(geometric mean, error bars represent the 95 % confidence intervals).
Statistical significance of the differences (Student’s t test):
p value \ 0.01 for all pairs except for the pair DSM43989—
ISS4746 (p value = 0.025) and the pair DSM44123—ISS3319
(p value = 0.083). d Schematic diagram illustrating the difference
between the visible (L) and the full (Lfull) contour length of a pilus.
B denotes the upper perimeter of the cell body

Table 1 Measured mean visible contour length (geometric mean L),! mean full contour length from the correction model, and persistence length
from method 2 (segment bending angles) for the pili from five different C. diphtheriae strains
Strain Mean visible contour Mean full contour Persistence length
length (nm) length (nm) (nm) (method 2)

DSM43989 780 [680, 880] 1,230 [1,070, 1,400] 220 [190, 250]
DSM44123 320 [270, 380] 640 [530, 760] 280 [230, 340]
ISS3319 260 [210, 310] 510 [430, 620] 280 [220, 350]
ISS4746 940 [840, 1,040] 1,390 [1,250, 1,540] 240 [210, 270]
ISS4749 1,590 [1,400, 1,790] 2,040 [1,800, 2,300] 260 [230, 280]
The values in parentheses indicate the respective 95 % confidence interval

differs from the full contour length by half of the upper Persistence length
perimeter of the cell body: L!full ! L! " B=2. In summary,
the mean full contour length can be approximated from the To obtain the persistence length P of the pili we used two
mean visible contour length by: different methods of analysis. The first method is based on
! the end-to-end distance (‘‘method 1’’) and has previously
! L\B=2
L; !
L!full " L! þ : ð8Þ been applied to biopolymers such as DNA (Rivetti et al.
B=2; L! $ B=2
1996; Mantelli et al. 2011), flagella (Trachtenberg and
Here we used B = 1 lm as a typical upper perimeter of the Hammel 1992), F-pili (Silverman and Clarke 2010), and
cell body for all strains analyzed. The results of the cor- filamentous bacteriophages (Wang et al. 2006). Using this
rection based on Eq. (8) are shown in Fig. 4c and Table 1. method we plotted, for each strain, the mean squared

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short pili ( Lfull < B ) N = 28

mean full contour length Lfull (µm)

a c
cell surface
2.0
pilus
N = 28
N = 29
L
L full 1.0
N = 29
N = 27
B

long pili ( Lfull > B )

b 0.0

89

23

19

46

49
39

41

33

47

47
M4

M4

ISS

ISS

ISS
DS

Fig. 4 One-dimensional model for estimating the mean full contour
length of the pili. a Short pili with a full contour length L!full smaller
than the upper perimeter B of the cell body. Only some of the pili
reach the substrate and become visible in the AFM images. On
average their visible contour length is half their full contour length:
L! $ L!full =2. b Long pili with a full contour length L!full larger than the
DS
upper perimeter B of the cell body. All pili reach the substrate and on
average the visible contour length differs from the full contour length
by half of the upper perimeter B: L!full ! L! $ B=2. c Strain-specific
mean full contour lengths (geometric mean, error bars indicate the
95 % confidence intervals)

visible end-to-end distance R2 of each pilus vs. its visible the bending angle, cos(#), were considered. However, as
contour length L. This is shown in Fig. 5a for strain can be seen from Eq. (2), the persistence length can also be
DSM44123 as an example; the measured values of R2 and directly obtained from the standard deviation of the bend-
L are labeled with different markers for a preparation on a ing angles, r#. This has particular benefits compared with
glass (‘‘?’’) or mica (‘‘9’’) as substrate. We then fitted method 1:
Eq. (4) to these data (Fig. 5a, solid line) to obtain the
persistence length P of the pili. For strain DSM44123, – first, because much more information is used, the sta-
using method 1, we obtained P = 520 ± 160 nm (fit tistical precision is much higher for the same number of
coefficient ±95 % confidence interval). The persistence polymers; and
lengths of the other strains were obtained similarly (for the – second, the statistical uncertainty follows directly from
other strains see Suppl. Fig. S2). The resulting persistence the statistics of the standard deviation.
lengths based on the end-to-end distance range from
The 95 % confidence interval of r2# after measuring Ns
280 to 520 nm and are plotted in Fig. 5b.
polymer segments is given by:
However, two problems arise from this method when
" #
applied to polymers of different contour length: first, long Ns ! 1 2 Ns ! 1 2
polymers have an overly high effect on the fit and hence on the s ; s : ð9Þ
v2Ns !1;0:975 # v2Ns !1;0:025 #
resulting persistence length. Second, the width of the confi-
dence interval obtained is only a lower estimate, because the Therefore, the 95 % confidence interval of the resulting
statistical fluctuation of R2 is not included. Furthermore, persistence length P is:
because only one value per polymer (the end-to-end distance) " #
is measured the statistical sample is relatively small. As can be DL v2Ns !1;0:025 DL v2Ns !1;0:975
; : ð10Þ
seen in Fig. 5b the differences obtained between strain-spe- s2# Ns ! 1 s2# Ns ! 1
cific persistence lengths are high (approx. a factor of two),
nevertheless they are not statistically significant (Student’s t Here, s2# is the measured sample variance of the bending
tests give p values [ 0.01 for all pairs). angle, v2Ns !1; 0:025 and v2Ns !1; 0:975 denote the 2.5th and 97.5th
To improve the statistical precision we therefore applied percentiles of the chi-squared distribution with Ns - 1
a second method that is based on the bending angle of short degrees of freedom.
polymer segments (‘‘method 2’’). This was possible Figure 5c shows the histogram of bending angles
because the paths of the pili were traced digitally. Similar using pili segments of length DL = 50 nm for strain
methods have been applied to DNA (Bednar et al. 1995; DSM44123 as an example (for the other strains see
Abels et al. 2005), for which the statistics of the cosine of Suppl. Fig. S3). The mean bending angle is zero within

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Fig. 5 Two methods for

persistence length analysis, both Method 1: Method 2:
shown for strain DSM44123 as end-to-end distance R segment bending angles
example (for the other strains
see Suppl. Figs. S2 and S3).
a Method 1: Squared visible
end-to-end distance R2 versus L
visible contour length L. The a c
persistence length P (±95 % 0.3
confidence interval) is DSM44123 DSM44123
determined by fitting Eq. (4) P = (520±160) nm 100 P = 280 [230,340] nm
(solid line). The dotted line 0.2

frequency
R 2 (µm2)

indicates the limit of infinitely

stiff pili, corresponding to
50
R2 = L2. Different markers 0.1
represent preparations on glass
(plus) or mica (cross) as
substrates. b Strain-specific
0.0 0
persistence lengths based on R2
0.0 0.1 0.2 0.3 0.4 0.5 -2.0 -1.0 0.0 1.0 2.0
versus L data. c Method 2:
Histogram of bending angles visible contour length L (µm) bending angle (rad)
using segments of length
b d
DL = 50 nm. The standard
deviation of the bending angles 0.8 N = 28 0.8
r# = 0.42 [0.35, N = 29
0.52] corresponds to a N = 28
0.6 0.6
persistence length of
P = 280 [230, 340] nm.
P (µm)

N = 29 N = 27
The gray trace shows the 0.4 0.4 Ns = 210 Ns = 137
Ns = 464 Ns = 531 Ns = 913
corresponding Gaussian
distribution from Eq. (2). 0.2 0.2
d Strain-specific persistence
lengths based on the segment
bending angles. Error bars 0.0 0.0
indicate the 95 % confidence
89

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46

49
89

23

19

46

49

39

41

33

47

47
39

41

33

47

47

intervals
M4

M4

ISS

ISS

ISS
M4

M4

ISS

ISS

ISS

DS

DS
DS

DS

the measurement precision, h#i = -0.03 [-0.09, 0.03], Discussion

and the standard deviation of the bending angles is
r# = 0.42 [0.35, 0.52]. The values in brackets indicate
the corresponding 95 % confidence intervals based on
Eq. (9). Furthermore, the distribution is Gaussian,
as indicated by the close match with the correspond-
ing distribution from Eq. (2) (gray trace) (for the other
strains see Suppl. Fig. S3). The standard deviation
of the bending angles corresponds to a persistence
length of P = DL/r2# = 280 [230, 340] nm. The persistence
lengths based on the bending angle statistics (method 2) are
summarized in Fig. 5d and are listed in Table 1. It can be
seen that the strain-specific persistence lengths from
method 2 are within a narrow range of 220–280 nm and
that the confidence intervals are much smaller than those
for method 1. Nevertheless, the differences between the
persistence lengths from the different strains are still not
significant (Student’s t tests give p values [ 0.05 for all
pairs). When we applied method 2 to the data from the
preparations on glass and mica separately, no significant
effect of the substrate on the measured persistence length
was detected.
Pili are crucial for adhesion to host cells, and for different
C. diphtheriae strains this process varies substantially (Ott
et al. 2010). Surprisingly, there seems to be no direct
correlation between adhesion efficiency and pili number or
length. Whereas ISS4746 and ISS4749 have long pili
(Fig. 3c) and strong adhesion, DSM43989 has long pili but
adheres much less strongly, and DSM44123 and ISS3319
have short pili but moderate adhesion.
One important question is whether features of the bac-
terial phenotype, for example pili contour length, can be
related to genetic information. Pili subunits SpaA and
SpaH have been identified as the main shaft proteins of
C. diphtheriae, and their existence or overproduction have
been found to increase the pili contour length
(Swierczynski and Ton-That 2006; Mandlik et al. 2007;
Guttilla et al. 2009). Strains ISS4746 and ISS4749 have a
large pili contour length (Fig. 3c) and high expression of
SpaA and SpaH (Ott et al. 2010). On the other hand, strains
DSM44123 and ISS3319, which have short pili, and
express no SpaH and comparatively little SpaA. Therefore,

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increased expression of SpaA and SpaH in combination or
alone might result in increased pili length.
To analyze the mechanical properties of the pili, we
applied the WLC model to determine the persistence
length. We used two different methods of analysis, based
on the end-to-end distance (method 1, Fig. 5b) and on the
bending angles of short segments (method 2, Fig. 5d). In
comparison, two differences can be seen between the
persistence lengths when using method 1 and when using
method 2.
– First, the confidence intervals from method 2 are
smaller than those from method 1.
– Second, on average, the persistence lengths from
method 2 seem to be smaller.
There are three main reasons for these differences.
– First, in method 1 only one value per pili is used. The
statistical sample of approximately 30 pili per strain is
therefore much smaller than for method 2 in which
approximately 100–900 segments per strain are
analyzed.
– Second, because the persistence length is positive by
definition, noise will, on average, increase the resulting
persistence length.
– Third, method 1 is more sensitive to directional bias.
These reasons imply that method 2 is both more accurate
and more precise. Therefore, we only consider the results from
method 2 (Table 1) in the following discussion.
By estimating statistical error we found that differences in
persistence length between the strains were not significant
(Student’s t test p values [ 0.05). In general, the persistence
length of C. diphtheriae pili seems to be significantly lower
than the persistence lengths of other bacterial polymers, for
example type IV pili (P & 5 lm; Skerker and Berg 2001),
F-pili (P & 5 lm; Silverman and Clarke 2010), or bacterial
flagella (P & 20–40 lm; Trachtenberg and Hammel 1992).
C. diphtheriae pili are also much more flexible than actin
(P & 20 lm) or microtubules (P & 5 mm; Gittes et al.
1993), but they are stiff in comparison with DNA
(P & 50 nm; Taylor and Hagerman 1990; Rivetti et al.
1996). Interestingly, the persistence length of filamentous
bacteriophages (P & 1 lm) is similar to that of C. diph-
theriae pili (Wang et al. 2006).
One limitation possibly affecting our measurements and
those previously obtained by electron microscopy is the use
of dried samples. It is known for electrically charged
polymers, for example DNA, that changing local electro-
static interactions have an effect on persistence length
(Baumann et al. 1997; Mantelli et al. 2011). C. diphtheriae
pili, however, are probably uncharged (Kang et al. 2009),
making the persistence length of the pili insensitive to
electrostatic conditions. Another possible limitation of our
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analysis is the assumption that the pili thermally equilibrate
in the substrate plane (Eq. 4) before they are immobilized
by the drying process (Wang et al. 2006; Silverman and
Clarke 2010). This is in contrast with another possible
scenario in which the pili are ‘‘kinetically trapped’’ on the
substrate, i.e. are immobilized immediately upon initial
contact with the substrate. For polymers without a charge
or with a low charge, for example C. diphtheriae pili,
however, this scenario is less likely (Bezanilla et al. 1995).
From the AFM images we estimated the diameter of the
pili as 1–2 nm. This is smaller than the value obtained by
electron microscopy (2.5–3.5 nm; Yanagawa and Honda
1976). A possible explanation of this discrepancy is that
AFM might underestimate the diameter because of the non-
zero imaging force that indents soft samples (Jiao and
Schäffer 2004). Electron microscopy, on the other hand,
might overestimate the diameter because of the conductive
coating required before imaging. From the crystal structure
of the pili subunit SpaA (Kang et al. 2009), we read off a
diameter of 2.5 nm. In the following discussion, we assume
a diameter of 2.5 nm to estimate the stiffness of the pili.
In combination with the persistence length, the pili
diameter can be used to estimate the stiffness (Young’s
modulus) of the pili by rearranging Eq. (3) and assuming
the pili are homogeneous, isotropic cylindrical rods of
diameter 2.5 nm. This gives a stiffness of 400–600 MPa
for the analyzed C. diphtheriae strains. In general, the
stiffness of C. diphtheriae pili is greater than that of DNA,
which is approximately 100–300 MPa, depending on base
composition (Hogan et al. 1983) and on the composition
(Wang et al. 1997) and ionic strength (Baumann et al.
1997) of the surrounding buffer solution. But it is smaller
than that of some other proteins, for example microtubules
(E & 1.2 GPa; Gittes et al. 1993) or actin filaments
(E & 2 GPa; Kojima et al. 1994; Huxley et al. 1994). It is
also smaller than that of electrically conductive microbial
nanowire pili (E & 1 GPa; Leung et al. 2011), which were
discovered recently (Reguera et al. 2005).
Our results also enable us to draw conclusions about the
mechanical properties of the pili subunits. The pili of
strains ISS4746 und ISS4749 consist of SpaA and SpaH
subunits whereas the pili of the other strains in this study
consist of SpaH only (Ott et al. 2010). Because the pili
persistence lengths of all investigated strains were similar
(within the precision of our measurement), we can con-
clude that SpaA and SpaH must contribute approximately
equally to the persistence length.
Conclusion
We have performed comprehensive AFM analysis of pili
contour length and persistence length for different

Eur Biophys J
C. diphtheriae strains. Measurements revealed significant
strain-specific differences among mean visible contour
length. Because each pilus lay partially on the cell body,
on which it could not be visualized, its visible contour
length underestimated its full contour length. By use of a
simple model we estimated mean full contour lengths for
the different strains as 510–2,060 nm, with significant
strain-specific variation. We also measured the persistence
lengths of the pili—as far as we are aware the first such
analysis for type III pili. We used two different methods
of analysis—one based on the end-to-end distance and
one based on the bending angles of short segments. The
bending angle analysis proved to be more precise.
The persistence lengths did not vary significantly among
the strains investigated, and were in the range
220–280 nm, which is relatively low compared with other
bacterial polymers, for example type IV pili, F-pili, or
flagella.
Acknowledgments ISS strains were kindly provided by
C. v. Hunolstein (Istituto Superiore di Sanita, Rome). LO and AB are
grateful for financial support of the Deutsche Forschungsgemeinschaft
(SFB796, B5). We thank Yves Muller (Biotechnology Division,
University of Erlangen-Nuremberg) for assistance with the Protein Data
Bank. We thank Asylum Research and Atomic Force F&E for support.
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3.7 Induction of the NFĸB signal transduction pathway in response

to Corynebacterium diphtheriae infection (Ott et al, 2012b)

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Induction of the NF-B signal transduction pathway in response to Corynebacterium

diphtheriae infection

Running title: NF-B induction by C. diphtheriae

Lisa Ott1), Brigitte Scholz2), Martina Höller1), Kristin Hasselt3), Armin Ensser2) and Andreas

Burkovski1)*

1)
Friedrich-Alexander-Universität Erlangen-Nürnberg, Lehrstuhl für Mikrobiologie, Staudtstr.

5, 91058 Erlangen, Germany

2)
Klinische und Molekulare Virologie, Virologisches Institut des Universitätsklinikums

Erlangen, Schlossgarten 4, 91054, Erlangen, Germany

3)
BioCer Entwicklungs-GmbH, Ludwig-Thoma-Str. 36c, 95447 Bayreuth, Germany

* Corresponding author: Andreas Burkovski, Lehrstuhl für Mikrobiologie, Friedrich-Alexander-

Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany. Phone: +49 9131 85

28086. Fax: +49 9131 85 28082. E-mail: aburkov@biologie.uni-erlangen.de

Keywords: host-pathogen interaction, internalization, invasion, MAMP, PAMP

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Summary (Abstract)

Corynebacterium diphtheriae, the causative agent of diphtheria, has been thoroughly studied

with respect to toxin production and pili formation, while knowledge on host responses to C.

diphtheriae infection is limited. In this study, we studied adhesion and invasion of epithelial

cells by different C. diphtheriae isolates. When NF-B reporter cell lines were used to monitor

the effect of C. diphtheriae infection on human cells, strain-specific differences were

observed. While adhesion to host cells had no effect, a correlation of invasion rate with NF-

B induction was found, which indicates that internalization of bacteria is crucial for NF-B

induction. Immune-fluorescence microscopy experiments used to support the reporter

assays showed that translocation of p65, as a hallmark of NF-B induction, was only

observed in association with cell invasion by C. diphtheriae. Our data indicate that the

response of epithelial cells to C. diphtheriae infection is determined by internalization of

bacteria and that invasion into these cells is an active process; tetracycline-treated C.

diphtheriae were still able to attach to host cells, but lost their ability to invade into the

cytoplasm. Recognition of pathogen-associated molecular patterns such as pili subunits by

membrane-bound receptors facing the outside of the cell is not sufficient for NF-B induction.

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Introduction

Corynebacterium diphtheriae is the etiologic agent of diphtheria, a toxaemic infection of

respiratory tract or skin. While diphtheria has almost been eradicated by vaccination programs

in many industrialized countries (Galazka, 2000; Hadfield et al., 2000; von Hunolstein et al.,

2003), the disease is still a global medical challenge and especially dangerous in many

resource poor countries. Furthermore, it has become evident that C. diphtheriae infections can

cause other manifestations besides diphtheria, such as bacteraemia, endocarditis, pneumonia,

osteomyelitis, septic arthritis and spleenic abscesses (Puliti et al., 2006 and references

therein; Hirata et al., 2008; Farfour et al., 2012). These systemic infections indicate that this

pathogen is not only able to attach to host epithelial cells, but must also be able to gain access

to deeper tissues by unknown portals of entry and to persist in these tissues. A possible

indication with regard to mechanisms of persistence of C. diphtheriae within the human body

came from comparative investigations of adherence and invasion of toxigenic and non-

toxigenic strains. Using a combination of gentamicin protection assays and thin-section

electron microscopy, Hirata and co-workers (Hirata et al., 2002) showed that toxigenic C.

diphtheriae were not only able to adhere to laryngeal HEp-2 cells, but also enter these cells

and survive after internalization. Similar observations were made for non-toxigenic strains,

showing that pharyngeal Detroit562 cells can be also invaded by C. diphtheriae (Bertuccini et

al., 2004; Ott et al., 2010a; Ott et al., 2010b).

With exception of the well documented effect of the diphtheria toxin on cellular

metabolism, responses of the human host to C. diphtheriae infection are almost not

investigated. Only a limited number of virulence factors important for host recognition and

attachment are known (for example see (Mandlik et al., 2007; Mattos-Guaraldi et al., 2000; Ott

et al., 2010a; Ott et al., 2010b). Mice were infected by intravenous injection of bacteria in order

to establish an experimental model of C. diphtheriae infection and development of septic

arthritis. Besides mortality and arthritogenic potential of different strains, cytokine production

was determined in this study (Puliti et al., 2006). Strain-specific differences were observed for

the production of interleukins and interferons, i.e. IL-1, IL-6, IFN- and TNF. On the one

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hand, production of these cytokines is known to be involved in NF-B signalling (Perkins,

2007; Tato & Hunter, 2002), a pathway which is frequently activated upon invasion of

pathogens. On the other hand it is known that pathogens have different strategies to interfere

with the NF-B pathway and some are even able to inhibit NF-B activation (Tato & Hunter,

2002). Since data were lacking for C. diphtheriae, we started an approach to investigate NF-

B induction in epithelial cells in response to C. diphtheriae infection, which is presented here.

Materials and methods

Bacterial strains and growth

Strains used in this study are listed in Table 1. C. diphtheriae was grown in Heart Infusion (HI)

broth at 37°C, Escherichia coli DH5aMCR were grown in Luria Broth (LB) (Sambrook et al.,

1989) at 37°C. If appropriate, kanamycin was added (30 μg ml-1 for E. coli; 50 μg ml-1 for C.

diphtheriae).

Cell cultures

Detroit562 cells were cultured in DMEM (Dulbecco’s  modified  Eagle’s  medium), high glucose

with L-glutamine and sodium pyruvate (PAA Laboratories, Austria), supplemented with 120 µg

ml-1 penicillin, 120 µg ml-1 streptomycin and 10 % heat inactivated fetal calf serum (FCS) in a

CO2 incubator. Cells were passaged at a ratio of 1:20 twice per week. HeLa cells were

cultured in DMEM, high glucose with L-glutamine (PAA Laboratories, Austria) supplemented

with 100 µg ml-1 gentamicin and 12 µg ml-1 ciprofloxacin and 10 % heat inactivated FCS in a

CO2 incubator. Cells were passaged at a ratio of 1:3 twice per week.

Scanning electron microscopy (SEM)

Infected monolayer cultures were first fixed with 0.1 % glutaraldehyde, 2 % paraformaldehyde,

5 % sucrose in 0.2 M sodium cacodylate solution for one hour at 37°C. Subsequently, samples

were fixed with 0.3 % glutaraldehyde, 3 % paraformaladehyde in 0.2 M sodium cacodylate

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solution for 1 h at 37°C. Samples were subsequently dehydrated using a graded series of

acetone, critical point dried and gold sputtered and examined using a FEI Quanta 200

scanning electron microscope.

Adhesion assays

Detroit562 and HeLa cells were seeded in 24 well plates (Nunc, Roskilde, Denmark) at a

density of 1 x 105 and 5 x 104 cells per well, respectively, 48 h prior to infection. Bacteria were

inoculated to an OD600 of 0.1 from overnight cultures and grown in HI broth to an OD600 of 0.4

to 0.6. Subsequently, the bacteria were harvested by centrifugation and cell density was

adjusted to an OD600 of 0.2. A master mix of the inoculum with a multiplicity of infection (MOI)

of 200 was prepared in DMEM and 500 µl per well were used to infect the cells. The plates

were centrifuged for 5 min at 500 rpm to synchronize infection and subsequently incubated for

90 min. The cells were washed with PBS three times, detached with 500 µl trypsin solution

(0.12 % trypsin, 0.01 % EDTA in PBS) per well (5 min, 37°C, 5 % CO 2, 90 % humidity) and

lysed with 0.025 % Tween 20 for 5 min at 37°C. Serial dilutions were made in pre-chilled 1 x

PBS and plated on Columbia agar with sheep blood (Oxoid, Wesel, Germany) to determine

the number of colony forming units (cfu).

Epithelial cell invasion model

Detroit562 and HeLa cells were seeded in 24 well plates (Nunc, Roskilde, Denmark) at a

density of 1 x 105 and 5 x 104 cells, respectively, per well 48 h prior to infection. Overnight

cultures grown in HI were re-inoculated to an OD600 of 0.1 in fresh medium and grown to an

OD600 of 0.4 to 0.6. An inoculum with a MOI of 200 was prepared in DMEM without antibiotics

and 500 µl per well were used to infect the cells. The plates were centrifuged for 5 min at 500

rpm to synchronize infection and subsequently incubated for 90 min (37°C, 5 % CO2, 90 %

humidity). The cells were washed three times with PBS to remove planctonic and loosely

attached bacteria. Subsequently, the cells were incubated for two hours in DMEM (500 µl per

well), containing 100 µg ml-1 gentamicin to kill remaining extracellular bacteria. After this

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incubation, the cell layers were washed three times with PBS, detached by adding 500 µl

trypsin solution (0.12 % trypsin, 0.01 % EDTA in PBS) per well (5 min, 37°C, 5 % CO2, 90 %

humidity), and lysed for 5 min at 37°C with 0.025 % Tween 20 to liberate the intracellular

bacteria. Serial dilutions of the inoculums and the lysates were plated out on Columbia agar

with sheep blood (Oxoid, Wesel, Germany) to determine the number of cfu.

Construction of an NF-B reporter cell line

The lentiviral NF-B reporter construct was generated as following: a fragment including an

eukaryotic elongation factor 1 alpha promoter (EF1)-driven puromycin resistance gene and the

Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was excised from

the vector pCDH-CMV-MCS-EF1-Puro  (System  Biosciences)  with  restriction  enzymes  NotI  (5’  

overhang filled in with Klenow DNA Polymerase) and KpnI. This fragment was ligated into the

vector pTRH-3xSIE-Luc (based on pTRH-MCS-Luc, System Biosciences) that has been cut

with   XbaI   (5’   overhang   filled   in   with   Klenow   DNA   Polymerase)   and   KpnI.   The   3X   SIE  

responsive element was then replaced with a synthetic linker containing 4x NFB responsive

elements. Correct insertion was verified restriction digests and by DNA sequencing. Lentiviral

vector stocks were produced in 293T cells (DSMZ ACC 635, Bennett & Pearson, 1993) that

were seeded in 80 cm2 flasks and transfected using Lipofectamine 2000 (Invitrogen) according

to  manufacturer’s  instructions  when  the  cells  were  60  %  confluent.  The  pTRH  reporter  vector  

plasmid  was  cotransfected  together  with  psPAX2  and  pMD2.G  in  a  ratio  of  4:3:1,  in  total  15  μg  

plasmid  with  12.5  μl  Lipofectamine  2000  per  80  cm²  flask.   pMD2G encodes for the vesicular

stomatitis virus G protein (VSV-G protein), a protein which mediates fusion of viral particles

with cellular membranes, ensuring a broad cell tropism. The plasmid psPAX2 encodes for the

HIV gag, pol, tat and rev genes, but is deleted for the env, vpr, vpu and nef genes.

Transfection of the different plasmids into 293T cells led to generation of VSV-G-pseudotyped

replication-defective viral particles that were released into the culture supernatant. The cell

free supernatants were further concentrated by ultracentrifugation and used for infection of T

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cells. Detroit562 and HeLa cells were transduced with the reporter lentivirus and selected with

1 µg ml-1 puromycin (Sigma) for 3 weeks, resulting in the corresponding NFB cell lines.

Luciferase activity measurements

Detroit562-NFB and HeLa-NFB cells were seeded in 48 well plates (Greiner bio-one

Cellstar, Frickenhausen, Germany) at a density of 5 x104 cells per well 24 h prior to infection.

Overnight cultures grown in HI were re-inoculated to an OD600 of 0.1 in fresh medium and

grown to an OD600 of 0.4 to 0.6. To test if dead bacteria can activate eukaryotic signalling

pathways, 10 ml of the overnight cultures were incubated for 3.5 h with 8 µg ml-1 tetracycline.

An inoculum with a MOI of 200 was prepared in DMEM and 300 µl per well were used to infect

the cells. In addition to the infected cells, a negative control (uninfected reporter cells) as well

as a positive control (reporter cells induced with 20 ng ml-1 Phorbol 12-myristate 13-acetate in

DMSO (1 mg ml-1), diluted 1:100 in DMEM) and 200 ng ml-1 calcimycin (1 mg ml-1 in DMSO

and diluted 1:100 in DMEM) was carried along. The plates were centrifuged for 5 min at 500

rpm to synchronize infection and subsequently incubated for 3 h (37°C, 5 % CO 2, 90 %

humidity). Afterwards cells were washed and DMEM supplemented with 8 µg ml-1 tetracycline

was added to avoid bacteria overgrowing the cells. Samples were taken after different time

points, cells were lysed with lysis buffer (100 mM potassium phosphate buffer (pH 7.8)

containing 0.1 % (v/v) Triton-X-100) for 20 min on a seesaw under light exclusion.

To determine the luciferase activity 100 µl luciferase buffer (15 mM potassium phosphate

buffer (pH 8.0), 25 mM glycylglycine, 4 mM EGTA, 15 mM MgSO 4, 2 mM ATP, 1 mM DTT, 75

µM D-luciferin) were added to 10 µl cell extract automatically and the luminal-dependent

chemiluminescence was measured using a Luminometer (Orion, Berthold, Pforzheim). The

unit of chemiluminescent activity is the relative light unit and was expressed as relative light

units per second (RLU sec-1). A Bradford protein assay was used to determine protein

concentrations in the cell extract. For this purpose 2 µl of the cell extract were added to 240 ml

deionised water and 60 ml Bradford reagent (Roti®-Quant, Roth, Karlsruhe) (10 min, room

temperature). The absorption at 595 nm was determined using a Spectra Fluor Plus (TECAN,

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Crailsheim). A calibration curve was prepared using bovine serum albumin (BSA) as reference

solution. The protein concentration was used to normalize the RLUs (RLU µg-1).

Immuno-fluorescence microscopy

HeLa cells were seeded on round coverslips in 24-well plates and after 24 h infected at a MOI

of 250 with GFP-expressing C. diphtheriae for two hours. Infected or uninfected HeLa cells

were fixed with 1 % paraformaldehyde at room temperature, permeabilized with 0.1 % Triton

X-100 in phosphate buffered saline without calcium and magnesium ions and stained. Primary

rabbit polyclonal anti-NFkB p65 antibody (C-20, sc-372; Santa Cruz Biotechnology) was used

at a dilution 1:250; this was detected with Alexa 647-labelled goat anti-rabbit secondary

antibody diluted 1:500 (Life Technologies). Cells were counterstained with the fixable dye

Pacific blue succimidyl ester (0.4 µg ml-1), visualized on a Leica TCS SP5 confocal microscope

and analyzed with the LAS software suite.

Cell viability assay

C. diphtheriae strains DSM43988 and DSM43989 were inoculated in 200 ml DMEM

(Dulbecco’s   modified   Eagle’s   medium,   PAA;;   high  glucose,   10   %   FCS,   2  mM   glutamine)   and  

cultivated overnight, at 37°C in an orbital shaker. Subsequently, the bacterial suspensions

were centrifuged (4500 x g, 10 min, 4°C) and the supernatants were filtered (syringe filter

Ministart, 0.45 µm pore size, Sartorius, Göttingen, Germany) to clear it from remaining

bacterial cells. 200 ml supernatant were concentrated to a volume of 15 ml by centrifugal filter

devices  (Ultracel  10K  membrane,  Merck  Millipore,  Darmstadt,  Germany)  after  manufacturer’s  

protocol and stored at 4°C. Detroit562 and HeLa cells were seeded in 96-well plates (bio-one

Cellstar, Greiner, Frickenhausen, Germany) at a density of 5 x 103 cells per well directly in 190

µl DMEM containing secreted proteins of DSM43988 and DSM43989, respectively. After 20 h

of incubation at 37°C, 5 % CO2 and 95 % humidity, alamar blue (AbD Serotec, Oxford, UK)

was added (10 % in culture medium) to each well and fluorescence was measured at 560 nm

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(excitation wavelength) and 590 nm (emission wavelength) using a microplate reader (Tecan

Infinite M200 Pro) at different time points.

Results

Adhesion of C. diphtheriae strains to epithelial cells

C. diphtheriae strains cannot only be isolated from human hosts but also from various animals,

for example from domestic cats (Hall et al., 2010), cows (Corboz et al., 1996) and horses

(Henricson et al., 2000; Leggett et al., 2010). Furthermore, adhesion of this bacterium to

several human cell lines was reported, e. g. pharyngeal Detroit562 (Bertuccini et al., 2004;

Mandlik et al., 2007; Swaminathan et al., 2007), laryngeal HEp-2 (Bertuccini et al., 2004;

Hirata et al., 2002; Hirata et al., 2008; Hirata et al., 2004; Mandlik et al., 2007), lung A596

(Mandlik et al., 2007), human U-937 macrophages (dos Santos et al., 2010) and erythrocytes

(Colombo et al., 2001), indicating that C. diphtheriae is able to bind to different cells as would

be expected based on the different host organisms and different clinical manifestations, i.e.

diphtheria of upper respiratory tract, cutaneous diphtheria or systemic infections.

Adherence assays with different C. diphtheriae isolates carried out before, showed the

highest adhesion to Detroit562 cells with a rate of about 8 % for strains ISS4746 and ISS4749

(Figure 1A, Ott et al., 2010b). While this cell line is close to the natural situation at least for the

upper respiratory tract diphtheria, HeLa cells are the most widespread model in host-pathogen

interaction studies used for a wide variety of organisms (for recent manuscripts, see Bose et

al., 2011; Corbett et al., 2011; Revez et al., 2011) and were therefore tested in this study. In

fact, C. diphtheriae was able to bind to this epithelial cell line and up to tenfold higher adhesion

rates compared to Detroit562 cells were observed (Ott et al., 2010a; Ott et al., 2010b). The

highest numbers of adherent bacteria to HeLa cells corresponding to adhesion rates of 50 to

70 % were observed for strains ISS3319, ISS4060 and DSM43988. The lowest adhesion with

a rate of only 1.25 ± 0.14 percent was observed for the toxin-producer DSM43989 (Figure 1B).

Interestingly, the ability to adhere to the host cells was not only strain- but also cell line-

specific: strains such as ISS4746 and ISS4749 carrying numerous pili showed always the

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higher adhesion rates to Detroit562 cells compared to the pili-free strain ISS4060. In contrast,

ISS4060 showed higher adhesion rates compared to these strains to HeLa cells.

Figure 1. Adhesion of C. diphtheriae wild type strains to epithelial cells.

Adhesion of C. diphtheriae strains to Detroit562 (A) and HeLa cell layers (B). Cell lines were
infected with different C. diphtheriae wild type strains, washed with PBS, detached with trypsin
solution, lysed with Tween 20, and the number of colony forming units (cfus) was determined.
Adhesion is expressed as percentage of the inoculum, showing means and standard
deviations of three independent measurements (biological replicates) with three samples each
(technical replicates). Statistically relevant differences between the strains (based on students
TTEST values below 0.05) are indicated by letters above columns.

The observed adhesion behaviour of the different strains to HeLa cells was validated

qualitatively by scanning electron microscopy (SEM) experiments. All samples of non-toxigenic

strains showed numerous bacteria attached to the HeLa cells (Figure 2A-E and 2G), while in

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case of the toxin producer, bacteria were hardly detectable (Figure 2F). Furthermore, in case

of infection with tox+ strain DSM43989 most HeLa cells were characterized by a ball-like shape

instead of the flat, extended form in case of infections with other strains, indicating that the

cells were undergoing apoptosis or necrosis (Figure 2F).

A B

C D

E F

Figure 2. Scanning electron microscopy of C. diphtheriae and HeLa cells.

Interaction of C. diphtheriae strains with HeLa cells viewed by scanning electron microscopy at
90 min post-infection. Infected monolayer cultures were fixed, dehydrated sputtered with gold
and examined using a FEI Quanta 200 scanning electron microscope. (A) ISS3319, (B)
ISS4060, (C) ISS4746, (D) ISS4749, (E) DSM43988, (F) DSM43989 (toxigenic) and (G)
DSM44123.

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Internalization of C. diphtheriae in eukaryotic cells

When gentamicin protection assays were carried out to analyze the number of invading

bacteria in Detroit562 cells, previous studies revealed strain-specific differences: for the pili-

free strains ISS4060 and DSM43988 the highest invasion efficiencies were observed (Figure

3A, see also Ott et al., 2010b). Strains ISS4060 and DSM43988 also showed the highest

invasion rates into HeLa cells with 3.97 ± 1.84 and 2.72 ± 1.13 %, followed by ISS3319 and

DSM44123 with invasion rates of 0.9 ± 0.07 % and 1.08 ± 0.35 %, respectively. For strains

ISS4746, ISS4749 and for the toxin-producer DSM43989 only negligible invasion rates were

observed (Figure 3B). The invasion rates obtained with HeLa cells were significantly higher

compared to those obtained with Detroit562 cells reported before (Ott et al., 2010b). A

correlation between adhesion and invasion rate was not apparent.

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Figure 3. Invasion of C. diphtheriae wild type strains into eukaryotic cells.

Detroit562 (A) and HeLa cells (B) were infected with different C. diphtheriae wild type strains,
washed, and incubated for two hours with 100 μg ml-1 gentamicin. Subsequently, cells were
washed, detached with trypsin solution, lysed with Tween 20, and the number of intracellular
cfus was determined. Invasion is shown as percentage of the inoculum internalized (means
and standard deviations of three independent biological replicates with three samples each
(technical replicates). Statistically relevant differences between the strains (based on students
TTEST values below 0.05) are indicated by letters above columns.

Analysis of NF-B reporter cell lines

Detroit562-NFB and HeLa-NFB reporter cells were used to study activation after exposure

to equivalent doses of the various C. diphtheriae strains at different time points post infection.

When these reporter cell lines were infected with different isolates of C. diphtheriae, strain-

specific differences in the cellular response were observed eight hours after infection by

luciferase activity measurements (Figure 4). For Detroit562-NFB cells only weak activation of

the NFB pathway was observed during infection with different C. diphtheriae strains (Figure

4A). The highest luciferase signals were detected when cells were infected with strains

ISS3319 and ISS4060. Infection of HeLa- NFB cells resulted in much stronger reporter

activation (Figure 4B). Interestingly, the strength of activation determined in the reporter assay

correlated with the invasion rates shown above (Figure 3, compare also data for Detroit562).

Strains ISS3319, ISS4060, ISS4749, DSM43988 and DSM44123 showed luciferase activity

significantly above background. Strongest light intensities were measured when the reporter

cells were infected with strain ISS4060 or DSM43988, which showed the highest invasion

rates. Luciferase activity was lower in the less invasive strains ISS3319 and DSM44123, while

no induction of luciferase in the NF-B reporter cell lines were observed in case of non-

invasive strains ISS4746 and DSM43989. The only exception observed was strain ISS4749,

which was also not invasive but showed significant luciferase activity for unknown reasons.

The luciferase signal declined below background level when HeLa cells were infected by the

toxigenic strain DSM43989 for 20 hours, indicating that this strain has a detrimental effect on

HeLa cells.

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3000 a, d, e, A
g, h
d, f,
2500 g, h
b, c, c b, d, h
c f, h b, c, h b, c, d,
2000
f, g

1500

1000

500

a b c d e f g h
rel. luciferase units

a, b, d,
4500 e, g, h B
4000
a, d, e,
3500 g, h
a, c,
3000
d, g
2500 a, c, d, a, c, d,
f, g f, g
2000 a, b, c, e,
f, g, h b, c, d,
1500 b, c, d,
e, f, h e, f, h
1000

500
a b c d e f g h
0

Strains
Figure 4. Activation of the NF-B pathway in eucaryotic cells after 8 h of infection with
C. diphtheriae wild type strains.
Detroit562-NFkB (A) and HeLa-NFkB cells (B) were infected with different C. diphtheriae wild
type strains (DSM43989 tox+, all others are non-toxigenic) for three hours, washed and
incubated for 5 h with DMEM containing 8 µg ml-1 tetracycline. Black bar: uninfected reporter
cells, grey bars: reporter cells infected with C. diphtheriae, white bars: reporter cells infected
with C. diphtheriae inactivated by tetracycline. Measurements of the luciferase units were
carried out on a Luminometer (Orion, Berthold, Pforzheim), expressed in RLU µg-1. Statistically
relevant differences between the strains (living bacteria, grey bars) (based on students TTEST
values below 0.05) are indicated by letters above columns.

When bacteria were killed by three hours of treatment with 8 µg ml-1 tetracycline

(Figure 4) or by 15 min of UV radiation (data not shown) no NF-B induction was observed.

Interestingly, the correspondingly treated bacteria were still found to be attached to the host

cells, when specimens were investigated for control by SEM (Figure 5), showing that adhesion

is a passive process. In addition, the inactivated tox+ strain DSM43989 was not able to kill the

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host cells (Figure 5B) as it was the case during infection with living bacteria (Figure 2F).

Furthermore, tetracycline-treated DSM43989 adhered in higher amounts to HeLa cells (Figure

5B) compared to the active strain (Figure 2F).

A B

Figure 5. Scanning electron microscopy of dead C. diphtheriae and HeLa cells.

Interaction of inactivated C. diphtheriae strains (incubation with 8 µg ml-1 tetracycline for 3.5 h)
with HeLa cells viewed by scanning electron microscopy at 90 min post-infection. (A) ISS4746
(non-toxigenic), (B) DSM43989 (toxigenic).

Investigation of NF-B induction by immuno-fluorescence microscopy

The increase of luciferase activity in the HeLa reporter strain upon invasion of C. diphtheriae

indicated an induction of the NF-B signal pathway. This was further studied using immuno-

fluorescence microscopy. Translocation of the NF-B subunit p65 into the nucleus served as a

marker for NF-B induction; this was tested using a p65-specific antibody and bacteria tagged

by GFP expression (Figure 6). In these experiments, p65 was labelled in red, the DNA within

the nucleus stained in blue and bacteria in green (Figure 6A). When the pictures of the

different fluorescence channels were merged, translocation of p65 was visualized by a change

of the nucleus colour from blue to pink due to translocation of p65 from the cytoplasm into the

nucleus. When bacteria were clearly inside the cell, as shown by orthogonal z stacks, a

translocation of p65 from the cytoplasm into the nucleus was detectable; bacteria attached to

the cell surface did not induce p65 translocation (Figure 6B) further supporting the idea that

invasion of C. diphtheriae is a prerequisite for NF-B induction.

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Figure 6. Investigation of NF-B induction by immuno-fluorescence microscopy.

(A) Overview: HeLa cells incubated 2 hours with invasive C. diphtheriae strain ISS4060
expressing a GFP marker protein (green) show nuclear translocation of NFB p65.
Permeabilized cells were counterstained with Pacific blue (blue), NFB p65 subunit (anti NFB
p65, C-20, 1:250) was detected with anti-rabbit Alexa 647 (1:500, red). Translocation of p65 is
indicated by pink coloring of the nucleus. (B) Non-adherent bacteria were removed by washing
and confocal image stacks were acquired on a Leica TCS SP5. Upper panel: representative
HeLa cell with intracellular C. diphtheriae DSM49388 pEPR1-P45gfp and nuclear translocation
of NFB p65 (arrow). Lower panel: HeLa cell with extracellular C. diphtheriae ISS4060

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pEPR1-P45gfp and predominantly cytoplasmatic localization of NFB p65. Left: DAPI, GFP
and NFB; Middle: GFP and NFB; Right: orthogonal sections of image stacks in x and y axis.

Effects of diphtheria toxin in eukaryotic cell cultures

As described above, the luciferase signal declined below background level when HeLa cells

were infected by the tox+ strain DSM43989 for 20 h. This led to the hypothesis that the

production of diphtheria toxin might have a detrimental effect on the cells after prolonged

incubation in this assay. To address this idea, cell viability assays based on mitochondrial

enzyme activity were performed (Figure 7). For this purpose Detroit562 and HeLa cells were

exposed to DMEM containing secreted proteins from strain DSM43988 and the toxin-

producing strain DSM43989, respectively. After 20 h alamar blue was added. In case of

undergoing apoptosis or necrosis, a redox reaction, in which alamar blue is reduced by the

cells, can be measured by fluorescence of 560 nm (excitation) and 590 nm (emission). Indeed,

the data revealed a cytotoxic effect of DMEM containing secreted proteins of the toxigenic

strain DSM43989. The fluorescence values of all cells incubated with this media declined to

that of cells lysed by 0.1 % Triton-X-100. This was not the case for cells exposed to the

supernatant of the non-toxigenic strain DSM43988, which values are up to five-fold higher than

the negative control. Our data indicate that strain DSM43989 might be able to produce the

toxin under cell culture conditions, which should be the reason for luciferase values declined

below background level, when cells were incubated for 20 h with the toxigenic strain.

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Detroit562 HeLa
50000 50000

45000 45000

Fluorescence 560/590nm
40000 40000

35000 35000

30000 30000

25000 25000

20000 20000

15000 15000

10000 10000

5000 5000

0 0
0 20 40 60 80 0 20 40 60 80

Time [h]

Figure 7. Cell viability assay of different eukaryotic cells exposed to diphtheria toxin.
Detroit562 and HeLa cells were seeded in 96-well plates at a density of 510 x 103 cells per
well directly in 190 µl DMEM containing secreted proteins of DSM43988 and DSM43989,
respectively. After 20 h of incubation at 37°C, 5 % CO2 and 95 % humidity, alamar blue was
added (10 % in culture medium) to each well and absorbance was measured at 560 nm
(excitation wavelength) and 590 nm (emission wavelength) using a microplate reader (Tecan
Infinite M200 Pro) at different time points. negative control: 0.1 % Triton-X-100, positive
control: cells without treatment, supernatant DSM43988, supernatant DSM43989
(toxigenic).

Discussion

In this study, we showed that HeLa cells are recognized by C. diphtheriae with extremely high

efficiency. This result might also explain, why HEp-2 cells are preferentially recognized

compared to Detroit562 cells in other studies (see for example Mandlik et al., 2007), since this

cell line carries HeLa markers according to the American Type Culture Collection (ATCC) and

the National Collection of Type Cultures (NCTC).

As mentioned above, adhesion of C. diphtheriae was described before for a variety of

human cell lines. All in all, this indicates that a general property or component of cells is

recognized by C. diphtheriae, besides the fact that minor pili subunits are involved in cell

specificity at least in vitro (Mandlik et al., 2008), and might explain the broad host and tissue

specificity of the bacterium.

The effect of C. diphtheriae infection on host cell signalling is not well understood and

besides this study only one further manuscript is available. Puliti and co-workers showed

strong differences in cytokine secretion in a mouse model in response to infection with various

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C. diphtheriae strains and it was concluded that the bacterial strains studied interact with the

host in a different manner (Puliti et al., 2006). The NF-B signalling pathway is involved in the

regulation of innate and adaptive immune responses, inflammation and various other cellular

processes (O'Dea & Hoffmann, 2009). It has a crucial role for resistance to infections (Tato &

Hunter, 2002) and controls cytokine excretion and consequently the fate of a pathogen inside

the host. During evolution, some pathogens have found mechanisms to inhibit NF-B

activation, while others even utilize and manipulate the pathway for their benefit (Tato &

Hunter, 2002).

In the study presented here, the influence of seven C. diphtheriae isolates on NF-B

induction in human epithelial cells was investigated. Recognition of pathogen-associated

molecular patterns such as pili subunits by membrane-bound receptors facing the outside of

the cell, as often found for pathogen host interaction (Hayden et al., 2006; Li & Verma, 2002),

was not crucial for NF-B induction. Piliation of the different (invading) strains and luciferase

expression were not correlated and also killed bacteria were not able to induce the NF-B

pathway despite adhesion. As shown recently (Ott et al., 2010b) the bold strain ISS4060 was a

strong inducer in this study, while DSM43989, a poor NF-B inducer, shows strong pili

formation. It might be argued that formation of diphtheria toxin and consequent inactivation of

protein synthesis might prevent luciferase expression, but also the non-toxigenic strain

ISS4749, which showed a high number of pili, caused only minor NF-B induction. In fact,

strong pili formation seems to have a negative effect on invasion into host cells (Ott et al.,

2010b) and subsequently on NF-B induction.

Our data indicate that the response of human epithelial cells to C. diphtheriae infection

is determined by the internalization of bacteria and that invasion into these cells is an active

process of the bacteria, since tetracycline-treated C. diphtheriae were still able to attach to

host cells, but lost their ability to invade into the cytoplasm. Since we found this correlation

between invasion and NF-B induction, it would be tempting to either manipulate an invasive

strain to become non-invasive or vice versa and to analyze NF-B induction in these mutants;

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unfortunately, the molecular background of C. diphtheriae internalization is almost unknown.

Only recently, in vitro experiments with protein-coated latex beads indicated that DIP0733,

encoding 67-72p hemagglutinin, contributes to invasion in HEp-2 cells (Sabbadini et al., 2012).

Once inside the cell, either a cell wall component of the internalized bacteria might be

recognized or a protein secreted by C. diphtheriae into the cytosol of the host cell. As shown

by a proteomics approach (Hansmeier et al., 2006), C. diphtheriae is able to export more than

one hundred proteins to the outside of the bacterium. These are prime candidates for putative

signals sensed by the host cell and future mutagenesis studies of the corresponding genes

might help to further understand the interaction of pathogen and host on a molecular level.

Acknowledgements

The  authors  wish  to  thank  C.  v.  Hunolstein  (Istituto  Superiore  di  Sanita’,  Rome)  for  providing  

strains ISS3319, ISS4060, ISS4746 and ISS4749. Lentiviral packaging plasmids were a

generous gift from D. Trono, Geneva. The financial support of AB, AE and LO by the Deutsche

Forschungsgemeinschaft for in frame of SFB796 (projects B1 and B5) is gratefully

acknowledged.

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Table 1. Bacterial strains and eukaryotic cell lines used in this study.

Strains Description Reference

C. diphtheriae

DSM43988 non-toxigenic, isolated from throat DSMZ, Braunschweig,

culture, no pili (Ott et al., 2010b, Germany

Rheinlaender et al., 2012)

DSM43989 tox+, unknown source, medium pili length DSMZ, Braunschweig,

and number (Ott et al., 2010b, Germany

Rheinlaender et al., 2012)

DSM44123 non-toxigenic isolate, type-strain, DSMZ, Braunschweig,

unknown source, medium number short Germany

spike-like pili (Ott et al., 2010b,

Rheinlaender et al., 2012)

ISS3319 C. diphtheriae var. mitis, non-toxigenic, (Bertuccini et al., 2004)

isolated from patients affected by

pharyngitis/tonsillitis, medium pili length

and number (Ott et al., 2010b,

Rheinlaender et al., 2012)

ISS4060 C. diphtheriae var. gravis, non-toxigenic, (Bertuccini et al., 2004)

isolated from patients affected by

pharyngitis/tonsilitis, no pili (Ott et al.,

2010b, Rheinlaender et al., 2012)

ISS4746 C. diphtheriae var. gravis, non-toxigenic, (Puliti et al., 2006)

isolated from patients affected by

pharyngitis/tonsillitis, medium number of

extended pili (Ott et al., 2010b,

Rheinlaender et al., 2012)

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ISS4749 C. diphtheriae var. gravis, non-toxigenic, (Puliti et al., 2006)

isolated from patients affected by

pharyngitis/tonsillitis, high number of

extended pili (Ott et al., 2010b,

Rheinlaender et al., 2012)

DSM43988 pEPR1- DSM43988 transformed with pEPR1- (Ott et al., 2012)

P45gfp P45gfp

ISS4060 pEPR1- C. diphtheriae var. gravis transformed (Ott et al., 2012)

P45gfp with pEPR1 carrying P45, gfpuv, KmR,

rep, per, T1, T2

E. coli

DH5MCR endA1 supE44 thi-1 - recA1 gyrA96 (Grant et al., 1990)

relA1 deoR (lacZYA-argF) U196

80lacZ M15mcrA (mmr

hsdRMSmcrBC)

Cell lines

Detroit562 human hypopharyngeal carcinoma cells (Peterson et al., 1968)

HeLa human cervical carcinoma cells (Gey et al., 1952;

Scherer et al., 1953)

Detroit-NFB reporter lentiviral transduced Detroit562 cells with This study

cells stable integrated NF-B reporter gene

construct pTRH-NFB FL-Luc-Puro

HeLa-NFB reporter lentiviral transduced HeLa cells with This study

cells stable integrated NF-B reporter gene

construct pTRH-NFB FL-Luc-Puro

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 Summary/Zusammenfassung

4 Summary/Zusammenfassung
4.1 Summary

Corynebacterium diphtheriae, the causative agent of respiratory diphtheria was generally

considered as an extracellular colonizer, with the diphtheria toxin as the main virulence factor.
But non-toxigenic strains are also able to adhere to the host cell and to internalize into it. To
date, not much is known about the factors, which are crucial for colonization of the host.
In the framework of this study, the characterization of the interaction of C. diphtheriae
with host cells revealed strain-specific differences in adhesion and invasion rates of seven wild
type strains and that adhesion and invasion are not strictly coupled processes. Atomic force
microscopic analysis showed that the studied strains differ significantly in their macromolecular
surface structure, especially in pili formation and length and number of pili structures (Ott et al,
2010b). Additionally, these pili structures show different biophysical properties which were
investigated in collaboration with Reinlaender and co-workers (Rheinlaender et al, 2012).
Interestingly, there is no correlation between pili formation, adhesion and invasion of the
different wild type strains. Furthermore, we were able to show that one of the three known pili
cluster (spaDEF) for C. diphtheriae is absent in the strains investigated. The work proved that
there must be other factors which are crucial for colonization if the host besides pili (Ott et al,
2010b).
To investigate the invasion process at a more molecular level, we generated insertion
mutants in gene DIP1281, whose gene product was annotated as an invasion-associated
protein. The resulting mutant strains were not able to separate the daughter cells after cell
division, to adhere to the host cell or to invade the cell. Further investigations of the mutants
demonstrated that this phenotype is an effect of rearrangements of the outer surface of
bacteria, and DIP1281 seems to be predominantly involved in the organization of the outer
surface protein layer (Ott et al, 2010a).
For further identification of virulence factors, we established an invertebrate infection
model for pathogenic corynebacteria for high throughput screening of mutant strains. The
nematode Caenorhabditis elegans gives differentiated results with regard to colonization of the
host, while the wax moth larvae Galleria mellonella will be useful with respect to innate
immune responses to pathogens because it offers a more complex cell-based immune system
compared to C. elegans (Ott et al, 2012a).
When we studied the host immune response during infection with C. diphtheriae we
were able to show that C. diphtheriae is capable of activating the  NFĸB  transduction  pathway  
in eukaryotic cells in a strain-specific manner; induction only occurs during incubation with
living bacteria and requires invasion of the bacteria into the epithelial cells (Ott et al, 2012b).

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 Summary/Zusammenfassung

In cooperation with Ochrombel and co-workers, we investigated potassium uptake

systems in corynebacteria (Ochrombel et al, 2011) and found that pathogenic corynebacteria,
such as C. diphtheriae and C. jeikeium, exhibit more potassium transporters than non-
pathogens, such as C. glutamicum. Furthermore, C. diphtheriae K+ transporter mutants were
eliminated faster by macrophages and are less stress-resistant, which make K+ uptake
systems of pathogenic corynebacteria potential virulence factors with regard to survival in an
acid environment, like the inside of macrophages.
In collaboration with Trost and co-workers, we participated in data evaluation of the
genome sequence of C. pseudotuberculosis FRC41 with the idea of a comparative analysis of
pathogenic corynebacteria. This led to the identification of an extended number of virulence
factors, indicating that the human isolate C. pseudotuberculosis FRC41 is equipped with a
distinct gene set promoting its survival under unfavorable environmental conditions (Trost et
al, 2010b).

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 Summary/Zusammenfassung

4.2 Zusammenfassung

Als Hauptvirulenzfaktor des Gram-positiven, aeroben Bakteriums Corynebacterium diphtheriae

wurde bisher das hochwirksame Diphtherietoxin betrachtet. Doch auch nicht-toxigene Stämme
sind in der Lage, sich an Wirtszellen anzuheften, in diese einzudringen und sie zu schädigen.
Über die Virulenzfaktoren, die dafür verantwortlich sind, ist noch wenig bekannt.
Im Rahmen dieser Arbeit wurde die Interaktion von C. diphtheriae mit eukaryotischen
Zellen näher charakterisiert. Adhäsions- und Invasionsanalysen von sieben verschiedenen
Wildtypstämmen an Epithelzellen ergaben, dass Adhäsion und Invasion keine strikt
gekoppelten Prozesse darstellen. Atomic force mikroskopische Untersuchungen zeigten, dass
sich die Stämme stark in der makromolekularen Oberflächenstruktur, hinsichtlich der
Ausbildung und Länge von Pili-Strukturen, voneinander unterscheiden (Ott et al, 2010b).
Diese Pili-Strukturen haben außerdem verschiedene biophysikalische Eigenschaften, was
durch Arbeiten in Kooperation mit der Arbeitsgruppe um Rheinlaender gezeigt werden konnte
(Rheinlaender et al, 2012). Interessanterweise konnte keine Korrelation zwischen Pili-
Formation, Adhäsion und Invasion gefunden werden. Es konnte auch gezeigt werden, dass
den untersuchten Stämmen eines von drei für C. diphtheriae bekannten Pili-Clustern
(spaDEF), wider Erwarten, fehlt. Diese Ergebnisse machen deutlich, dass es weitere Faktoren
neben den Pili geben muss, die für Adhäsion an Wirtszellen verantwortlich sind (Ott et al,
2010b).
Um die Invasion auf molekularer Ebene zu untersuchen, wurden Insertionsmutanten im
Gen DIP1281 hergestellt, dessen Genprodukt als ein Invasions-assoziiertes Protein annotiert
ist. Die daraus resultierenden Mutanten waren nicht mehr in der Lage, sich nach der
Zellteilung von einander zu separieren, an Wirtszellen anzuheften oder in diese zu invadieren.
Weiterführende Studien der Mutanten zeigten, dass dieser Phänotyp auf eine Umgestaltung
der Oberfläche der Bakterien zurückzuführen ist. DIP1281 scheint daher vorwiegend in die
Organisation der äußeren Proteinschicht involviert zu sein (Ott et al, 2010a).
Für zukünftige Arbeiten konnte ein Infektionsmodell für pathogene Corynebakterien
etabliert werden, welches ermöglicht Mutanten-Banken von C. diphtheriae in großem Maßstab
hinsichtlich der Virulenz zu testen. Dabei erhält man mittels Infektion des Nematoden
Caenorhabditis elegans detaillierte Ergebnisse bezüglich der Wirtskolonisation von C.
diphtheriae, während die Wachsmottenlarve Galleria mellonella hilfreich bei der Aufklärung der
Immunantwort gegenüber Pathogenen ist, da sie über ein komplexeres Immunsystem verfügt
als C. elegans (Ott et al, 2012a).
Außerdem konnte gezeigt werden, dass C. diphtheriae den  NFĸB-Signalweg in HeLa-
Zellen auslösen kann und dass dafür die Invasion der Bakterien notwendig ist. Die bloße

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 Summary/Zusammenfassung

Adhäsion der Bakterien an die Wirtszellen führte zu keiner intrazellulären Wirtsantwort (Ott et
al, 2012b).
In Kooperation mit der Arbeitsgruppe um Ochrombel wurden unterschiedliche Kalium-
Aufnahmesysteme in Corynebakterien untersucht. Es konnte gezeigt werden, dass pathogene
Corynebakterien wie C. diphtheriae und C. jeikeium über mehr Kaliumtransporter verfügen als
nicht-pathogene. Außerdem werden K+-Transportermutanten von C. glutamicum und C.
diphtheriae schneller von Makrophagen eliminiert und sind weniger Stress-resistent
(Ochrombel et al, 2011; Ott, unveröffentlicht).
Für eine vergleichende Analyse pathogener Corynebakterien, habe ich mich in einem
weiteren Kooperationsprojekt an der Datenauswertung der Genom-Entschlüsselung des
humanen Isolats Corynebacterium pseudotuberculosis FRC41 maßgeblich beteiligt. Dies
führte zur Identifikation einer großen Anzahl von Virulenzfaktoren, die zeigen, dass C.
pseudotuberculosis mit einem enormen Genset ausgestattet ist, welches sein Überleben in
einer nachteiligen Umgebung sichert (Trost et al, 2010b).

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 References

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134
 Curriculum Vitae

6 Curriculum Vitae

Lisa Ott
Schronfeld 61
91054 Erlangen
0177 3885373
lisaott@biologie.uni-erlangen.de

Persönliches

geboren am 05.01.1982 in Nürnberg

ledig, keine Kinder

Ausbildung

Doktorarbeit
1/2009 - 2012 Titel: “Interaction  of  Corynebacterium diphtheriae with  host  cells“
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität
Erlangen-Nürnberg, Prof. Dr. A. Burkovski

Mitglied des Sonderforschungsbereichs 796 und des integrierten

Graduiertenkollegs des SFB796

Diplomarbeit
03/2008 - 12/2008 Titel: „Adhäsion  und  Invasion  von  Corynebacterium diphtheriae“  
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität
Erlangen-Nürnberg, Prof. Dr. A. Burkovski

Diplom
10/2003 - 01/2008 Biologie, Friedrich-Alexander Universität Erlangen-Nürnberg
Diplomprüfungen in: Mikrobiologie (Hauptfach), Molekulare
Pflanzenphysiologie, Humangenetik und Biochemie

Vordiplomprüfungen in: Bio I, Bio II, Bio III, Bio IV, Physikalische
Chemie, Grundlagen der anorganischen und organischen Chemie

Abitur
09/1992 - 07/2001 Ohm-Gymnasium Erlangen

135
 Curriculum Vitae

Lehrerfahrung

seit 01/2009 Durchführung  und  Betreuung  des  Mastermoduls  „Pathogenität“  und  

des  Fachmoduls  „Mikrobiologie“  sowie  Betreuung  von  Studenten  in  
Bachelor- und Masterarbeiten

Publikationen

2012 Ott L., Scholz B., Höller M., Hasselt K., Ensser A., and Burkovski A.
(2012). Induction of the NFk-B signal transduction pathway in
response to Corynebacterium diphtheriae infection, akzeptiert.

2012 Rheinlaender, J., Gräbner, A., Ott, L., Burkovski, A. & Schäffer, T. E.
(2012). Contour and persistence length of Corynebacterium
diphtheriae pili by atomic force microscopy. Eur. Biophys. J. 41, 561-
570.

2012 Ott, L., McKenzie, A., Baltazar, A. T., Britting, S., Bischof, A.,
Burkovski, A. & Hoskisson, P. A. (2012). Evaluation of invertebrate
infection models for pathogenic corynebacteria. FEMS Immunol.
Med. Microbiol. 65, 413-421.

2011 Ochrombel, I., Ott, L., Krämer, R., Burkovski, A. and Marin, K. (2011).
Impact of improved potassium accumulation on pH homeostasis,
membrane potential adjustment and survival of Corynebacterium
glutamicum. Biochim Biophys Acta. 1807, 444-450.

2010 Trost, E., Ott, L., Schneider, J., Schröder, J., Jaenicke, S., Goesmann,
A., Husemann, P., Stoye, J., Alves Dorella, F., Rocha, F. S., de Castro
Soares,  S.,  D‘Afonseca,  V.,  Miyoshi,  A.,  Ruiz,  J.,  Silva,  A.,  Azevedo,  V.,  
Burkovski, A., Guiso, N., Join-Lambert, O. F., Kayal, S. and Tauch, A.
(2010). The complete genome sequence of Corynebacterium
pseudotuberculosis FRC41 isolated from a 12-year-old girl with
necrotizing lymphadenitis reveals insights into gene-regulatory
networks contributing to virulence. BMC Genomics 11, 728.

2010 Ott, L., Höller, M., Rheinlaender, J., Schäffer, T. E., Hensel, M. and
Burkovski, A. (2010). Strain-specific differences in pili formation and
the interaction of Corynebacterium diphtheriae with host cells. BMC
Microbiol 10:257.

2010 Ott, L., Höller, M., Gerlach, R. G., Hensel, M., Rheinländer, J.,
Schäffer, T. E. and Burkovski, A. (2010). Corynebacterium diphtheriae
invasion-associated protein (DIP1281) is involved in cell surface
organization, adhesion and internalization in epithelial cells. BMC
Microbiol 10:2.

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2008 Amon, J., Bräu, T., Grimrath, A., Hänßler, E., Hasselt K., Höller, M.,
Jessberger, N., Ott, L., Szököl, J., Titgemeyer, F. and Burkovski, A.
(2008). Nitrogen control in Mycobacterium smegmatis: nitrogen-
dependent expression of ammonium transport and assimilation
proteins depends on the OmpR-type regulator GlnR. J Bacteriol 190,
7108-7116.

Buchkapitel

2012 Corynebacterium diphtheriae and related toxigenic species.

A. Burkovski (ed.), Springer, Dortrecht, NL
Kapitel: Adhesion, Invasion and Host Response, in Vorbereitung.

Präsentationen

07/2012 4th Annual Retreat, Erlangen School of Molecular Communication,

Kloster Banz, Bad Staffelstein
Vortrag:  “Evaluation  of  invertebrate  infection  models  for  pathogenic  
corynebacteria”

03/2012 Jahrestagung der Vereinigung für Allgemeine und Angewandte

Mikrobiologie, Tübingen
Poster: “Evaluation  of  invertebrate  infection  models  for  pathogenic  
corynebacteria”

07/2011 3rd Annual Retreat, Erlangen School of Molecular Communication,

Kloster Banz, Bad Staffelstein
Vortrag und Poster:  “Interaction  of  Corynebacterium diphtheriae
with  host  cells”

05/2011 Mol Micro Meeting, Würzburg

Poster:  “Strain-specific differences in pili-formation and interaction
of Corynebacterium diphtheriae with  host  cells”

04/2011 Jahrestagung der Vereinigung für Allgemeine und Angewandte

Mikrobiologie, Karlsruhe
Poster:  “Strain-specific differences in pili-formation and interaction
of Corynebacterium diphtheriae with  host  cells”

09/2010 2nd Annual Retreat, Erlangen School of Molecular Communication,

Kloster Banz, Bad Staffelstein, Germany
Vortrag:  “Adhesion  and  Invasion  of  Corynebacterium diphtheriae”

03/2010 3th joint Conference DGHM and VAAM, Hannover, Germany

Poster: “Strain-specific differences in the interaction of
Corynebacterium diphtheriae with  host  cells”

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09/2009 First Annual Retreat, Erlangen School of Molecular Communication,

Schloss Atzelsberg, Atzelsberg, Germany
Vortrag: “Adhesion  and  Invasion  of  Corynebacterium diphtheriae“

Forschungsreisen

05/2011 Strathclyde Institute of Pharmacy and Biomedical Sciences,

Glasgow, Scottland; Infektion von Caenorhabditis elegans und
Galleria mellonella mit pathogenen Bakterien

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 Danksagung

7 Danksagung
Mein größter Dank gilt Prof. Dr. Andreas Burkovski. Ohne seine uneingeschränkte
Unterstützung für mein Projekt wäre die Arbeit in diesem Umfang nicht möglich gewesen. Ich
danke Andreas für das entgegengebrachte Vertrauen, die wichtigen Impulse für meine Arbeit
und dass ich immer meine Ideen verwirklichen durfte. Auch für die stets ausführlichen
Korrekturvorschläge und die immer offene Tür möchte ich mich ganz herzlich bei ihm
bedanken. Die Zusammenarbeit war auf wissenschaftlicher sowie persönlicher Ebene immer
fair und unkompliziert und hat mir sehr viel Spaß gemacht.

Weiterhin möchte ich Prof. Dr. Uwe Sonnewald für die Übernahme des Zeitgutachtens für
diese Arbeit danken.

An dieser Stelle möchte ich mich auch bei der besten und nettesten Sekretärin der Welt
bedanken: Frau Donata Wehr! Sie ist eine sehr liebenswürdige und zuverlässige Person!

Mein Dank gilt weiterhin allen Mitgliedern des Burki-Labs: Nadine, Kristin, Nadja, Eva und
Elena: ihr alle habt mir die Zeit im Labor extrem angenehm und kurzweilig gemacht und ich bin
sehr froh, dass daraus so tolle Freundschaften entstanden sind! Eva danke ich ganz
besonders für die unendliche Geduld und Unterstützung, wenn ich mal wieder an Word, Clone
manager oder Vector NTI verzweifelt bin. Ich danke auch meinen beiden Lieblingsmasteretten
Susi und Kerstin für die schöne und lustige Zeit.

Zu guter Letzt möchte ich mich bei meinem Freund Daniel, meiner Familie und ganz
besonders bei meinen Eltern bedanken. Sie haben mich immer bei meinen Plänen unterstützt
und mir vollstes Vertrauen entgegengebracht.

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