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ARTICLE IN PRESS

International Journal of Medical Microbiology 298 (2008) S1, 325332


www.elsevier.de/ijmm

Changes in the expression pattern of structural proteins after exposure


of Borrelia burgdorferi to penicillin G and doxycycline
Klaus-Peter Hunfelda,, Sebastian Burga, Christa Hanssen-Hubnera, Michael Karasb,
Volker Bradea, Peter Kraiczya
a

Institute of Medical Microbiology & Infection Control, University Hospital of Frankfurt, Paul-Ehrlich Str. 40,
D-60596 Frankfurt/Main, Germany
b
Institute of Pharmaceutical Chemistry, Johann-Wolfgang-Goethe University, Frankfurt/Main, Germany
Accepted 7 December 2007

Abstract
The numerous genes and proteins encoded in the borrelial genome have been shown to undergo differential
expression in response to environmental cues. To gain a better understanding of possible interactions between
antimicrobial agents and Borrelia, we investigated here the effects of increasing concentrations of penicillin G and
doxycycline on the protein expression of the Borrelia burgdorferi s.s. isolate LW2 after 24 and 48 h of incubation. For
14 protein spots in Borrelia exposed to penicillin G at 0.25 and 0.5 mg/ml and for 5 protein spots in Borrelia exposed to
doxycycline at 0.5 and 1 mg/ml, differences in spot intensity were identied by use of high resolution two-dimensional
electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-ight mass spectrometry (MALDITOF-MS). At concentrations of both antimicrobial agents around the median minimal inhibitory concentration
(0.5 mg/ml), all but one of the detected spots showed a considerable down-regulation as revealed by a X50% decrease
of spot intensity in comparison to untreated controls. Most of the spots identied thus far belong to proteins that are
encoded by genes localized on the borrelial chromosome and are known to be involved in the different pathways of
bacterial cell metabolism. Interestingly, one spot, identied as triosephosphate isomerase, was clearly up-regulated in
the presence of doxycycline. Our data provide for the rst time scientic evidence that B. burgdorferi s.l., although it
possesses a small genome and extremely limited biosynthetic capabilities, shows a variable but distinct physiological
response to exposure with penicillin G and doxycycline.
r 2008 Elsevier GmbH. All rights reserved.
Keywords: Borrelia burgdorferi; MALDI-TOF MS; Antimicrobial susceptibility; Penicillin; Doxycycline; 2D electrophoresis; Protein
expression

Introduction
Borrelia burgdorferi s.l., the causative agent of Lyme
borreliosis, is used to adapt to rapidly changing
Corresponding author. Tel.: +49 69 6301 6441;
fax: +49 69 6301 5767.
E-mail address: K.Hunfeld@em.uni-frankfurt.de (K.-P. Hunfeld).

1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2007.12.001

environmental conditions as it successfully moves


between arthropod vector and its diverse mammalian
hosts (Nowalk et al., 2006). It is well known that in the
course of evolutionary processes bacteria have evolved
elaborate strategies that enable them to survive periods
of starvation and exposure to antimicrobial agents
(Siegele and Kolter, 1992; Graumann et al., 1996;
Martinez-Martinez et al., 1996; Alban et al., 2000).

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Correspondingly, the numerous genes and proteins


encoded in the borrelial genome have been demonstrated
to undergo differential expression in response to in vitro
environmental cues, which simulate signicant changes in
pH, temperature, and other aspects of the host milieu as
encountered by these spirochetes during transmission
(Alban et al., 2000; Brooks et al., 2003; Nowalk et al.,
2006). Some authors even speculate that Borrelia can
overcome unfavorable environmental conditions or the
presence of antibiotics by undergoing morphological
changes into a physiologically dormant state or the
formation of so-called spheroblast-L-form variants (Preac-Mursic et al., 1989, 1996; Brorson and Brorson, 1997,
1998, 1999; Alban et al., 2000). From a theoretical point of
view, such instruments of evasion would offer protection
from both the host defense and antimicrobial agents
(Hunfeld and Brade, 2006). At present, there is no
convincing scientic evidence for the existence of such
mechanisms in vitro or in vivo. In comparison with
common bacterial pathogens, however, relatively little is
known to date about the means B. burgdorferi s.l. may
have developed to evade unfavorable conditions in general
and the presence of bactericidal or bacteriostatic antimicrobial agents in particular (Hunfeld and Brade, 2006).
Proteomics is a snapshot-like analysis of a protein
composition regarded representative for the functional
status of a dened biological compartment and is a
rapidly growing eld of research that encompasses both
genetic and environmental factors. As the protagonist
macromolecules in bacterial cells, proteins can serve as
prominent targets for a better understanding of infectious
diseases (Brooks et al., 2003). High resolution, twodimensional electrophoresis (2-DE) can separate proteins
from complex mixtures, such as cells or tissues (Klose and
Kobalz, 1995), and, as such, represents a very promising
approach for providing additional insights into physiological processes of the bacterial cell metabolism. Unfortunately, investigations concerning a more detailed analysis
of the changing expression pattern of the structural
proteins of B. burgdorferi s.l. in the presence of
antimicrobial agents via application of proteomics are
lacking. Here, to better characterize the physiological
response and the possible interactions between antimicrobial agents and Borrelia, we investigated the effects of
increasing concentrations of penicillin G and doxycycline
on the protein expression of Borrelia by use of 2-DE and
matrix-assisted laser desorption/ionization time-of-ight
mass spectrometry (MALDI-TOF-MS).

Materials and methods


Borrelial strain and culture conditions
B. burgdorferi s.s. isolate LW2 (skin isolate, Germany)
was grown at 33 1C for 56 days up to cell densities of

1  107/ml in modied Barbour-Stoenner-Kelly (BSK)


medium as described previously (Hunfeld et al., 2000;
Kraiczy et al., 2001). The density of spirochetes
was determined using dark-eld microscopy and a
Kova counting chamber (Hycor Biomedical, Garden
Grove, CA).

Determination of minimal inhibitory concentration


(MIC)
For susceptibility testing of penicillin G and doxycycline (Sigma-Aldrich, Steinheim, Germany), a colorimetric assay was performed using a standardized
inoculum of 2.5  107 Borrelia/ml, and MICs for LW2
were determined under the same standard conditions as
described previously (Hunfeld et al., 2000; Kraiczy et al.,
2001). The test ranges were 0.00516 mg/ml for penicillin
G and 0.01532 mg/ml for doxycycline, respectively.
Growth control experiments were undertaken under
the same test conditions except for the addition of
antibiotics.

Protein preparation from antibiotic-exposed


borrelial samples and controls
For preparation of proteins from antibiotic-exposed
Borrelia, cultures (inoculum: 2.5  107 cells/ml) were
grown in 10 ml BSK supplemented with increasing
concentrations of penicillin G (range of concentration:
0.0150.5 mg/ml) and doxycycline (range of concentration: 0.0151 mg/ml) for 24 and 48 h. Single experiments
for each concentration were performed on 3 different
days. For control purposes each time, borreliae were
grown under the same condition in 10 ml BSK medium
without antimicrobial agents. Borrelial cells from
cultures exposed to antimicrobial agents and the
corresponding controls were then harvested by centrifugation at 5000 rpm and 4 1C for 30 min after incubation periods of 24 and 48 h. The pellets were washed 3
times in 10 ml PBS at pH 7.0. The resulting pellets were
suspended in 100 ml lysis buffer (7 M urea, 2 M thiourea,
1% dithiothreitol and 4% CHAPS [3-(3-chloramidopropyl)-dimethylammonio-1-propane-sulfonate]), supplemented with 1 ml TBP (tributylphosphine) (Bio-Rad,
Munich, Germany) and 0.2 mM AEBSF (4-[2-]-aminoethylbenzolsulfonyl uoride, Sigma-Aldrich), and the
cells disrupted by 5 times sonication for 30 s at 4 1C with
90 s pauses between each sonication period. DNA and
RNA were degraded by adding 0.1 volume of nuclease
buffer (100 mM Tris, 50 mM MgCl2, 1 mg/ml [w/v],
DNase I, 0.5 mg/ml [w/v] RNase, pH 7.0) to the cell
lysates. After incubation for 1 h at 4 1C, the protein
suspensions were sedimented by a nal centrifugation at
14,000g and 20 1C for 1 h, and the resulting supernatants
that contained the bacterial proteins were then stored at

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327

directly for 10% Tris/tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis or stored at 80 1C.
The focused strips used for the second dimension were
rst reduced in 50 mM Tris (pH 8.8) equilibration buffer
containing 6 M urea, 30% glycerol, 2% SDS, 1%
dithiothreitol, and traces of bromophenol blue for
15 min and then alkylated in the same equilibration
buffer containing 260 mM iodoacetamide. The complemented rst-dimension strips were separated on a 10%
Tris/tricine-sodium dodecyl sulfate-polyacrylamide gel
electrophoresis in a Protean II Xi chamber (Bio-Rad)
together with a broad-range protein marker (Precision
Protein standard, Bio-Rad). Running conditions were
0.5 h at 40 V and 17.5 h at 85 V. Protein spots were
detected by silver-staining (analytical gels, Fig. 1) or
Coomassie-staining (preparative gels).

80 1C until use. To determine the protein concentrations of the different samples, a modied Bradford
protein assay (Bio-Rad) was used.

Immobilized pH gradient-2D electrophoresis


Protein samples were analyzed by immobilized pH
gradient two-dimensional electrophoresis (IPG-2-DE)
using the Protean IEF cell from Bio-Rad. For the rst
dimension, a 20 mg protein sample was solubilized to a
nal volume of 330 ml in rehydration buffer containing
8 M urea, 2% CHAPS, 0.5% carrier ampholytes and a
trace of bromphenol blue (pH 310, Bio-Rad). After an
initial incubation for 30 min at room temperature, the
samples were loaded by in-gel rehydration for 12 h at
250 V using immobilized linear pH gradient strips (pH
310) (Bio-Rad). The isoelectric focusing parameters
were as follows: (i) 1 h at a 150-V gradient, (ii) 1 h at a
300-V gradient, (iii) 1 h at a 600-V gradient, (iv) 1 h at a
1200-V gradient, (v) 1 h at a 2400-V gradient, (vi) 1 h at
a 7000-V gradient, (vii) 7000 V for 20,000 Vh, and
15 min at 500 V with a 50-mA-per-strip maximum setting
at 20 1C. The focused IPG strips were either used

Scanning and spot detection


The PDQuests software (V. 6.1.1, Bio-Rad) and a
densitometer model GS-710 (Bio-Rad) was used for the
computer-assisted image analysis of gels. To detect
qualitative and quantitative differences between the gels,
all spots were matched as follows: After each of the

SDS-PAGE

IEF
P-4802 P-4803 P-4804
P-3703
P-2702

75

D/P-4703
D/P-4705
P-5702
P-7602

50

P-7603

D-0504

37

Mr [kD]

P-4702
P-4706

25
P-7103

D-4101

D/P-7001

15
3

10

pH

Fig. 1. Representative two-dimensional gel analysis of Borrelia burgdorferi s.s. isolate LW2. Twenty micrograms of protein derived
from a culture in the log-phase of growth were focused in a pH-gradient ranging between pH 3 and 10. After isoelectric focusing
(IEF), the sample was separated through a 10% Tris/tricine SDS-PAGE and subsequently silver stained. Location and molecular
mass of differentially expressed proteins in borrelial samples exposed to penicillin G and doxycycline at concentrations around the
MIC are indicated. Such protein spots were then cored from preparative gels and further analyzed by MALDI-TOF MS (see Table 1
and Fig. 2). The positions of molecular mass standard in kDa are indicated on the right. The linear pH gradient ranging from pH 3
to 10 is indicated at the bottom of the photograph.

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three experiments, protein preparations extracted after


24 and 48 h from: (i) penicillin G-exposed Borrelia,
(ii) doxycycline-exposed Borrelia, and (iii) untreated
controls 2-DE gels were run and stained on the same
day. A total of three gels were then used for computerassisted matching to reduce articial spot-detection
caused by the staining procedure. To further substantiate the differences in spot intensity, we also performed
software-assisted image analysis of the matched gels to
quantify the corresponding differences in protein
expression of Borrelia exposed to antimicrobial agents
and untreated controls. Differences were regarded

relevant only if a reduction or increase of X50% in


intensity became obvious in spots of gels loaded with
antibiotic-exposed samples compared to the corresponding control preparations (Fig. 2).

Sample preparation for mass spectrometry


The selected protein spots were cored from gels and
subjected to in-gel digestion protocols as described
previously (Jungblut et al., 1996; Hartmann et al., 2006).
For mass spectrometric analysis the samples were solved
in 5 ml 50% ACN/1% (v/v) TFA (Fluka, Buchs,

2500

2500

2500

250

2000

2000

2000

200

1500

1500

1500

150

1000

1000

1000

100

500

500

500

50

0
P-3703

P-2702
750

1500
1250
1000
750
500
250
0

600
450
300
150
0

P-4706
1500

600
500
400
300
200
100
0
P-4803

P-4802
3000
2400
1800
1200
600
0

0
P-4702

900
600
300
0
P-5702

P-4804

2000

800

1600

600

1200

1200

400

800

200

400

P-7603

P-7602

P-7103
200

350

600

150

280

450

210

100

300

140

50

150

70
0

D/P-4703

D/P-4705

800

1600

600

1200

400

800

200

400

D/P-7001

0
D-0504

D-4101

Fig. 2. Comparative analyses of protein synthesis in antibiotic-exposed Borrelia. Protein preparations of Borrelia exposed to 0.25
and 0.5 mg/ml of penicillin G or to 0.5 and 1 mg/ml of doxycyline for 24 and 48 h and untreated controls were subjected to 2 DE and
subsequent software-assisted densitometry. Bars display the differences in spot intensities of the proteins affected by penicillin G (P-)
and doxycycline (D-) as measured by densitometry and are expressed as OD values (black bars indicate untreated controls; white
bars indicate antibiotic-exposed samples). Proteins affected by both penicillin G and doxycyline (D/P-4703, D/P-4705, and D/P7001) are summarized in a single diagram where bars on the right indicate penicillin-treated samples and bars on the left represent
doxycycline-treated samples. The error bars represent the standard error of the mean (SEM) calculated for the means of spot
intensity obtained from three independent gel runs for antibiotic-exposed samples and untreated controls.

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K.-P. Hunfeld et al. / International Journal of Medical Microbiology 298 (2008) S1, 325332

Switzerland). 0.5 ml of the sample was mixed with 0.5 ml


of matrix (2 mg/ml a-cyano-4-hydroxycinnamic acid
[Bruker, Bremen, Germany] in 50% ACN/0.5% [v/v]
TFA) directly on a stainless steel MALDI target
(Applied Biosystems [ABI], Darmstadt, Germany) and
dried under ambient conditions.

MALDI-TOF-MS
Delayed extractionTM (DE) MALDI time of ight
mass spectra were recorded on a Voyager-DE STR
instrument (ABI) using a nitrogen laser (l 336 nm,
repetition rate 20 Hz) for desorption and ionisation
with an acquisition mass range from 600 to 5000m/z and
the low mass gate set to 550m/z. The total acceleration
voltage was 20 kV with 68.5% grid voltage on the rst
grid, 0.02% guide wire voltage, 150 ns delay and a
mirror voltage ratio of 1.12. Spectra were externally
calibrated with SequazymeTM Peptide Mass Standards
Kit (ABI). Between 1000 and 2000 laser shots were
accumulated for each mass spectrum. All spectra were
smoothed, noise-ltered and deisotoped using Data
Explorer (V. 4.3, ABI). Deisotoped peaks were labeled
by the software and the 100 most intense peaks were
used for database searching. Autolytic tryptic peptides
or peptides resulting from the identied protein were
used for internal calibration.

Protein database queries


Proteins were identied using Spectrum Mill (V. 3.0,
Agilent Technologies, Waldbronn, Germany) installed
on a local server using the the MASCOT (TM) Search
Engine, Matrix Science (http://www.matrixscience.com)
for comparison with the B. burgdorferi s.s. strain B31
genome sequence (http://www.tigr.org).

Results
In vitro susceptibility testing results
MICs of penicillin G and doxycycline for B. burgdorferi s.s. isolate LW2 ranged from 0.5 to 1 mg/ml (median
MIC: 0.5 mg/ml for both substances), as determined by a
colorimetric assay in independent experiments performed on 3 different days (data not shown).

Variability of protein expression in borrelia after


exposure to penicillin G and doxycycline
Based upon our MIC ndings in isolate LW2,
borrelial cells were then exposed to increasing concentrations of penicillin G and doxycycline for 24 and 48 h
to test whether their protein expression level was

329

inuenced signicantly by sub-inhibitory and inhibitory


concentrations of these antimicrobial agents. Visual
interpretation of the 2-DE gels of proteins extracted
from borrelial cells growing at concentrations of 0.015,
0.031, 0.0625, and 0.125 mg/ml of penicillin G and
doxycycline after 24 and 48 h of incubation indicated
that the protein patterns of antibiotically treated cells
revealed no differences in comparison to that of
untreated controls (data not shown).
In the presence of penicillin G at half the median MIC
(0.25 mg/ml) and at the median MIC (0.5 mg/ml),
however, a reduction of intensity was detected for
several protein spots by visual examination of the 2-DE
gels. Similarly, differences in spot intensity became
obvious in the presence of doxycycline at the median
MIC (0.5 mg/ml) and at one log2 unit dilution above the
MIC (1 mg/ml). To further substantiate the analytical
relevance of the observed decrease or increase of
spot intensity, computer-aided analysis of the 2-DE gels
(Fig. 2) was performed to quantify the differences in
expression levels of the selected proteins. Softwareassisted densitometry of spot intensity in matched gels
of antibiotic-exposed borrelial culture preparations
clearly revealed a relevant reduction or over-expression
compared to untreated controls with regard to 16
protein spots after 24 and 48 h of antibiotic exposure
(Fig. 2, Table 1). Changes in spot intensity for these
variably expressed proteins did not differ signicantly at
time point 24 h compared to 48 h of exposure. Fourteen
protein spots detected on the 2-DE gels of the penicillin
G-treated samples (P2702 to D/P-7001, Table 1) and
four protein spots identied on the 2-DE gels of the
doxycycline-treated samples (D/P-4703, D/P-4705, D/P7001, and D-0504, Table 1) were down-regulated as
revealed by a X50% decrease of spot intensity in
comparison to untreated controls (Fig. 2). In three spots
(D/P-4703, D/P-4705, and D/P-7001; Fig. 2, Table 1),
both penicillin G and doxycycline exposure of Borrelia
led to such relevant down-regulation of protein synthesis. Remarkably, after 24 and 48 h, one protein
(D-4101, Fig. 2, Table 1) could also be identied which,
compared to untreated controls, was 50% over-expressed after cultivation of Borrelia in the presence of
doxycycline at the MIC and at one log2 unit dilution
above the MIC.

Identication of variably expressed proteins


All 16 protein spots that showed up- or downregulation in their expression levels as identied on the
2-DE gels (Fig. 2) were marked and then excised from a
preparative gel for further analysis by in-gel digestion
followed by MALDI-TOF-MS. Eleven out of the 16
proteins were identied unambiguously after matching
the data obtained by MALDI-TOF-MS analysis. For

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Table 1. Protein spots and corresponding proteins showing up- or down-regulation after exposure of Borrelia burgdorferi s.s. strain
LW2 to increasing concentrations of penicillin G, doxycycline, or both
Spot

pI
(estimated
from gel)

Mol. mass
(estimated
kDa from gel)

Up- or
downregulation

Mol. mass
(calculated kDa
from database)

Identied protein (by MALDI-TOF MS)

P-2702
P-3703
P-4702
P-4706
P-4802
P-4803

5.9
6.2
6.4
6.7
6.4
6.5

65
65
63
63
75
75

k
k
k
k
k
k

68.13
68.13
63.496
NA
NA
80.298

P-4804
P-5702

6.6
7.0

75
57

k
k

NA
50.061

P-7103
P-7602
P-7603
D/P-4703
D/P-4705
D/P-7001
D-0504
D-4101

8.1
7.8
7.9
6.5
6.6
7.9
5.2
6.4

25
50
49
65
63
23
42
26

k
k
k
k
k
k
k
m

30.417
55.856
53.227
NA
NA
26.569
42.372
27.984

Membrane-associated protein p66 (BB0603)


Membrane-associated protein p66 (BB0603)
Ribosomal protein S1 (rpsA) (BB0127)
No match found
No match found
Polyribonucleotide nucleotidyltransferase (pnpA)
(BB0805)
No match found
Pyrophosphate-fructose-6-phosphate-1phosphotransferase (pfp) (BB0727)
Predicted coding region (BB0238)
Glycerol kinase (glpK) (BB0241)
Pyruvate kinase (pyk) (BB0348)
No match found
No match found
pfs protein (pfs-1) (BB0375)
Cell division protein (ftsZ) (BB0229)
Triosephosphate isomerase (BB0055)

Proteins were identied by MALDI-TOF MS after preparative isolation from 2-DE gels.
pI: isoelectric point; P: proteins affected by penicillin G exposure; D: proteins affected by doxycycline exposure; NA: not available.

ve proteins (P-4706, P-4802, P-4804, P/D-4703, and


P/D-4705), no signicant match could be found for
B. burgdorferi s.s. proteins in the database. Molecular
masses, isoelectric points (pI), and identities of the
corresponding proteins are given in Table 1. All spots
identied thus far belong to proteins that are encoded by
genes localized on the borrelial chromosome and most
of the proteins that display a variable expression after
exposure of borrelial cells to penicillin G and doxycycline are known to be involved in different pathways of
bacterial cell metabolism (Table 1). Interestingly, a
membrane-associated protein (p66) was also involved
and is obviously down-regulated in the presence of
penicillin G. According to our results, this protein
appears in two distinct conformations as documented by
the occurrence of two different spots (P-2702, P-3703)
on the 2-DE-gels of both penicillin-exposed samples and
untreated controls. The only protein that was strongly
over-expressed (D-4101) in the presence of doxycycline
could be identied as triosephosphate isomerase (TIM).

Discussion
The occurrence of changes both in the morphology
and the protein expression pattern of B. burgdorferi s.l.
have been reported in response to a variety of adverse
environmental conditions (Barbour and Hayes, 1986;

Preac-Mursic et al., 1996; Brorson and Brorson, 1997,


1999; Alban et al., 2000). Such response mechanisms in
spirochetes were shown to occur in cells exposed to
physiological stress, such as changes in pH, depletion of
metabolites, presence of antimicrobial agents, and aging
(Barbour and Hayes, 1986; Preac-Mursic et al., 1996;
Brorson and Brorson, 1997, 1999; Alban et al., 2000;
Kraiczy et al., 2001; Brooks et al., 2003; Hartmann
et al., 2006; Nowalk et al., 2006). The exposure of
microorganisms to antimicrobial agents represents a
very intense stress factor and in common Gram-negative
and Gram-positive bacteria elaborate response mechanisms and a complex enzymatic armentarium is required
to avoid immediate bacterial cell death in the presence of
active antimicrobial substances (Martinez-Martinez
et al., 1996; Nitzan et al., 2002; Bandow et al., 2003).
Here, we studied the variability of the protein expression
pattern in Borrelia after exposure of the pathogen to
increasing concentrations of penicillin G and doxycycline antimicrobial agents frequently used in the
treatment of Lyme borreliosis under dened test
conditions. Fourteen protein spots detected on the 2-DE
gel of the penicillin G-treated samples (P-2702 to D/P7001) and four protein spots identied on the 2-DE gels
of the doxycycline-treated samples (D/P-4703, D/P4705, D/P-7001, and D-0504) were down-regulated as
revealed by a X50% decrease of spot intensity in
comparison to untreated controls (Fig. 2; Table 1).

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Eleven out of these proteins could be unambiguously


identied after matching the data obtained by MALDITOF MS with amino acid sequences in the database.
However, for ve proteins (P-4706, P-4802, P-4804,
D/P-4703, and D/P-4705), no signicant match could be
found in the database. All spots identied thus far
belong to proteins that encode genes localized on the
borrelial chromosome. Most of the proteins that display
a variable expression after exposure of borrelial cells to
penicillin G and doxycycline are involved in several
different metabolic pathways of the cell (Table 1), e.g.,
in glycolysis (P-5702, P-7602, P-7603), salvage of
nucleotides (P-4803), cell division (D-0504, D/P-7001),
translation (P-4702), and energy metabolism (D-4101).
It is interesting to note, though, that in addition to
proteins involved in the nucleotide and energy metabolism of Borrelia the membrane-associated protein
p66, was also involved and, obviously, is signicantly
down-regulated in the presence of penicillin G at
sub-inhibitory concentrations as well as at the MIC.
Our observation of two different spots (P-2702, P-3703)
for this protein on the 2-DE gels of both penicillinexposed samples and untreated controls parallel similar
ndings by Skare et al. (1997) demonstrating two
different conformations for p66 by fast-performance
liquid chromatography. This outer membrane protein is
known to function as a borrelial porin and adhesin with
channel-forming activity and integrin-binding ability
(Pinne et al., 2007; Skare et al., 1997). Under mammalian, host-adapted conditions, transcription of p66 is
up-regulated, implying that p66 may play an important
role in adhesion and/or nutrient acquisition in the
infected host (Brooks et al., 2003). In common Gramnegative bacteria, porins are also known to be of
importance for the inux of antimicrobial agents into
the cytoplasm (Nitzan et al., 2002) and the absence of
porins is associated with resistance to b-lactams, which
occurs in porin-decient mutants of Klebsiella pneumoniae with increased resistance to extended spectrum
cephalosporins (Martinez-Martinez et al., 1996).
Whether down-regulation of p66 by Borrelia is simply
part of cell degeneration or whether decreased expression of the protein in the presence of b-lactams may be
part of an adaptive process to hinder the inux of these
drugs through the outer membrane into the periplasmic
space should be explored further. Additional investigations will be necessary to elucidate whether such
mechanisms indeed play a signicant role in the
physiological response that antimicrobial agents evoke
in B. burgdorferi s.l.
To date, it is well established that the up- or downregulation of enzymes belonging to the energy metabolism of bacteria is a frequently observed means of
adaptation to a starvation process. For example,
borreliae are known to react to the depletion of essential
components of BSK medium by a complex response of

331

its protein expression that involves more than 20


proteins (Alban et al., 2000). Similarly, E. coli responds
to fatty acid starvation of the culture medium by
accumulation of spoT-dependent guanosin tetraphosphate (ppGpp) and inhibition of rRNA synthesis
(Seyfzadeh et al., 1993). In the present study, exposure
of Borrelia to doxycycline led to down-regulation of
proteins involved in cell division (pfs-1, ftsZ) but also
induced signicant up-regulation of TIM (D-4101), a
key enzyme of the glycolytic carboanhydrate metabolism (Fig. 2, Table 1). This observation is new and
resembles previous ndings in Bacillus subtilis where the
induction of a cold shock resulted in increased expression of proteins. Besides glyceraldehydes dehydrogenase
(Gap), cysteine synthase (CysK), and ketol-acid reductoisomerase ([IlvC], which catalyze the second step in
valine and isoleucine synthesis), TIM again was one of
the enzymes whose de novo synthesis was signicantly
induced after a temperature shift from 37 to 15 1C
(Graumann et al., 1996). Very recently, TIM also has
been identied as being up-regulated up to 244-fold
during the stationary growth phase of Staphylococcus
epidermidis cultures (Vandecasteele et al., 2004). These
observations indicate that glycolysis and the synthesis of
amino acids are of critical importance for bacterial
pathogens to keep up a minimal level of metabolic
activity under such unfavorable growth conditions
(Graumann et al., 1996; Vandecasteele et al., 2004).
Correspondingly, the up-regulation of TIM in Borrelia
in the presence of doxycycline may be interpreted as a
counteracting induction of increased enzymatic activity
owing to a potentially adaptive or evasive metabolic
mechanism on the part of the pathogen being exposed to
this bacteriostatic drug. Although further research is
clearly needed to elucidate and corroborate such
speculations, the data presented here provide for the
rst time scientic evidence for a variable but distinct
physiological response by Borrelia to antimicrobial
agents. Moreover, our ndings indicate that although
B. burgdorferi s.l. possesses a small genome and
extremely limited biosynthetic capabilities, it can respond to antibiotic exposure within 2448 h of incubation by inducing a metabolic reaction pattern that
includes 14 proteins in the presence of penicillin G and
ve proteins in the presence of doxycycline at concentrations of these substances around the MIC.

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