FEMS Microbiology Letters 253 (2005) 151–154

www.fems-microbiology.org

Sortase A contributes to pneumococcal nasopharyngeal

colonization in the chinchilla model
Shu Chen a, Gavin K. Paterson b, Hua Hua Tong a,

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Timothy J. Mitchell b, Thomas F. DeMaria a,*
a
Division of Otologic Research, College of Medicine and Public Health, The Ohio State University, Room 4331, Cramblett Hall,
456 W. 10th Avenue, Columbus, OH 43210, United States
b
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland G 12 8 QQ, UK

Received 6 September 2005; accepted 22 September 2005

First published online 13 October 2005

Edited by J.-I. Flock

Abstract

Sortase A (SrtA) is required to anchor neuraminidase, b-galactosidase, and possibly other LPXTG motif proteins to the pneu-
mococcal cell surface. We examined the role of SrtA in Streptococcus pneumoniae nasopharyngeal (NP) colonization in the chinchilla
model. The srtA mutant colonized the nasopharynx at a significantly lower level than the D39 parent strain during the second and
third week of the carriage, and was eliminated from nasopharynx one week earlier than the D39 pneumococci. Our data indicate
that SrtA contributes to pneumococcal NP colonization in this animal model.
Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Streptococcus pneumoniae; Sortase A; Colonization

1. Introduction the subsequent induction of disease. The asymptomatic

Otitis media (OM) (middle ear inflammation/infec-
tion) in its various clinical forms, is recognized as one
of the most common childhood diseases. The preva-
lence, medical cost and hearing related morbidity of
OM are significant. Streptococcus pneumoniae remains
one of the major pathogens of OM and accounts for
30% of the cases of acute OM, and 5% of the cases of
chronic OM with effusion [1]. The natural course of
OM is thought to proceed from nasopharyngeal (NP)
colonization, to retrograde ascension of the pathogen
via the eustachian tube, and invasion of middle ear with
*
Corresponding author. Tel.: +1 614 293 8103; fax: +1 614 293
5506.
E-mail address: demaria.2@osu.edu (T.F. DeMaria).
carrier state in children is believed to be the reservoir
of the pathogen [2]. The regulation of colonization and
interrelationship between S. pneumoniae virulence fac-
tors that promote carrier state remains an important
research question.
Sortases are bacterial enzymes that mediate the cova-
lent attachment of specific surface proteins of gram-
positive bacteria to the cell wall. These enzymes typically
cleave a LPXTG motif (leucine, proline, X, threonine,
and glycine, where X is any amino acid) between the
threonine and glycine residues and covalently attaches
the threonine to the amino group of the pentaglycine cell
wall cross bridge resulting in cell wall attached protein
[3,4]. S. pneumoniae strains vary in their content of
sortase genes but all S. pneumoniae strains examined con-
tain sortase A (srtA) [5,6]. A previous study demonstrated

0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.09.052
152 S. Chen et al. / FEMS Microbiology Letters 253 (2005) 151–154
that SrtA is required to anchor neuraminidase, b-galac-
tosidase and presumably other LPXTG motif proteins
to the pneumococcal cell surface [7]. A S. pneumoniae
srtA mutant has been developed which exhibits a re-
duced concentration of neuraminidase A (Nan A) asso-
ciated with the cell surface. It demonstrates decreased
adherence to human pharyngeal cells in vitro [7]. An-
other recent study indicates that a S. pneumoniae srtA
mutant was attenuated in murine models of pneumonia,
bacteremia and NP colonization [6]. The purpose of this
study was to compare the ability of a srtA mutant with
that of the parent strain in the chinchilla NP coloniza-
tion model in order to further define the role of
LPXTG-mediated cell anchoring of proteins in coloni-
zation by S.pneumoniae.
2. Materials and methods
2.1. Bacterial strains, preparation of inocula
S. pneumoniae serotype 2, strain D39 (ATCC 7466)
and an isogenic srtA derivative of D39 deficient in Srt
A have been described in detail previously [6]. Briefly,
srtA was deleted in D39 by allelic replacement and con-
firmed by PCR as described. In order to maintain selec-
tion pressure, erythromycin (1 lg/ml) was added to the
blood agar when D39srtA was cultured. Following
growth on plates, colonies were transferred to Todd-
Hewitt broth (Difco Laboratory, Detroit, MI) containing
erythromycin (5 lg/ml). Prior to inoculation of the chin-
chillas, both strains were passed once in mice as previ-
ously described [8]. Approximately four colonies from
the mouse-passed culture were grown on blood agar
and transferred to Todd-Hewitt broth. After an over-
night incubation at 37 °C, the cultures were centrifuged
at 3500g for 20 min, washed twice with and resuspended
in DulbeccoÕs phosphate-buffered saline (D-PBS) and
used for intranasal (i.n.) inoculation described below.
The S. pneumoniae concentration in the inoculum was
confirmed by standard colony plate count.
2.2. Assessment of NP colonization and invasion of the
middle ear
Forty chinchillas (Chinchilla lanigera) (400–600 g),
free of middle ear diseases as determined by otoscopy
and tympanometry, were randomly assigned by sex
and weight to two cohorts. Twenty chinchillas from
each group were inoculated intranasally with 0.5 ml of
a suspension containing approximately 107 CFU of S.
pneumoniae D39 or D39 srtA/ml, respectively. Four
chinchillas from each cohort, pre-selected and random-
ized, were evaluated by tympanocentesis and NP lavage
on days 1, 7, 14, 21 and 28 post inoculation with D39 or
srtA as previously described [8]. Tympanocentesis was
performed on both ears of the chinchillas by aspiration
with a tuberculin syringe fitted with a 25-gauge needle. If
no middle ear fluid (MEF) was present, the bullas were
lavaged with 0.5 ml sterile saline. Subsequent to tympa-
nocentesis, NP lavage was performed on each chinchilla
as described previously [8]. Chinchillas were not sub-
jected to repeat tympanocentesis or nasal lavage. Tym-
panocentesis and bulla lavage were always performed
before NP lavage to prevent contamination of the mid-
dle ear. The MEF or lavage, and nasal lavage were cul-
tured on blood agar, incubated overnight at 37 °C in a
humidified atmosphere with 5% CO2 and the number
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of CFU per ml were determined by standard dilution
and plate count.
2.3. Phenotypic analysis
Organisms recovered from NP lavage and MEF or
middle ear lavage of chinchillas were tested for erythro-
mycin resistance by standard macrodilution broth
method. For neuraminidase activity assays, representa-
tive colonies were inoculated into 10 ml of BHI with
or without erythromycin (5 lg/ml), and incubated in a
humidified atmosphere with 5% CO2 overnight at
37 °C. The cultures were centrifuged and neuraminidase
activity was measured in both the bacterial cell pellets
and cell free culture supernatants as described by
Winter et al. [9]. Assays were performed in duplicate
on two separate occasions.
2.4. Statistical analysis
Data are expressed as means ± standard error from
at least duplicate samples. Bacterial culture results be-
low the detection limit of the viable count assays
(10 CFU/ml NP lavage fluid) were ascribed values just
below the detection limit (9 CFU/ml). Differences of
S. pneumoniae concentration in NP lavage fluid
between the D39 and the srtA mutant cohorts were
analyzed by the Mann Whitney Rank Sum Test. A
P value of <0.05 was accepted as the minimal level
of significance.
3. Results
3.1. Effect of srtA gene disruption on NP colonization
The relative ability of the parent and the srtA-defi-
cient mutant to colonize and persist in the nasopharynx
for up to 28 days post i.n. challenge is shown in Fig. 1.
There was no significant difference in the concentration
of the parent or mutant in the lavage samples obtained
on days 1 and 7 post inoculation. However, a statisti-
cally significant reduction in the concentration of the
srtA mutant, compared with the D39 parent, was
 S. Chen et al. / FEMS Microbiology Letters 253 (2005) 151–154
8 processed by sortase [10]. A srtA mutant has been used
7 D39
Log CFU/ml in nasal lavage fluid
6
5 *
4
3 sule [7]. However, a recent report by Paterson and
2
1 mouse model. In order to obtain a complete assessment
0
0 5 10 15
Days post intranasal inoculation
Fig. 1. Nasopharyngeal colonization dynamics in chinchillas chal-
lenged i.n. with S. pneumoniae D39 or the srtA mutant. Each data
point represents the geometric mean number of CFU of S. pneumoniae
bacteria ± standard error of the mean per milliliter of nasal lavage
fluid from duplicate samples from four animals. *P < 0.05 compared
to the srtA mutant inoculated group. The dashed line represents the
detection limit of the assay.
evident by day 14. The srtA S. pneumoniae were elimi-
nated from 3 of the 4 chinchillas sampled at this time
point. By day 21 no srtA S. pneumoniae were detected
in the lavage fluid, whereas the D39 parent persisted in
the nasopharynx of all four animals. By 28 days post
inoculation both the D39 parent and the srtA mutant
were eliminated from the nasopharynx. Two chinchillas
from the D39 cohort and none from the srtA cohort
developed OM with culture positive MEFs.
3.2. Phenotypes of D39 and its srtA-deficient mutant
Bacteria recovered from the NP lavage samples of
animals challenged with the srtA mutant were erythro-
mycin resistant, MIC > 32 lg/ml, while those from the
animals challenged with D39 were erythromycin sensi-
tive, MIC < 1 lg/ml. Cell associated neuraminidase
activity for the srtA mutant was below the lower limit
of detection of the assay (0.5 mU/lg of cell protein),
while D39 expressed 10.5 mU/lg of cell protein.
4. Discussion
S. pneumoniae express a number of virulence factors
on the cell surface, which are involved in the pathogen-
esis of pneumococcal diseases. Several important viru-
lence factors such as choline binding proteins are
attached to the cell surface by choline residues of the tei-
choic acids through non-covalent binding. However,
like other gram-positive bacteria, S. pneumoniae also
use covalent attachment of some of the surface proteins.
A genome analysis of S. pneumoniae R6 revealed 13 sur-
face proteins with C-terminal LPTGX motifs that are
153
for evaluation of its virulence in different models both
srtA mutant
in vitro and in vivo [6,7].
A prior investigation into the role of SrtA in the
mouse model of intraperitoneal infection conducted by
Kharat and Tomasz indicated no clear role of SrtA in
* the virulence of strain R36A expressing a type III cap-
Mitchell [6] demonstrated that a srtA mutant of D39
was attenuated during competitive infections in the
of bacterial clearance kinetics data from nasopharynx,
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we used a chinchilla NP colonization model to evaluate
20 25 30
the role of SrtA in the persistence of bacteria in naso-
pharynx. The chinchillas were monitored for up to 28
days, when S. pneumoniae were no longer present in
the lavage fluids. Our results demonstrate that the extent
and duration of NP colonization is altered significantly
by disruption of the srtA gene during the second and
third weeks post i.n. challenge. The mechanisms respon-
sible for the differences between the parent and the srtrA
mutant are not clear.
The possibility of localization defects in the 13 pro-
teins that are the substrates of sortase could contribute
to the lower level of NP colonization of the srtA mutant
compared to that of D39. Two surface proteins b-galac-
tosidase and NanA have shown drastic changes in the
localization from the cell wall to the culture medium
in a S. pneumoniae srtA mutant [7]. b-Galactosidase
has been reported to alter pneumococcal adherence
and invasion of human pharyngeal cells, which could
play a possible role in this animal model of NP coloni-
zation [7].
Finally, changes in the localization of NanA in the
srtA mutant might also be involved in its defect of NP
colonization. We have previously demonstrated that dis-
ruption of nanA diminishes the ability of S. pneumoniae
to colonize and immunization with native or recombi-
nant neuraminidase affords a degree of protection in
the chinchilla OM model [8]. Consistent with the results
of Kharat and Tomasz [7], we could not detect any neur-
aminidase activity from srtA NP or middle ear isolate cell
pellets in vitro. However, neuraminidase activity was
detectable in the culture supernatant (data not shown),
which indicates that neuraminidase is secreted by S.
pneumoniae srtA. It is assumed that the extracellular
neuraminidase would be available in the nasopharynx
in vivo and possibly plays a significant role in the coloni-
zation of S. pneumoniae in the nasopharynx during the
first week of carriage. Whereas differences in NP coloni-
zation between the srtA mutant and parent were not
apparent for 7 days post i.n. inoculation, the altered col-
onization by the srtA-deficient mutant was evident by
significantly lower bacterial counts on days 14 and 21.
In contrast, a comparison of the bacterial clearance
kinetics data of srtA with the nanA mutant from our
154 S. Chen et al. / FEMS Microbiology Letters 253 (2005) 151–154
previous report, which showed a rapid clearance starting
from day1 post i.n. inoculation of a nanA mutant of D39,
is of interest [8]. The lesser effect of the srtA mutation in
comparison to the nanA deletion demonstrates the subtle
role played by cell anchoring of LPXTG-proteins in this
model and reinforces previous findings in the mouse
model [6]. This is in marked contrast to the role of SrtA
in other Gram positive organisms such as Staphylococcus
aureus and Listeria monocytogenes, where SrtA plays a
much larger role in virulence [5]. This may relate to the
fact that most LPXTG anchored proteins in the pneumo-
coccus are enzymes and they are still functionally active
when released from the cell surface. This is in contrast to
the anchorage of adhesins and invasins in Staphylococcus
aureus and Listeria monocytogenes which presumably
need to be anchored to the cell surface to perform their
function. In conclusion, the data from the present study
indicate that disruption of srtA diminishes the ability of
S. pneumoniae to colonize and persist in the chinchilla
nasopharynx.
Acknowledgments
This study was supported, in part, by a grant from
the National Institute on Deafness and Other Commu-
nication Disorders, National Institute of Health (RO1
DC3105-09) to TFD. Work in Glasgow was supported
by the Wellcome Trust.
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