Identification of novel adhesins from Group B streptococci by use of phage display reveals that C5a peptidase mediates fibronectin binding.
ABSTRACT Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenic scpB mutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by in trans complementation of the mutant with the cloned wild-type scpB gene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.
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ABSTRACT: Streptococcus agalactiae (group B Streptococcus or GBS) is a leading cause of neonatal sepsis and meningitis, peripartum infections in women, and invasive infections in chronically ill or elderly individuals. GBS can be isolated from the gastrointestinal or genital tracts of up to 30% of healthy adults, and infection is thought to arise from invasion from a colonized mucosal site. Accordingly, bacterial surface components that mediate attachment of GBS to host cells or extracellular matrix represent key factors in colonization and infection of the human host. We identified a conserved GBS gene of unknown function that was predicted to encode a cell wall-anchored surface protein. Deletion of the gene and a co-transcribed upstream ORF in GBS strain 515 reduced bacterial adherence to VK2 vaginal epithelial cells in vitro and reduced GBS binding to fibronectin-coated microtiter wells. Expression of the gene product in Lactococcus lactis conferred the ability to adhere to VK2 cells, to fibronectin and laminin, and to fibronectin-coated ME-180 cervical epithelial cells. Expression of the recombinant protein in L. lactis also markedly increased biofilm formation. The adherence function of the protein, named Bacterial surface adhesin of GBS (BsaB), depended both on a central BID1 domain found in bacterial intimin-like proteins and on the C-terminal portion of the BsaB protein. Expression of BsaB in GBS, like that of several other adhesins, was regulated by the CsrRS two-component system. We conclude that BsaB represents a newly identified adhesin that participates in GBS attachment to epithelial cells and extracellular matrix.Infection and immunity 12/2013; · 4.16 Impact Factor
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ABSTRACT: Group B Streptococcus (GBS), is currently the leading cause of neonatal meningitis. This is due to its ability to survive and multiply in the bloodstream, and interact with specialized human brain microvascular endothelial cells (hBMEC), which constitute the blood-brain barrier (BBB). The exact mechanism(s) of GBS-BBB penetration are still largely unknown. We and others have shown that GBS interact with components of the extracellular matrix. In this study, we demonstrated that GBS of representative serotypes bind immobilized and cell surface fibronectin, and identify a putative fibronectin binding protein, streptococcal fibronectin binding protein A (SfbA). Allelic replacement of sfbA in the GBS chromosome resulted in a significant decrease in ability to bind fibronection and invade hBMEC compared with the WT parental strain. Expression of SfbA in the non-invasive strain, Lactococcus lactis, was sufficient to promote fibronectin binding and hBMEC invasion. Furthermore, the addition of an anti-fibronectin antibody or an RGD peptide which blocks fibronectin binding to integrins, significantly reduced invasion of the WT but not the sfbA deficient mutant strain, demonstrating the importance of a SfbA-fibronectin-integrin interaction for GBS cellular invasion. Using a murine model of GBS meningitis, we also observed that WT GBS penetrated the brain and established meningitis more frequently than did the ΔsfbA mutant strain. Our data suggest that GBS SfbA plays an important role in bacterial interaction with BBB endothelium and the pathogenesis of streptococcal meningitis.Infection and immunity 03/2014; · 4.16 Impact Factor
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ABSTRACT: Background Since 2009, large-scale Streptococcus agalactiae infections have broken out in cultured tilapia farms in China, resulting in considerable economic losses. Screening of the surface proteins is required to identify virulence factors or protective antigens involved in piscine S.agalactiae infections in tilapia. Pre-absorbed immunoproteomics method (PAIM) is a useful method previously established in our laboratory for identifying bacterial surface proteins.ResultsA serine-rich repeat protein family 1 (Srr-1), designated XF, was identified by PAIM in piscine S. agalactiae isolate GD201008-001. To investigate the role of XF in the pathogenesis of piscine S. agalactiae, an isogenic xf mutant strain (¿xf) and a complemented strain (C¿xf) were successfully constructed. The ¿xf mutant and C¿xf showed no significant differences in growth characteristics and adherence to HEp-2 cells compared with the wild-type strain. However the 50% lethal dose of ¿xf was increased (4-fold) compared with that of the parental strain in a zebrafish infection model.Conclusions The findings demonstrated that XF is a virulence-related, highly immunoreactive surface protein and is involved in the pathogenicity of S. agalactiae infections in fish.BMC Veterinary Research 10/2014; 10(1):259. · 1.74 Impact Factor
INFECTION AND IMMUNITY, June 2002, p. 2869–2876
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 6
Identification of Novel Adhesins from Group B Streptococci by Use of
Phage Display Reveals that C5a Peptidase Mediates
Christiane Beckmann, Joshua D. Waggoner, Theresa O. Harris, Glen S. Tamura,
and Craig E. Rubens*
Division of Infectious Disease, Children’s Hospital and Regional Medical Center
and University of Washington, Seattle, Washington
Received 26 November 2001/Returned for modification 14 January 2002/Accepted 11 February 2002
Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and
infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence
of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization
(G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We
have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A
shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity
selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with
homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic pro-
cesses, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology
to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the
complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demon-
strated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the
peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays
to fibronectin were performed, comparing an isogenic scpB mutant to the wild-type strain. Approximately 50%
less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully
restored by in trans complementation of the mutant with the cloned wild-type scpB gene, providing further
evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional
protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin
has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a
second important role for this surface protein in the pathogenesis of GBS infections.
Group B streptococci (GBS) are important human patho-
gens which are responsible for a broad range of diseases in
human newborns (2) and immunocompromised hosts (33). In-
fections in newborns occur in the first 6 days of life (early-onset
disease) or in the first weeks (weeks 1 to 12) of life (late-onset
disease). The pathogenesis of GBS infections is a multifacto-
rial process that includes the ability to adhere, colonize, and
invade epithelial and endothelial cells and then replicate and
evade host immune defenses (29, 41). Adherence is thought to
be the initiating step for entry into host cells, promoting inva-
sion of deeper tissues and ultimate dissemination of the bac-
teria to the bloodstream and multiple organ systems (42).
The process of colonization involves microbial and host re-
ceptor-ligand interactions. Many streptococci express proteins
that bind specifically to proteins of the extracellular matrix
(ECM) and/or serum. The ECM surrounds cells in connective
tissue such as skin, bones, and cartilage. It is heterogeneous,
and various ECM proteins, including fibronectin (Fn), fibrin-
ogen, laminin, collagen, and integrins are utilized as adhesin
receptors by several different pathogens (47). The most widely
described interaction is the binding to Fn, which is a large
dimeric glycoprotein present in the ECM in a fibrillar form. Fn
contains repeats of a characteristic amino acid triplet se-
quence, arginine-glycine-aspartic acid [RGD]), which can
function as the binding site for some bacteria (39). Fn is also
one of the major adherence targets for group A streptococci
(GAS) (35). Adhesins include protein F/SfbI (13, 40), serum
opacity factor (18), and Fn binding protein FBP54 (10). Fn
binding proteins, such as SfbI from Streptococcus pyogenes,
have also been demonstrated to act as invasins (26). Further-
more, SfbI induces a protective immune response against S.
pyogenes in the sera and lungs of mice after intranasal vacci-
nation (12), emphasizing the significance of Fn binding for
pathogenesis of streptococcal infections.
Unlike GAS, Streptococcus dysgalactiae, and Staphylococcus
aureus, GBS do not bind Fn in its soluble form (43), suggesting
that Fn binding of GBS is a low-avidity interaction that re-
quires multiple GBS adhesins (29). So far the GBS surface
structure(s) that mediates binding to Fn has not been identi-
To identify Fn adhesins of pathogenic GBS, we have em-
ployed a shotgun phage display approach to screen a GBS
genomic library for sequences which encode peptides that bind
to solid-phase Fn. Using this screen, we have identified gene
fragments from GBS with homology to other gram-positive
* Corresponding author. Mailing address: Division of Infectious
Disease, Children’s Hospital and Regional Medical Center/University
of Washington, 4800 Sand Point Way N.E. CH-32, Seattle, WA 98105.
Phone: (206) 526-2073. Fax: (206) 527-3890. E-mail: cruben@chmc
adhesins and virulence genes, confirming the feasibility of our
approach. One of the isolated Fn binding clones revealed ho-
mology to the C5a peptidase (ScpB), a highly specific endo-
peptidase found in GAS (48), GBS (15), and group G strep-
tococci (GGS) (8). It cleaves and inactivates C5a, a component
of the human complement system, thereby contributing to the
ability of GBS to evade phagocytosis (3).
Based on the identification of a C5a peptidase-related pep-
tide, we hypothesized that this protein is bifunctional, serving
as a protease and mediating binding to Fn. In this report we
demonstrate that GBS C5a peptidase binds to immobilized Fn
and serves as a bacterial adhesin.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The bacterial strains and
plasmids used for this study are listed in Table 1. A derivative of phagemid vector
pHEN1 (16), pG3H6, which contains a six-histidine tag, a c-Myc tag, and a SmaI
site for blunt-end cloning, was kindly provided by L. Frykberg (Swedish Univer-
sity of Agricultural Sciences, Uppsala, Sweden). Helper phage VCSM13 was
purchased from Stratagene. Escherichia coli BL21(DE3) was used for expression
and purification of recombinant ScpB (rScpB) and rScpB-PDF in expression
vector pGEX-4T3. E. coli XL-1 Blue was used as a recipient for cloning the
phagemid library. Allelic replacement experiments with the temperature-sensi-
tive vector pVE6007 were performed with E. coli DH5?. E. coli MC1061 was
used as the host strain for shuttle vector pDC123.
Streptococcal strains were grown in Todd-Hewitt broth at 37°C with aeration,
on Todd-Hewitt agar (THA) plates, or on blood agar plates at 37°C; mutant
TOH97 was grown with the addition of 1 mg of kanamycin (KAN) per ml. E. coli
strains were cultured in 2? YT medium supplemented with 100 ?g of ampicillin
(AMP) per ml (XL-1 Blue/pG3H6 and BL21/pGEX-4T3) or on Luria-Bertani
(LB) agar (DH5?) with 10 ?g of chloramphenicol (CHL) per ml (MC1061/
pDC123) and 50 ?g of XP (5-bromo-4-chloro-3-indolylphosphate) (Sigma) per
ml for blue-white selection.
Construction of scpB mutant TOH97. For allelic replacement mutagenesis of
scpB, the broad-host-range vector pVE6007, which mediates CHL resistance and
replicates at 30°C but not at 37°C, was used. A 2.5-kb fragment of scpB was
amplified from chromosomal DNA of GBS strain 78-471 (7) and cloned directly
into pT7Blue (Novagen). The resultant plasmid was digested with KpnI and
HindIII, and the scpB-containing fragment was cloned into pVE6007 digested
with the same enzymes. A unique BglII site in scpB was used to insert the 2.3-kb
BamHI fragment of pCIV2 containing ?-kan2 (30). The omega-KAN cassette
terminates translation of ScpB at Glu-154. The resulting construct, pTH14, was
transformed into DH5? and grown on selective LB agar plates containing CHL
(5 ?g/ml) and KAN (40 ?g/ml) at 30°C. Plasmid DNA prepared from E. coli
DH5? was then transformed into GBS strain COH1, and transformants were
selected by plating on THA plus CHL (10 ?g/ml) and growing at 30°C. Allelic
exchange mutagenesis was carried out as described by Yim and Rubens (50).
Complementation of scpB mutant TOH97. C5a peptidase was PCR amplified
with the Long Template Expand PCR system (Boehringer Mannheim) by using
the primers CBP6_C5a and CBP17_C5a. The PCR product contained the ribo-
somal binding site and the cell wall-anchoring LPTTND motif inserted into a
T-vector derivative of pDC123 (5). The ligation was used to transform E. coli
MC1061 and selected on solid medium containing 10 ?g of CHL per ml and 50
?g of XP per ml to monitor alkaline phosphatase activity. The resultant comple-
mentation vector, pBEC101, was sequenced, and the integrity and orientation of
the insert were confirmed. Three micrograms of pBEC101 was transformed into
competent scpB mutant TOH97. Competent GBS were derived by the method of
Framson et al. (11). Transformants, designated BEC97, were grown on THA
containing 10 ?g of CHL per ml and 1 mg of KAN per ml.
TABLE 1. Bacterial strains, plasmids, and primers used in this study
Strain, plasmid, or primerGenotype, phenotypea, or sequence Reference or source
S. agalactiae strains
Wild type, serotype Ia
Wild type, serotype III, Tetr
Wild type, serotype II
COH1, scpB::?-Kan2, Tetr, Kanr
TOH97, pBEC101 (scpB?), Tetr, Kanr, Cmr
E. coli strains
XLI-Blue recA1 endA1 gyrA96 thi hsdR17(rK
F?ompT gal (dcm) (lon) hsdSB(rB
supE44 thi-1 recA1 gyrA(Nalr) relA1 ?(lacZYA-argF)169 [?80dlac?(lacZ)M15]
F?araD139 ?(araABC-leu)7696 ?(lac)X74 galU galK hsdR2(rK
?) supE44 relA1 ??lac [F?proABStratagene, La Jolla, Calif.
?) endA1 hsdR17(rK
Gibco BRL, Inc.
Apr, expression vector, 4.9 kb
Apr, phagemid vector, 4.5 kb
ori (Ts), Cmr, temperature-sensitive shuttle vector, 3.4 kb
phoZ, Cmr, shuttle-expression vector with blue-white
Apr, 2.9 kb, T-overhang PCR cloning vector
?-kan2, Kanr, 5.9 kb
pDC123 with scpB, Cmr
5?-GTT ATT ACT CGC GGC CCA-3?
5?-GGC CCC ATT CAG ATC CTC-3?
5?-CAG CTG CAG CAC CAC CAC-3?
5?-GCC CGT TTG ATC TCG AGG T-3?
5?-AGC TAC TAA TCC CAA GAA G-3?
5?-ACC AGG AGA GAA TCG TTT G-3?
5?-CAT GAA AGG ATC CGA CAC ATT GCG-3?
5?-GAT CGT TTC TCT CGA GAG TAC GAG-3?
5?-GAT GGA TCC GTC AAA ACC CTG CAG-3?
5?-GTT CTC GAG ACA CGC ATC AAA AGC A-3?
aKanr, kanamycin resistance; Tetr, tetracycline resistance; Apr, ampicillin resistance; Cmr, chloramphenicol resistance.
2870BECKMANN ET AL.INFECT. IMMUN.
Phenotypic assays. The quantification of type III capsule expression in COH1
and the mutant TOH97 was analyzed as described by Chaffin et al. (6). Beta-
hemolysin and CAMP factor analysis was performed as described by Nizet et al.
Construction of phagemid library. Genomic DNA was isolated from Fn bind-
ing Streptococcus agalactiae strain A909 as described previously (19). The DNA
was sonicated, and fragments of between 100 and 1,000 bp were isolated by
preparative gel electrophoresis, treated with T4 DNA polymerase to generate
blunt ends, and ligated into the SmaI site of the phagemid vector pG3H6.
AMP-resistant transformants were harvested, grown in liquid culture (2? YT) at
37°C, and, in the logarithmic growth phase (optical density at 600 nm of 0.6),
superinfected with 2.5 ? 109of PFU helper phage VCSM13. After 1 h of
incubation at 37°C, the culture was pelleted and resuspended in 2? YT contain-
ing 100 ?g of AMP per ml, 10 ?g of tetracycline per ml, 50 ?g of KAN per ml,
and 1 mM IPTG (isopropyl-?-D-thiogalactopyranoside). Induction of expression
continued overnight at 30°C (phage rescue).
Phage particles were isolated from the supernatants of the expression cultures
by polyethylene glycol precipitation. The phage titer was determined by reinfec-
tion of log-phase E. coli XL1-Blue cells and plating on LB agar containing 100 ?g
of AMP per ml as described by Scott and Smith (34).
Panning of the phagemid library. Seven wells of a 96-well microtiter plate
were coated overnight at 4°C with highly purified human plasma Fn (Sigma) at
a concentration of 5 ?g/well in phosphate-buffered saline (PBS) (150 mM NaCl,
10 mM sodium phosphate, pH 7.2) or with 5% nonfat dry milk in PBS as a
negative control. Each well was blocked with 200 ?l of 5% nonfat dry milk per
ml in PBS for 1 h at room temperature and washed with 200 ?l of PBS plus
0.05% Tween. Fifty microliters of the streptococcal phage display library, con-
taining 5 ? 1010phagemid particles, was added to each well and incubated for 2
to 3 h at room temperature. After intensive washing of the wells with PBS–0.05%
Tween, the bound phage was used to directly infect cells of E. coli XL1-Blue in
the microtiter plate. Therefore, 100 ?l of log-phase E. coli XL1-Blue cells per
well was added to the wells and incubated for 1 h at 37°C. Ten microliters from
each well was used to determine the titer of the bound phage particles by 10-fold
dilutions and plating on LB agar with 100 ?g of AMP per ml. The remaining 90
?l/well was plated on LB agar plus AMP and used for an additional cycle of
phage rescue and panning on Fn.
Sequencing of displayed inserts. Individual clones were isolated after each
panning cycle, and the insert DNA was amplified by standard PCR. Primers used
for amplifying the insert hybridized on the plasmid pG3H6 to a region at the 5?
end of the insert (CBP1_pG3H6) and at the 3? end of the insert (CBP2_pG3H6).
The obtained PCR products were purified with the Qiagen PCR purification kit,
and the DNA was sequenced from both ends by using the Big Dye terminator
sequencing kit (PE Biosystems, Foster City, Calif.) with the primers
CBP3_pG3H6 and CBP4_pG3H6. Analysis of the DNA and protein sequences
was done with the BLAST (Basic Local Alignment Search Tool) programs of the
National Center for Biotechnology Information, National Institutes of Health
(1), to compare the GBS sequences with those in the GenBank database.
Construction and purification of fusion proteins. The scpB gene and the scpB
gene fragment (scpB-PDF) were amplified by PCR from chromosomal DNA of
GBS strain COH1. Primers CBP19_C5a and CBP20_C5a contained BamHI and
XhoI restriction sites for cloning of scpB into the expression vector pGEX-4T3.
Primers CBP25_C5a and CBP26_C5a were used to amplify scpB-PDF and also
contained BamHI and XhoI restriction sites. Amplified products were purified
using the Qiagen PCR purification kit, digested with BamHI and XhoI, and
ligated separately into the BamHI- and XhoI-digested expression vector pGEX-
4T3. The ligated DNA was transformed into E. coli BL21(DE3). Recombinant
clones were analyzed by restriction enzyme analysis. The rScpB used in this study
contains amino acids (aa) 1 to 1090 of the protein and is missing the cell wall
anchor. rScpB-PDF consists of the 112 aa (aa 116 to aa 227) found in the phage
display screen. Production and purification of the two glutathione S-transferase
(GST) fusion proteins from E. coli were performed according to the instructions
of the manufacturer (Amersham Pharmacia), and they were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (12%) followed by Western
blot as described by Laemmli (21). The recombinant fusion proteins were de-
tected with a goat anti-GST antibody (Amersham Pharmacia) followed by a
horseradish peroxidase (HRP)-conjugated rabbit anti-goat immunoglobulin G
(heavy plus light chains) antibody (Pierce). GST protein alone was prepared as
Binding of recombinant proteins to Fn. The binding activities of purified
rScpB and rScpB-PDF to immobilized Fn were measured by enzyme-linked
immunosorbent assay (ELISA). Fn (2 ?g/ml) was applied at 4°C overnight to
96-well U-bottom microtiter plates. After blocking with 5% BSA for 2 h, purified
rScpB, rScpB-PDF, or rGST at various concentrations was added to the wells
and incubated at 37°C for 2 h, and the wells were washed with PBS. A goat
anti-GST antibody was added and allowed to bind for 1 h at room temperature.
The wells were incubated with a secondary HRP-conjugated rabbit anti-goat
immunoglobulin G antibody for 1 h at room temperature. The plate was washed
three times with PBS and developed by adding substrate solution (ortho-phenyl-
diamine) (Sigma) according to the instructions of the supplier. After 15 min the
reaction was stopped by addition of 10% sulfuric acid, and the color development
was measured at 490 nm with an ELISA plate reader (Dynatech MR5000).
Radiolabeling of bacteria and adherence assays. GBS were grown to log phase
and labeled with L-[4,5-3H]leucine (Amersham Pharmacia) as described previ-
The adherence assays with radiolabeled GBS were performed as described by
Tamura and Rubens (43) with the following modifications: Fn (10 ?g/ml) was
applied to U-bottom microtiter plates (Falcon) overnight, and the bacteria re-
mained on the plates for 2 h at 4°C after a centrifugation step. All determinations
were performed in triplicate. To calculate the percent adherence of bacteria to
Fn, the output counts per minute minus background (adherence to uncoated
wells) were divided by the input counts per minute minus the background and
multiplied by 100. CFU per milliliter were determined for each bacterium by
plating dilutions of the radiolabeled bacteria, and the ratio of CFU to counts per
minute was determined to verify comparable uptake of label for the wild-type
and mutant strains in each experiment.
Derivation of a GBS genomic phage display library. The
expression of proteins as fusions with phage envelope proteins,
called phage display, has been shown to be a powerful ap-
proach to identify and isolate peptides with specific binding
properties from genomic libraries (17, 22, 27, 45, 51). To gen-
erate a GBS genomic phage display library, we isolated chro-
mosomal DNA from the GBS type Ia strain A909 and then
sonicated the DNA into 100- to 1,000-bp fragments. These
fragments were cloned into the phagemid vector pG3H6 5? to
the gene encoding the phage coat protein g3p (gene 3 protein).
Since sonication leads to random fragmentation of DNA, in
contrast to restriction enzyme digestion, it ensures the gener-
ation of an almost complete representation of the genome
within the library. DNA fragments which, after insertion, cre-
ate a translational fusion with the g3p lead to the production of
chimeric proteins that are presented on the capsid of the re-
To ensure that the library displays all potential reading
frames, a particular GBS sequence has to be present multiple
times in different lengths and fused in different reading frames.
Our library contained 4 ? 106individual clones with inserts
ranging from 100 to 1.000 bp. Given that GBS has an estimated
genome size of 2.2 Mb, the library represents the GBS genome
at least 200 times.
Affinity selection for clones with Fn binding activity. To
identify phage expressing Fn binding peptides, we affinity
screened the phage library by panning on Fn-coated plates.
Bound phage were subsequently enriched by several cycles of
binding and elution; in each cycle 3.5 ? 1011phagemid parti-
cles were used to ensure that several copies of each specific
phage in the library were present. The phage titer increased
with each cycle of binding, which is indicative of a specific
enrichment. Four cycles of panning on Fn-coated plates re-
sulted in an approximately 1,000-fold enrichment of eluted
phage (Fig. 1). As a negative control, the phage library was also
panned against 5% nonfat dry milk in PBS; 1,000-fold less
binding was observed with this control experiment (data not
shown), confirming that enrichment of the library for Fn bind-
ing phage was specific.
VOL. 70, 2002C5a PEPTIDASE IS A FIBRONECTIN BINDING PROTEIN 2871
Identification of Fn binding phage. To identify the genes
within specific phagemid clones encoding Fn binding activity,
clones from each cycle of panning were isolated, and the
genomic inserts were amplified by PCR and sequenced. A
group of 23 clones did not display any homology to sequences
in the public databases, including the database of unfinished
microbial genomes. Homology to 19 known genes in the public
databases was observed, and 13 of them had homology to
putative or hypothetical genes (Table 2). Several genes were
similar to those from other gram-positive bacteria, such as
streptococci (mainly S. pyogenes) and staphylococci. Interest-
ingly, a gene fragment with low homology to a region of a Fn
binding protein (SFS) from Streptococcus equi (22) was iden-
tified, and it is listed in Table 2 because of its known Fn binding
activity. DNA from clone CB 3-25 displayed high homology
(98%) to a glutamine permease (GlnP) from GBS. glnP is
transcribed in an operon with the glutamine transport gene
glnQ, which is required for maximum Fn binding in GBS (un-
published data). Since studies with Campylobacter jejuni re-
vealed that a GlnP homolog acts as an adhesin (31), these data
provide additional evidence that the GBS GlnP binds Fn.
One of the more interesting clones contained DNA with
significant homology (98% identity over 112 aa) to the GBS
C5a peptidase gene, scpB. The phagemid insert encoding the
putative Fn binding fragment (ScpB-PDF) (Fig. 2) lies in the N
terminus of the protein (aa 116 to 227) and includes two of the
active residues of this serine protease catalytic site, aspartic
acid 130 and histidine 193.
Binding of recombinant C5a peptidase and peptides to Fn.
C5a peptidase (Scp) is a surface-associated peptidase pro-
duced by GAS, GBS, and GGS, which cleaves complement
chemotaxin C5a and is thought to play an important role in
immune evasion by attenuating recruitment of polymorphonu-
clear leukocytes to the site of infection (15). scpB from GBS is
expressed by representative strains from all nine serotypes
(15). The identification of a peptide from ScpB which bound
Fn, as expressed in the GBS phage display library, led to our
hypothesis that ScpB may also serve as a bacterial adhesin.
We further characterized the role of ScpB in Fn binding by
using the type III strain COH1 rather than A909 (type Ia),
from which the phage display library was created, since the
adherence of COH1 to Fn had been fully characterized previ-
ously (43) and GBS strain COH1 was also shown to express
FIG. 1. Enrichment of the phage display library as a consequence
of subsequent cycles of Fn binding (panning). Enrichment is indicated
by the amplification of the number of bound phage with each panning,
which also indicates specific binding. Peptide-carrying phages have
been enriched by a factor of 10 to 100 in each cycle.
TABLE 2. Sequence analysis of GBS DNA isolated from phagemids encoding Fn binding fusion proteins
% Amino acid identity
Genes with known functionCB 5–8
DNA-directed RNA polymerase subunit (S. pyogenes M1)
C5a peptidase (S. agalactiae)
GlnP (S. agalactiae)
Phosphoglycerate kinase (S. pyogenes M1)
ABC transporter (S. pyogenes M1)
Fn binding protein (S. equi)
Genes with hypothetical or
Putative phosphoglucomutase (S. pyogenes M1)
Putative pyruvate formate lyase 2 (S. pyogenes M1)
Putative CTP-synthetase (S. pyogenes M1)
Conserved hypothetical protein (S. pyogenes M1)
Putative aminodeoxychorismate lyase (S. pyogenes M1)
Putative transcription factor (S. pyogenes M1)
Putative phosphotransferase system, enzyme II, A component (S. pyogenes M1)
Conserved hypothetical protein (S. pyogenes M1)
ORFID (SA2098 hypothetical protein similar to glycerate dehydrogenase) (S. aureus)
Putative integral membrane protein (S. pyogenes M1)
Probable tail protein (Streptococcus phage ?01205)
Putative C3-degrading proteinase (S. pyogenes M1)
Hypothetical protein (S. pyogenes M1)
aThe clone number indicates the number of phage rescued and the number of the clone.
bHomology scores with a sum probability of ?e?10were considered significant. Homology scores of ?e?10were classified as having no significant database match.
Only one individual mutant of particular interest with low homology is listed (CB 5-6).
cThe span is the length of the region, in amino acids, to which percent similarity and identity refer. Twenty-three clones showed no homology to any sequence present
in the public database or to any unfinished genome sequence and are not listed.
2872BECKMANN ET AL.INFECT. IMMUN.
ScpB (4). The DNA sequence of the COH1 scpB gene frag-
ment is identical to the one from A909 (data not shown), and
an insertion mutation in COH1 scpB had already been con-
structed (see below).
To generate rScpB and rScpB-PDF encoded by the DNA
insert of the isolated phagemid clone, chromosomal COH1
DNA was amplified by PCR and cloned into the pGEX-4T3
expression vector. These plasmid constructs created GST fu-
sion proteins allowing expression and affinity purification of
each protein with GST-Sepharose. The expected molecular
weight and purity of each recombinant protein were confirmed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and the nature of the fusion proteins was confirmed by West-
ern blotting using an anti-GST antibody (data not shown).
Increasing concentrations of the purified fusion proteins
were incubated on ELISA plates containing 2 ?g of immobi-
lized Fn per ml, and the binding characteristics were quantified
using anti-GST antibody (Fig. 3). Purified rGST proteins
served as a negative control and bound poorly in comparison to
the fusion proteins rScpB and rScpB-PDF. The recombinant
proteins bound to Fn in a concentration-dependent manner,
although adherence of rScpB-PDF was 50% lower than that of
rScpB. These results confirmed the observations from the
phage display library experiments, specifically, that the peptide
spanning the region of ScpB from aa 116 to 227 contains an Fn
binding domain. We next sought to confirm that ScpB could
mediate adherence of GBS bacterial cells to immobilized Fn.
Generation and characterization of an scpB isogenic mu-
tant. We hypothesized that ScpB would mediate adherence of
GBS to immobilized Fn. To address this hypothesis, we utilized
an isogenic mutant of COH1, designated TOH97. This mutant
was made by allelic replacement mutagenesis utilizing the
?-kan2 cassette, which was inserted within the scpB gene at
Glu-154. This insertion truncates the amino-terminal end and
terminates transcription distal to the ?-kan2 insertion site,
including the region encoding the Fn binding peptide de-
scribed above. Southern blot analysis with COH1 genomic
DNA and PCR confirmed that the wild-type gene indeed had
been replaced in TOH97 by scpB::?-kan2 (data not shown).
The absence of C5a peptidase expression by TOH97 was
verified by Western blot analysis of mutanolysin surface ex-
tracts with C5a peptidase antiserum (monoclonal antibody F1)
(kindly provided by J. F. Bohnsack, University of Utah), con-
firming the interruption of the C5a peptidase reading frame
(data not shown). The ?-kan2 cassette contains a transcrip-
tional terminator preventing transcription of downstream
genes. Sequence analysis using the Genetics Computer Group
software package revealed a putative terminator at the 3? end
of scpB. The lmb gene, which codes for the laminin binding
protein, is positioned downstream of scpB and is under the
control of its own promoter (36). Therefore, it was unlikely
that insertion of the ?-kan2 cassette affects Lmb expression.
Furthermore, serotype III strain COH1 has been shown not to
bind to laminin (43).
Further phenotypic analysis of the mutation showed no ef-
FIG. 2. Schematic presentation of ScpB. Shaded area, Fn binding fragment (ScpB-PDF, aa 116 to 227); black area, cell wall anchor motif
LPTTND at the C terminus; S, signal sequence; Asp-130, His-193, and Ser-512, amino acids in the serine protease active site. The insertion site
of the ?-kan2 cassette for construction of the mutant TOH97 is indicated as a large arrowhead above the protein.
FIG. 3. Binding of purified rScpB (I) and rScpB-PDF (}) fusion
proteins and the control rGST protein (F) to immobilized Fn. Fn-
coated microtiter plates were blocked with 5% BSA in PBS, and
various concentrations of recombinant proteins were allowed to bind
for 2 h. Detection of bound proteins was performed with an anti-GST
antibody and a secondary HRP-conjugated anti-goat IgG antibody (see
Materials and Methods). The plates were developed with ortho-phe-
nyldiamine, and color development was analyzed by optical density
(OD) at 490 nm. Points represent means of triplicates, and standard
deviations are indicated. Recombinant proteins bound in a concentra-
VOL. 70, 2002 C5a PEPTIDASE IS A FIBRONECTIN BINDING PROTEIN2873
fect on bacterial growth in Todd-Hewitt broth, RPMI plus 5%
Casamino Acids, or plasma. Colony morphology and expres-
sion of ?-hemolysin, CAMP factor, hippuricase, hyaluronidase,
and caseinase were indistinguishable from those of the wild
type, as were the amounts of type III capsule (6).
Analysis of Fn binding of the mutant. To address whether
C5a peptidase mediates Fn binding of GBS, we compared the
binding of COH1 and TOH97 as described previously (43). All
experiments were performed at 4°C to prevent growth of the
bacteria and to reduce metabolic activity, since bacterial met-
abolic enzymes such as proteases can influence adherence.
In agreement with our previous results (43), the wild-type
strain COH1 adheres to Fn at 30 to 50% of the input inoculum
for Fn concentrations of 20 to 100 ?g/ml (Fig. 4). Coating wells
with Fn concentrations of ?20 ?g/ml did not increase the
binding significantly, whereas adherence dropped to 5 to 10%
at a concentration of 2 ?g/ml. The ScpB?mutant TOH97
showed a 50% decrease in adherence compared to COH1.
Both COH1 and TOH97 showed less than 0.01% adherence to
either uncoated wells or wells coated with BSA, indicating that
the adherence to immobilized Fn was specific. These data
suggest that C5a peptidase contributed significantly to the ad-
herence of GBS. However, since binding was not completely
abolished, the bacteria may produce other Fn binding adhesins.
Complementation of TOH97 with scpB in trans restores Fn
binding. We hypothesized that cloning of the wild-type scpB
gene into the scpB mutant, TOH97, would restore Fn binding.
The recombinant plasmid pBEC101 containing the scpB gene,
including its ribosomal binding site and the carboxy-terminal
cell wall-anchoring LPTTND motif, was transformed into the
mutant TOH97. Clones growing on THA plates with CHL and
KAN were isolated, and the presence and integrity of the
plasmid were confirmed by vector- and insert-specific PCR
amplification and restriction enzyme analysis (data not shown).
Expression of ScpB by pBEC101 on individual transformants
was confirmed by extracting cell surface proteins with mutano-
lysin and subsequent Western blot analysis with anti-C5a pep-
tidase monoclonal antibody F1. All analyzed transformants
containing pBEC101 expressed surface C5a peptidase. Figure
5 shows a representative Fn binding assay comparing one of
the complemented clones, BEC971, to wild-type COH1 and
the mutant TOH97. As expected, TOH97 demonstrated 50%
less binding to Fn than COH1, and BEC971 bound to Fn at
wild-type levels. Binding of all clones containing pBEC101 was
comparable to or better than that of the wild-type strain.
These data demonstrate in trans complementation by the
recombinant scpB gene, restoring Fn binding, and provide fur-
ther evidence that ScpB mediates GBS adherence to Fn.
Attachment of GBS to host cell receptors is hypothesized to
be an important initial step in colonization and subsequent
infection. However, the adhesins contributing to the binding of
epithelial cell and/or host ECM molecules are poorly under-
stood. To date the only characterized adhesin of S. agalactiae is
the laminin binding protein (Lmb) (36), although binding to
laminin is not observed with all strains (43).
Some of the host molecules that serve as substrates for
bacterial binding have recently been identified. They include
cytokeratin 8 (44), laminin (36), and solid-phase (but not sol-
uble) Fn (43).
In this study, we have used genomic peptide libraries dis-
played on the surface of bacteriophage to isolate Fn binding
FIG. 4. Adherence of radiolabeled COH1 and TOH97 to immobi-
lized Fn. Microtiter plates were coated with various concentrations of
Fn, and [3H]leucine-labeled COH1 (I) and TOH97 (}) were added.
Shown is the percentage of adherent bacteria relative to the input.
Points represent means of triplicates, and bars represent the standard
deviation. TOH97 adhered to Fn approximately 50% less than wild-
FIG. 5. Fn binding of radiolabeled COH1 (I), TOH97 (}), and
mutant BEC971 containing the complementation plasmid pBEC101
(Œ). Microtiter plates were coated with four different concentrations of
Fn, and adherence of radiolabeled bacteria was performed as de-
scribed in Materials and Methods. Points represent means of tripli-
cates, and standard deviations are indicated. pBEC101 expressed ScpB
in the complementing strain BEC971 and restored the binding to
immobilized Fn to at least wild-type levels.
2874 BECKMANN ET AL.INFECT. IMMUN.
proteins of GBS. Phage display has been successfully used to
isolate and characterize surface proteins of other gram-positive
organisms, such as S. aureus (17, 51), S. dysgalactiae (45), S.
epidermidis (27), and S. equi (22). One advantage of phage
display compared to other types of expression libraries is that
the screening process is replaced by panning, which allows fast
identification of peptides from a complex library that demon-
strate binding to a desired molecule.
We constructed a phage display library of a type Ia strain of
GBS and isolated those phages which contained GBS peptides
that mediate binding to immobilized Fn (Fig. 1). Some of the
isolated peptides shared homology to other gram-positive pro-
teins which have been previously implicated in adherence and
virulence (Table 2). However, this approach also picks unre-
lated molecules in addition to potentially relevant ones. One
particular clone, though, showed high homology to the C5a
peptidase of S. agalactiae. The scpB gene is 97% identical
between GAS and GBS (7), and furthermore, a homolog has
been identified in GGS (8). C5a peptidase is a serine protease
which cleaves the complement component C5a between His-67
and Lys-68 in the polymorphonuclear leukocyte binding site,
destroying its chemoattractant function. Many GBS express
the surface-associated serine protease, although it is not func-
tional in all strains (4). Due to its ubiquitous expression, Bohn-
sack et al. (4) suggested that C5a peptidase might have a
second, unknown function. Stafslien and Cleary (37) hypothe-
sized a potential role for C5a peptidase from GAS (ScpA) as
an adhesin or invasin, based on its relatively large size and very
limited substrate specificity. For example, the Hap serine pro-
tease in Haemophilus influenzae has been shown to mediate
bacterial adherence to epithelial cells (14, 38), providing a pre-
cedent for a serine protease to exhibit more than one function.
Our in vitro binding studies with the recombinant C5a pep-
tidase protein and the peptide fragment expressed as recom-
binant GST fusion proteins indicated that ScpB does bind to
immobilized Fn. However, rScpB-PDF (peptide fragment) did
not display the same high binding affinity for Fn as the whole
rScpB (Fig. 3). This could be explained by a different protein
conformation of the fragment when fused with GST compared
to the whole protein. Alternatively, it is also possible that a
second binding domain exists, which is absent in the fragment
but confers higher affinity on the entire protein.
Using the C5a peptidase mutant TOH97, we confirmed the
role of this protease in binding Fn. This nonpolar scpB muta-
tion was shown previously to destroy peptidase activity against
C5a (J. F. Bohnsack, personal communication). The location
of the mutation within scpB specifically interrupted the pre-
dicted Fn binding domain. Here we show that this mutant
binds Fn 50% less than the wild-type strain. However, binding
in the presence of recombinant ScpB had no effect on Fn
binding of the wild-type or mutant strain (data not shown). The
mutation had no effect on other phenotypic characteristics
known for GBS, including capsular polysaccharide. Comple-
mentation of the mutation in trans with the wild-type gene
expressed on a plasmid restored Fn binding. Since the scpB
mutation did not completely inhibit adherence to Fn, our re-
sults also suggest that other bacterial factors may be involved
in Fn binding.
Our experiments with truncated C5a peptidase suggest that
a peptide domain (aa 116 to 227) within ScpB mediated bind-
ing to Fn. This sequence is part of the catalytic domain of scpB,
including two of the active residues, Asp-130 and His-193 (Fig.
2). Ser-512 was not found to be part of the Fn binding domain.
Sequence analysis of the C5a peptidase revealed that the pro-
tease contains two amino acid triplet sequences Arg-Gly-Asp
(RGD), one in the N-terminal half and one in the C-terminal
half of the protein. This tripeptide has been shown to mediate
cell adhesion to many proteins and was originally identified in
Fn (32). However, our data show that neither of the RGD
motifs is part of the binding peptide identified by phage dis-
play. This result is in agreement with a previous report by
Tamura and Rubens (43) showing that peptides containing the
sequence RGD do not inhibit the binding of GBS to Fn.
Therefore, adherence of GBS C5a peptidase to Fn must be
mediated by another mechanism which is independent of the
Fn is a large glycoprotein of eukaryotic extracellular matri-
ces and plasma and has been shown to be the binding substrate
for a variety of pathogenic bacteria, such as S. pyogenes (9) and
S. aureus (20). Respiratory epithelial cells, such as A549, pro-
duce Fn on their surface (data not shown), and the lung is one
of the main sites of GBS colonization. If Fn is important for
the binding of GBS to epithelial cells, the ScpB?mutant
TOH97 should display a reduced level of adherence to this cell
line. In preliminary experiments TOH97 demonstrated re-
duced adherence to A549 cells compared to the wild type (data
The results of a study by Cheng et al. (6) show that recom-
binant ScpB binds Fn and may mediate invasion of A549 cells
in part, confirming our results. Taken together, these observa-
tions indicate that GBS adherence to epithelial cells is medi-
ated in part by the interaction of C5a peptidase and Fn.
Based on our observations, we speculate that ScpB may
facilitate S. agalactiae persistence and colonization of epithelial
surfaces, which is a prerequisite for subsequent disease. Hen-
drixson and St. Geme (14) have demonstrated that the Hap
serine protease in H. influenzae promotes bacterial interaction
with epithelial cells and possibly degrades host proteins. Our
report and that of Cheng et al. (6) demonstrate a second
example of a serine protease showing two functions: in this
case, adherence to a host molecule and evasion of the host
immune system. We are continuing to characterize the Fn
binding domain of ScpB and pursuing the role of this protease
in the pathogenesis of GBS infections.
This work was supported by a Feodor-Lynen Fellowship from the
Alexander von Humboldt Foundation (Germany) to C.B. and by the
National Institutes of Health Streptococcal Initiative, grants N01-AI-
75326 and AI-30068 to C.E.R.
We thank Patrick Cleary and Qi Cheng for helpful discussions of the
results and Anne Clancy for critical review of the manuscript.
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Editor: E. I. Tuomanen
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