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1β1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells
1 Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115, USA
2 Department of Medicine, Brigham and Women's Hospital, 75 Fransis Street, Boston, MA 02115, USA
Correspondence
Gilles R. Bolduc
grbolduc{at}comcast.net
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ABSTRACT |
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1β1-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds 1β1-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind 1β1-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.
Present address: Cequent Pharmaceuticals, Inc., One Kendall Square, Building 700, Cambridge, MA 02139, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Manning et al., 2004). The organism colonizes the vagina, rectum and urethra of 10–35 % of adults without causing disease. Neonates acquire GBS either in utero or during birth, aspirating GBS-containing fluid. GBS enters the lungs, binds to and invades the alveolar epithelial cells, and enters the blood, allowing the organisms to infect multiple organs. Invasive disease occurs within hours of birth, with 4–6 % mortality (Baker & Edwards, 1995; Schrag et al., 2000). In a survey in the USA, the incidence of early-onset disease (within the first week of life) declined by 65 % (from 1.7 per 1000 live births to 0.6 per 1000) from 1993 to 1998, due to intrapartum antimicrobial prophylaxis (Schrag et al., 2000) and stabilized at 0.35 per 1000 in 2004 . The incidence of late-onset disease (>1 week to 3 months of age) remained stable, with an average of 0.35 per 1000 live births during the same time period (CDC, 1997, 2005).
GBS was more recently found to be pathogenic in the elderly, and in adults with underlying medical conditions, including diabetes (Dahl et al., 2003; Jackson et al., 1995; Tyrrell et al., 2000). Adult invasive GBS diseases result in bacteraemia, meningitis, skin and soft-tissue infection, and bone and joint infections. The rate of invasive diseases is significantly higher in neonates than in adults. However, the case fatality rates are greater in adults (Schuchat, 1999), with the incidence of GBS disease increasing with advanced age (Farley et al., 1993). Despite the clinical importance of these diseases, little is known about the events that lead up to GBS invasion and its virulence factors. Several studies suggest that surface proteins play a major role in GBS binding to and invasion of human mucosal surfaces (Bulgakova et al., 1986; Tamura et al., 1994). We previously reported that the alpha C protein (ACP) is one such protein (Bolduc et al., 2002).
ACP is the prototype of a family of surface proteins known as alpha-like proteins (Alps) found on 90 % of GBS clinical isolates, as well as other Gram-positive organisms (Lachenauer et al., 2000; Shankar et al., 1999; Stalhammar-Carlemalm et al., 1999; Turner et al., 2003). ACP consists of an N-terminal domain (NtACP; 170 amino acids) followed by a variable number of tandem repeats (82 amino acids each) and a C-terminal domain (45 amino acids) containing an LPXTG peptidoglycan-anchoring motif (Michel et al., 1992). Nearly all strains of serotype Ia, II and most of Ib express ACP on their surface (Madoff et al., 1991). In addition, some human serotype III, IV and NT strains and bovine V, VII and non-typable strains contain the ACP gene (bca) (Creti et al., 2004).
The biological function(s) of the Alp family of proteins remains unclear (Lindahl et al., 2005). Deleting bca attenuates the virulence of GBS in the neonatal mouse model by approximately sevenfold (Li et al., 1997). The lack of ACP expression in vitro reduces the ability of GBS to invade ME180 cells, a human cervical carcinoma epithelial cell line (Bolduc et al., 2002). ME180 cells resemble stratified ectocervical squamous epithelial cells and are an established cellular model for studying group A and B streptococcal binding and invasion (Soriani et al., 2006; Stalhammar-Carlemalm et al., 1999; Sykes et al., 1970). The group A Streptococcus Alp R28 promotes binding to ME180 cells and is essentially identical to GBS Alp3 (Lachenauer et al., 2000). Similarly, adherence of Enterococcus faecalis, lacking expression of the Alp Esp, to urinary epithelium is reduced (Shankar et al., 2001). Together, these data provide strong evidence for the role of Alps in the adherence to and invasion of epithelial cells by Gram-positive micro-organisms.
We solved the tertiary structure of NtACP by X-ray crystallography (Auperin et al., 2005). NtACP can be further divided into two structurally distinct domains, D1 and D2. D1, the more distal (amino-terminal) portion, consists of a beta sandwich with strong structural homology to fibronectin's integrin-binding region (FnIII10). D2 consists of three antiparallel alpha helix coils containing a portion of the glycosaminoglycan (GAG)-binding domain. A partial putative GAG-binding site was mapped within domain 2 (D2), adjacent to the repeat region, consistent with our data showing that NtACP binds to heparin and GAGs only when it is covalently associated with the adjacent repeat region (Baron et al., 2004). However, exogenous soluble NtACP alone (without the repeat region) can still competitively bind to ME180 cells and reduce internalization and translocation of GBS (Bolduc et al., 2002), suggesting that NtACP binds to another non-GAG receptor. The structural data also revealed a potential integrin-binding site located on the most distal amino-terminal portion of NtACP (domain 1; D1), opposite the GAG-binding site on D2. We sought to determine if ACP binds to integrins present on the surface of ME180 cells and, if so, to identify the specific intergrin heterodimeric molecules. Secondly, we tested the hypothesis that the binding of ACP's D1 domain to integrin plays a role in GBS invasion.
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+, β+) strain A909 and the ACP-deficient mutant JL2053 have been described (Li et al., 1997). A909 was obtained from Dr Rebecca Lancefield (Lancefield et al., 1975). JL2053 was obtained from Dr Jing Li. ME180 cells (HTB33) (Sykes et al., 1970) were purchased from the American Type Culture Collection (Manassas, VA, USA).
Cloning and site-directed mutagenesis of ACP-specific proteins.
The amino acid sequence of ACP is in the NCBI Protein Database under accession number AAA26848 (Auperin et al., 2005; Michel et al., 1992). The DNA sequence encoding the ACP (pCL1), NtACP (pDEK14), repeat region RR' (pET24RR') and D2R has been cloned previously (Auperin et al., 2005; Bolduc et al., 2002; Gravekamp et al., 1996; Kling et al., 1997). The aspartic acid residue (Asp146) of the recombinant NtACP was substituted with an alanine residue (D146A) using the Quik-Change site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Forward primer 5'-CCA CAT GTA AAG ACT GCT GGA CAA ATT GAT-3' and reverse primer 5'-ATC AAT TTG TCC AGC AGT CTT TAC ATG TGG-3' and the pDEK14 template were used, resulting in the pGB3 construct. The underlined nucleotides indicate the mutated codon.
The DNA sequence encoding the D2 region (Ser161–Leu225) was PCR amplified from pDEK14 using forward primer 5'-C GCT AGC ACA ACC TTG AGG GAT AAG ATT GAA-3' and reverse primer 5'-G GAA TTC TTA CAA TAC TAA CAA TTT CTC TAA TTC ATT AAC CTC-3'. The 209 bp PCR product was cloned into pCR2.1-TOPO, transformed into Top10 cells (Invitrogen), excised with NheI and EcoRI, and recloned into pTrcHisA. The resulting construct is pGB4. The DNA sequence encoding the Inv497 protein was PCR amplified from pRI203 (Hamburger et al., 1999) using forward primer 5'-C GCT AGC AGC GTC ACC GTT CAG CAG CC-3' and reverse primer 5'-C CGA ATT CTT ATA TTG ACA GCG C-3'. The 1494 bp PCR product was cloned into pCR2.1-TOPO and transformed into TOP10 cells. The plasmid was isolated and digested with NheI, EcoRI and SphI. The inv497 NheI/EcoRI fragment was gel purified and cloned into pTrcHisC. The resulting construct pGB5 was transformed into Escherichia coli strain BLR.
Protein expression and purification.
Plasmids pET200/D-TOPO : : D2-R, pDEK14 and pGB3 were transformed into E. coli BL21(DE3). Plasmids pGB4, pGB5 and pCL1 were transformed into E. coli BLR. Plasmid pET24RR' was transformed into BL21(DE3). Expression and purification of each protein was performed as previously described (Bolduc et al., 2002). Briefly, protein expression was induced with 1 mM IPTG. The cells were harvested, lysed and the lysates loaded onto an 18 ml Fractogel (M) (Novagen) column charged with 50 mM NiSO4. Fractions containing recombinant protein were pooled and dialysed in 20 mM HEPES, pH 7.2.
ME180-binding assay.
ME180 cells were grown to confluence in 96-well titre plates (Falcon Microtest 96 353072) in RPMI 1640 with L-glutamine containing 10 % Newborn Bovine Serum (26010-074; Invitrogen), 100 U penicillin ml–1 and 100 µg streptomycin ml–1 (15140-122; Invitrogen) at 37 °C with 5 % CO2. Purified ACP, NtACP, NtACP D146A, D2-R and RR' were biotin-labelled using the EZ-Link Sulfo-NHS-Biotinylation kit (Pierce). The moles of biotin per mol protein were determined by the automatic HABA calculator available on the Pierce website (http://www.piercenet.com). The proteins were incubated with the cells for 4 h at 37 °C with 5 % CO2. Unbound proteins were removed by gently washing three times with Dulbecco's phosphate-buffered saline (PBS), pH 7.1 (Gibco) containing 0.1 mM CaCl2 (PBS+CaCl2). The cells were fixed with 2 % paraformaldehyde in PBS+CaCl2 and incubated in 0.5 % BSA in PBS+CaCl2 for 18 h at 4 °C. Bound proteins were detected with avidin–peroxidase conjugate (1 : 100 000; Sigma) in 0.5 % BSA in PBS+CaCl2 and developed with o-phenylenediamine tablets (Sigma P-6662) dissolved in phosphate/citrate buffer. The absorbance was read at 490 nm and 650 nm and recorded as 
A. The molar ratios of biotin to ACP, NtACP, NTACP D146A, D2-R, and RR' were 2 : 1, 1 : 1, 1 : 1, 1 : 1 and 1 : 1, respectively. Therefore, the values obtained for ACP were divided by 2 to compare with the values obtained for the other proteins.
Inhibition assays were performed similarly with the exception that 0–500 µM unlabelled NtACP, NtACP D146A and RR' was added to the wells containing 20 µM biotinylated NtACP. The percentages of inhibition of bound biotinylated NtACP were calculated with the formula [(uninhibited A – inhibited A)/unihibited A]x100. Statistical analysis was performed using the paired t-test (http://www.graphpad.com/quickcalcs/ttest1.cfm). A P value equal to or less than 0.050 is considered statistically significant.
1β1-Integrin-binding assay.
Purified human 1β1-integrin (5 µg ml–1; Chemicon International) in 50 mM carbonate/biscarbonate buffer, pH 9.6, was allowed to bind to the wells of 96-well titre plates (NUNC flat-bottom MicroWell plate, MaxiSorp surface-treated) during an incubation of 2 h at 37 °C. The wells were blocked with 2 % BSA in 50 mM Tris/HCl pH 7.4, 150 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 0.1 % Triton X-100 overnight at 4 °C. Antibodies to human 1- or β1-integrin subunits were added to the wells along with 0–20 µM of NtACP, NtACP D146a mutant, RR' or BSA, in 2 % BSA in 50 mM Tris/HCl pH 7.4, 150 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 0.1 % Triton X-100 to allow binding to the immobilized 1β1-integrin for 1 h at room temperature. Antibodies used were: 1 : 400 dilution of rabbit anti-human 1-integrin antibodies (AB1934; Chemicon International), 1 : 400 dilution of rabbit anti-human β1-integrin antibodies (AB1952; Chemicon International) and 1 : 500 dilution of mouse anti-human 1-integrin I-domain monoclonal antibody (MAB1973Z; Chemicon International). The wells were washed with 50 mM Tris/HCl pH 7.4, 150 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 0.1 % Triton X-100 to remove unbound ACP and antibodies. Binding of rabbit polyclonal antibodies was detected by adding a 1 : 500 dilution of anti-rabbit IgG–peroxidase conjugated antibody (A6154; Sigma) followed by OPD substrate (P6662; Sigma). Absorbance was recorded at 490 nm and 650 nm. The results are reported as A.
Confocal microscopy.
ME180 cells were grown in 0.5 ml RPMI medium 1640 with L-glutamine, 10 % fetal calf serum, 25 mM HEPES, 0.4 M CaCl2 on a 12 mm diameter transwell-COL (3.0 µm pore size, Costar) membrane suspended in wells of a 12-well plate containing 1.5 ml of similar medium, as previously described (Bolduc et al., 2002). ACP, BSA and Inv497 (Hamburger et al., 1999) were labelled with AlexaFluor 568 using the AlexaFluor 568 Protein Labelling kit (Molecular Probes; A-10238). Aliquots of 0.25 ml of 1 µM labelled proteins were added to the apical side of the cells for 4 h at 37 °C with 5 % CO2. Unbound protein was removed by gently washing the cells three times with PBS. The cells were fixed by suspending the transwells in 2 % paraformaldehyde at 4 °C for 16 h. The membrane inserts were removed from the transwell and washed twice with PBS, cells facing up on a glass slide. The cells were permeated by incubating the membrane in 200 µl of 0.1 % Triton X-100 in PBS for 10 min at 22 °C. The cells were washed once in PBS and incubated in 0.5 % BSA in PBS for 30 min at 22 °C to block non-specific binding. Primary antibodies to -integrins (Chemicon International; ECM445) and β-integrins (Chemicon International; ECM440) were diluted 1 : 500 in 0.5 % BSA in PBS. MAB1951Z (mouse anti-human integrin β1) was diluted in 0.5 % BSA in PBS containing 1 M CaCl2, as its epitope binding is Ca2+-dependent; 200 µl of the antibodies was added to the cells for 1 h at 22 °C. The cells were washed three times with PBS containing 1 M CaCl2. AlexaFluor 488 donkey anti-mouse IgG (H+L) (Molecular Probes; A-21202) or AlexaFluor 488 donkey anti-rabbit IgG (H+L) (Molecular Probes; A-21206) was diluted 1 : 200 in 0.5 % BSA in PBS containing 1 M CaCl2 and added to the cells for 1 h at 22 °C. AlexaFluor 568 goat anti-rabbit IgG (H+L) (Molecular Probes; A-11011), diluted 1 : 200, was used to stain -integrins when determining colocalization of subunits with β1-integrin. Rabbit anti-integrin alpha-1 polyclonal antibody (Chemicon International; AB1934, 1 : 500 dil.) and AlexaFluor 660 goat anti-rabbit IgG (H+L) (Molecular Probes; A-21073, 1 : 500 dil.) were used to stain 1-integrin when determining colocalization of 1β1-integrin with ACP. The cells were washed three times in PBS containing 1 M CaCl2. A drop of Fluorotec (Accurate Chemical) was added to the cells before they were covered with a cover glass. The edges were sealed with clear nail varnish.
Internalization assay.
The assay was performed as previously described (Bolduc et al., 2002). Briefly, ME180 cells were grown to confluence in 24-well culture plates containing 1 ml RPMI medium (RPMI 1640 with L-glutamine, 10 % fetal calf serum, 25 mM HEPES). On the day of the assay, the medium was removed from the wells and 0.4 ml fresh RPMI medium was added to the cells. Soluble proteins (0.1 ml of NtACP, NtACP D146A, D2, D2-R, RR' and BSA) were added to the cells 1 h before adding 0.5 ml of 2x106 c.f.u. ml–1 of A909 and JL2503 (m.o.i.=5 bacteria cell–1). The cells were incubated at 37 °C with 5 % CO2 for 2 h. The monolayers were washed three times with PBS before adding 1 ml RPMI medium containing 100 µg gentamicin ml–1 and 5 µg penicillin G ml–1 per well for 2 h at 37 °C with 5 % CO2. The cells were then washed with PBS, lysed in 0.4 ml of 0.025 % Triton X-100, and serial dilutions of the epithelial lysates plated on THA plates. The percentage of internalized bacteria was calculated as [(c.f.u. per well after antibiotic treatment)/(c.f.u. originally added to the well)]x100. Percentage inhibition was calculated as [(c.f.u. of internalized GBS)/(c.f.u. of original inoculum)]/[(c.f.u. of internalized GBS uninhibited)/(c.f.u. of original inoculum of GBS uninhibited)]x100. Statistical analysis was performed using the unpaired t-test (http://www.graphpad.com/quickcalcs/ttest1.cfm). A P value equal to or less than 0.050 is considered statistically significant.
Translocation inhibition assay.
The assay was performed as previously described with the additional step of preincubating the cell membranes with soluble protein before adding GBS (Bolduc et al., 2002). Before GBS strain A909 or JL2053 was added to the cells, the medium was removed from the top chamber and replaced with 0.1 ml of 1, 5, 10, or 20 µM of repeat protein (RR'), NtACP, ACP, or BSA in RPMI medium 1640 with L-glutamine, 10 % fetal calf serum, 25 mM HEPES, 0.4 M CaCl2. The cells were incubated at 37 °C for 1 h. Without removing the soluble protein, 0.4 ml of approximately 2x105 c.f.u. ml–1 of GBS strain A909 or JL2053 was added to the top chamber, and the cells were incubated for up to 4 h. E. coli DH5 served as a non-invasive control.
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) were previously determined to bind qualitatively to ME180 by confocal microscopy and flow cytometry (Baron et al., 2004; Bolduc et al., 2002). In the current study, the proteins were biotin-labelled and quantitatively assessed for their ability to bind ME180 cells in an ELISA-based binding assay. The labelled proteins were allowed to interact with the cells for 4 h. The cells were then washed with saline to remove unbound protein, and the remaining proteins were detected with avidin conjugated to horseradish peroxidase. ACP and NtACP bound similarly to ME180 cells, whereas NtACP D146A and D2-R demonstrated less binding. RR' did not bind (Fig. 1b), suggesting that the observed binding of ACP to ME180 cells is through the NtACP region.
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). Unable to express the isolated D1 region in a sufficient quantity, we chose to mutate D1 in an NtACP background and compare its ability to bind ME180 cells with that of wild-type NtACP. The integrin-binding activity of type III fibronectin (FnIII) requires the presence of the Arg1493-Gly1494-Asp1495 (RGD) motif on FnIII module 10 (FnIII10) and Arg1379 and Asp1373 on FnIII module 9 (FnIII9) (Hamburger et al., 1999). A structurally similar triad of charged residues required for integrin binding is present in Yersinia pseudotuberculosis Inv497. Inv497 is a non-RGD protein that competes with FnIII by binding to 5β1-integrin (Isberg et al., 2000). NtACP's D1 region contains a K144-T145-D146 (KTD) motif, located within a loop region that is structurally analogous to the loop containing the RGD integrin-binding motif in FnIII10. D1 also contains residues Arg110 and Asp118, forming a triangular arrangement of charged residues similar to those found in fibronectin and Y. pseudotuberculosis Inv497. If NtACP binds integrin, we hypothesize that a (KT)D146A substitution (NtACP D146A) may reduce its ability to bind ME180 cells.
Biotinylated NtACP along with either unlabelled NtACP or NtACP D146A was added to the cells for 4 h. We hypothesize that the labelling of NtACP with biotin may have increased the protein's ability to bind ME180 cells, since it required 25-fold more unlabelled NtACP to inhibit binding of biotin-labelled NtACP by 60 %. Nevertheless, NtACP D146A inhibited biotin-labelled NtACP binding significantly less than NtACP (P=0.0152 at 350 µM, P=0.0211 at 500 µM) (Fig. 2). Purified D2 was also tested for its ability to inhibit NtACP binding to ME180 cells; the inhibition was substantially less than that seen with NtACP (P=0.0204 at 250 µM, P=0.0054 at 500 µM). The data suggest that NtACP binds to an ME180 surface receptor via D1. The loss of inhibition due to a single mutation in the hypothetical integrin-binding KTD motif suggests that the receptor is an integrin heterodimer.
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1β1-integrin- and β-integrin subunits were probed with antibodies specific to the subunits, followed with AlexaFluor 488-labelled secondary antibodies, and examined by confocal microscopy. The cells stained with equally strong intensity for β1 and β2 subunits (Fig. 3a). Staining for β3, β4 and β5 subunits was near background levels and not detectable. ME180 cells also stained with strong positive intensity for 1, 2 and 3 subunits, but not for 4, 5 and V subunits (Fig. 3b).
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3β1-integrin and was included as a control (Van Nhieu & Isberg, 1991). Both ACP and Inv497 colocalized with β1 but not with β2 subunits (Fig. 4a, b). Interestingly, the cells with the most intense staining for ACP also demonstrated the highest fluorescent staining for the β1 subunit, with little to no staining for the β2 subunit. One of two possible explanations is that the interaction between ACP and ME180 cells results in a clustering of β1 subunits. The other possibility is that, upon interaction with ACP, β1 expression increases while β2 expression decreases. AlexaFluor 568-labelled BSA served as a negative control (Fig. 4c). ACP also colocalized with the 1 subunit (Fig. 4d) and not with 2 or 3 subunits. The 1 and β1 subunits colocalize with ACP on ME180 cells (Fig. 4e), identifying the 1β1-integrin heterodimer known as the very late antigen 1 (VLA1; CD49a/CD29; laminin and collagen receptor) (Hemler et al., 1986).
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1β1-integrin was measured by ELISA. Purified 1β1-integrin was used to coat the wells of a 96-well microtitre plate. Mono- and polyclonal antibodies to the 1 subunit were added to the wells containing various concentrations of the proteins. The proteins' abilities to inhibit binding of the antiserum were recorded as percentage inhibition. NtACP inhibited the 1 subunit polyclonal antibodies from binding, while NtACP D146A showed significantly less inhibition (P=0.0140 at 5 µM, P=0.0042 at 10 µM and P=0.0243 at 20 µM) (Fig. 5), suggesting that NtACP binding to 1β1-integrin requires the intact KTD motif. RR', D2-R and BSA showed no significant difference in inhibition (P>0.1) from that observed with NtACP D146A. The monoclonal antibody MAB1973 recognizes an epitope located in the 1-integrin I domain (Fabbri et al., 1996). It was included in the assay since it is known to inhibit binding of 1-integrin to laminin and collagen. MAB1973 did not affect ACP binding to 1β1-integrin. Similar assays were performed using polyclonal antibodies to the β1 subunits, but no inhibition was observed (data not shown). NtACP may bind to an epitope(s) on β1 that is not detectable with the polyclonal antibodies used, or it may bind only to the 1 subunit.
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). In addition, structural data suggest that NtACP may bind integrin via a KTD motif in the D1 domain (Auperin et al., 2005). Based on these results and the data presented above, we hypothesize that soluble NtACP may compete for binding to host cell 1β1-integrin, thus preventing GBS-associated ACP from binding and inhibiting its contribution to the internalization of GBS. Therefore, in the current study we re-examined the internalization of A909 and JL2053 within ME180 cells in the presence of soluble NtACP D146A, D2 and D2-R to isolate the region within NtACP needed for maximal internalization.
Confluent ME180 cells were incubated with the soluble proteins for 2 h before GBS was added and the cells were allowed to internalize the bacteria. Both NtACP and NtACP D146A reduced A909 internalization, but did not affect the internalization of JL2053 (Fig. 6a). Pretreating cells with 20 µM NtACP reduced the internalization of A909 by 82 % (5.55 times less) compared to that observed with non-treated cells. The single mutation within the KTD motif significantly attenuated NtACP's ability to inhibit A909 internalization in a dose-dependent manner. Cells treated with 20 µM NtACP D146A internalized 61 % less GBS than the non-treated cells.
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). In addition to FnIII10's RGD integrin-binding motif, FnIII requires two charged residues (Arg1379 and Asp1373) located on module 9 (FnIII9) for maximal integrin binding (Hamburger et al., 1999). D1 residues (Arg110 and Asp118) occupy analogous positions in relation to the KTD motif, suggesting that these residues may also play a direct role in binding ACP to 1β1-integrin. Therefore, mutations within all three residues KTD146, Arg110 and Asp118 may be required to completely prevent the competitive binding of exogenous NtACP and the subsequent entry of wild-type GBS into epithelial cells. The data support the hypothesis that the D1 domain of GBS-associated ACP is required for full GBS internalization into ME180 cells.
The addition of D2 (Fig. 6b) and D2-R (Fig. 6c) resulted in an increase in the percentage of internalized A909 (147.2±7.8 and 158.3±27.3 %, respectively). A 30.3 % increase in internalized ACP-deficient mutant JL2053 was also observed following the addition of D2-R to the cells. The causes for these observed increases are not understood. We previously demonstrated that GAGs bind D2-R (Auperin et al., 2005). It is possible that D2-R binding to GAGs expressed on the surface of ME180 cells acts as a bridge to stabilize GBS binding and its subsequent internalization within ME180 cells. Alternatively, it is possible that the D2 domain stimulates the cells via an unknown mechanism resulting in the observed increase of GBS internalization.
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DISCUSSION |
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). Higher frequency is seen among sexually active individuals (Manning et al., 2004; Meyn et al., 2002). GBS colonizing these mucosal sites can traverse the mucosal barriers, resulting in invasive disease. GBS adhesion is required for colonization and subsequent invasion. Several GBS surface proteins bind host extracellular matrix components and serum proteins (Beckmann et al., 2002; Gutekunst et al., 2004; Schubert et al., 2002; Spellerberg et al., 1999). ACP, a virulence factor and prototype for the Alp family of proteins, is the first surface protein identified to bind integral host cell-surface receptors, GAGs and 1β1-integrin. Structural analysis of the GBS C5a peptidase (SCPB) suggests that it too may bind integrins via the RGD motif (Brown et al., 2005), but this has not been demonstrated experimentally.
GBS invasion of epithelial membranes involves internalization within membrane-bound vacuoles and paracellular translocation (Soriani et al., 2006; Valentin-Weigand et al., 1997). GBS internalization, but not translocation, requires actin polymerization and Rho GTPase activity (Baron et al., 2004; Soriani et al., 2006). Capsular expression also attenuates GBS internalization but not translocation (Gibson et al., 1993; Soriani et al., 2006). The different requirements for the two biological events suggest that they may be independent of each other. In contrast, expression of ACP appears to play a role during both events.
We initially reported that ACP binds heparin and epithelial cell-associated GAGs (Baron et al., 2004). NtACP and the repeat region separately do not bind heparin. However, the D2-R peptide consisting of NtACP's D2 domain covalently associated with a single repeat binds heparin, suggesting that the heparin- and host GAG-binding domain is located within the junction between the D2 domain and the adjacent repeat. The NtACP tertiary structure maps a portion of the putative heparin-binding domain to BR2, a positively charged cluster within the D2 domain, adjacent to the first repeat region (Auperin et al., 2005). We now report that ACP binds to a second cell-surface receptor, 1β1-integrin, via the D1 domain located on the opposite side of the molecule from the putative GAG-binding domain. The 1β1-integrin is one of four collagen-binding I-domain-containing integrins (Gullberg & Lundgren-Akerlund, 2002). It preferentially binds to collagen IV but also binds collagen I and laminin via the I domain (Calderwood et al., 1997; Riikonen et al., 1995). The monoclonal antibody MAB1973 recognizes an epitope specific to the human 1-integrin I domain and inhibits binding of activated human lymphocytes to these extracellular matrix components (Fabbri et al., 1996). MAB1973 does not affect ACP binding to 1β1-integrin. However, the presence of a polyclonal antibody to 1-integrin does reduce ACP binding to 1β1-integrin by 42 %, suggesting that ACP binding to 1-integrin differs from that of collagen and laminin. One possible explanation is that ACP binds to 1-integrin via a unique non-I-domain interaction.
Proteins of other micro-organisms have similarly been reported to bind GAGs and integrin as co-receptors. For example, fibronectin acts as a bridge binding OpA, a Neisseria gonorrhoeae outer-membrane protein, to 5β1-integrin and is essential for N. gonorrhoeae internalization (van Putten et al., 1998). GAG forms a complex with fibronectin and integrin for maximal internalization. The Borrelia burgdorferi surface lipoprotein BBK32 also binds fibronectin and GAG (Fischer et al., 2006). ACP differs such that it binds directly to the integrin molecule without the use of a bridge. Pretreating ME180 cells with sodium chlorate, an inhibitor of sulfate incorporation, or with heparinase inhibits binding of ACP to the cells by 84–98 % (Baron et al., 2004). We currently report that a single mutation within the KTD motif (D146A), present in the D1 domain, reduces NtACP binding to 1β1 and ME180 by 20–25 %. Therefore, we sought to determine the contribution of integrin binding to GBS internalization within host cells.
Mutagenesis of NtACP, generating a KTD146A single mutation within the putative KTD integrin-binding motif, reduced its ability to inhibit GBS internalization. In similar assays where ME180 cells were preincubated with D2-R, containing the putative GAG-binding domain, internalization was not diminished, but instead increased. This effect does not require the expression of ACP on GBS. The RR' peptide had no measurable effect on GBS internalization. The data suggest that binding to both GAG and integrin may be needed for maximal binding of ACP to ME180 cells. However, the direct interaction between D1 of ACP and 1β1-integrin plays a key role in GBS internalization.
Structural analysis of the D1 domain, in particular the region containing the putative integrin-binding loop and KTD motif, shares a strong structural homology with the FnIII10's integrin-binding region. Amino acid sequence alignment of Alps indicates that KTD is highly conserved (Auperin et al., 2005; Creti et al., 2004), suggesting biological importance. The RGD motif present on FnIII10 interacts directly with integrin and is the canonical integrin-binding motif. Sixteen other motifs, not including KTD, were identified on short disintegrins (Sanz et al., 2006). Each motif binds to a specific integrin heterodimer. The first two residues of the triads dictate the specificity of the subunit (Sanz et al., 2006). The motif KTS is expressed by obtustatin and viperistatin and binds 1β1, in a I-domain-independent manner, similar to ACP (Marcinkiewicz et al., 2003). We show that ACP interacts with the 1 subunit of 1β1-integrins. Also, the KT(D146A) substitution reduces the binding of NtACP to 1β1-integrins. ACP did not alter binding of a polyclonal antibody specific to the β1 subunit in similar experiments (data not shown). ACP may alter binding of a different source of poly- or monoclonal antibodies to β1-integrin. Therefore, we cannot rule out the possibility that ACP also binds to the β1 subunit.
The 1β1-integrin is mainly expressed by mesenchymal cells and activated T lymphocytes and monocytes (Ben-Horin & Bank, 2004; Duband et al., 1992; Hemler, 1990; Rubio et al., 1995). However, it is also detected on epithelial and endothelial cells (Atsuta et al., 1997; Miettinen et al., 1993). Integrins in general can be involved in cell migration, tissue morphogenesis and immune response. The 1β1-integrin plays a role in chronic inflammation, fibrosis and angiogenesis (Cosgrove et al., 2000; de Fougerolles et al., 2000; Senger et al., 1997). It remains to be determined what biological effects GBS may have on the host, when binding to 1β1. The current study suggests that ACP binding to 1β1-integrin leads to the clustering of β1-integrin subunits. Prior to adding ACP to the cells, β1 and β2 subunits are equally dispersed on the surface of the ME180 cells. Following ACP treatment, the distribution of the subunits is altered. The two subunits segregate, resulting in the colocalization of the β1 subunits with ACP. Clustering activates integrins, which form focal adhesions, and in turn activate a cascade of downstream intracellular signal pathways by a process known as ‘outside-in’ signalling. The cytoplasmic tails of the integrin subunits interact with cytoplasmic proteins, causing cytoskeletal rearrangements, phosphorylation and/or induction of gene expression (Loster et al., 2001). Active 1β1-integrins may phosphorylate and activate focal adhesion kinase (FAK), resulting in the phosphorylation of other proteins necessary in signal transduction. FAK is capable of binding to the cytoplasmic tail of the 1 subunit of 1β1-integrin.
ACP may also bind other integrins. The effect could broaden GBS tissue tropism and/or effects on cellular pathways. ME180 cells express β2-integrin subunits, to which ACP does not appear to bind. However, GBS can induce tumour necrosis factor alpha (TNF-) release from monocytes and macrophages, enhancing CD11b/CD18 (Mβ2-integrin)-dependent cell activation (Levy et al., 2003). Therefore, further studies of β2-integrin may reveal its possible relation to GBS disease. Structural analysis of SCPB suggests that it binds integrin via one or more RGD motifs (Brown et al., 2005), which would give GBS the ability to bind other integrin heterodimers and possibly activate multiple signal transduction pathways.
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ACKNOWLEDGEMENTS |
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Edited by: T. Msadek
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REFERENCES |
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Atsuta, J., Sterbinsky, S. A., Plitt, J., Schwiebert, L. M., Bochner, B. S. & Schleimer, R. P. (1997). Phenotyping and cytokine regulation of the BEAS-2B human bronchial epithelial cell: demonstration of inducible expression of the adhesion molecules VCAM-1 and ICAM-1. Am J Respir Cell Mol Biol 17, 571–582.
Auperin, T. C., Bolduc, G. R., Baron, M. J., Heroux, A., Filman, D. J., Madoff, L. C. & Hogle, J. M. (2005). Crystal structure of the N-terminal domain of the group B Streptococcus alpha C protein. J Biol Chem 280, 18245–18252.
Baker, C. J. & Edwards, M. S. (1995). Group B streptococcal infections. In Infectious Diseases of the Fetus and Newborn Infant, pp. 980–1054. Edited by J. Remington & J. O. Klein. Philadelphia: W. B. Saunders.
Baron, M. J., Bolduc, G. R., Goldberg, M. B., Auperin, T. C. & Madoff, L. C. (2004). Alpha C protein of group B Streptococcus binds host cell surface glycosaminoglycan and enters cells by an actin-dependent mechanism. J Biol Chem 279, 24714–24723.
Beckmann, C., Waggoner, J. D., Harris, T. O., Tamura, G. S. & Rubens, C. E. (2002). Identification of novel adhesins from Group B streptococci by use of phage display reveals that C5a peptidase mediates fibronectin binding. Infect Immun 70, 2869–2876.
Ben-Horin, S. & Bank, I. (2004). The role of very late antigen-1 in immune-mediated inflammation. Clin Immunol 113, 119–129.[CrossRef][Medline]
Bolduc, G. R., Baron, M. J., Gravekamp, C., Lachenauer, C. S. & Madoff, L. C. (2002). The alpha C protein mediates internalization of group B Streptococcus within human cervical epithelial cells. Cell Microbiol 4, 751–758.[CrossRef][Medline]
Brown, C. K., Gu, Z. Y., Matsuka, Y. V., Purushothaman, S. S., Winter, L. A., Cleary, P. P., Olmsted, S. B., Ohlendorf, D. H. & Earhart, C. A. (2005). Structure of the streptococcal cell wall C5a peptidase. Proc Natl Acad Sci U S A 102, 18391–18396.
Bulgakova, T. N., Grabovskaya, K. B., Ryc, M. & Jelinkova, J. (1986). The adhesin structures involved in the adherence of group B streptococci to human vaginal cells. Folia Microbiol (Praha) 31, 394–401.[Medline]
Calderwood, D. A., Tuckwell, D. S., Eble, J., Kuhn, K. & Humphries, M. J. (1997). The integrin alpha1 A-domain is a ligand binding site for collagens and laminin. J Biol Chem 272, 12311–12317.
CDC (1997). Decreasing incidence of perinatal Group B streptococcal disease – United States, 1993–1995. MMWR Morb Mortal Wkly Rep 46, 473–477.[Medline]
CDC (2005). Early-onset and late-onset neonatal group B streptococcal disease – United States, 1996–2004. MMWR Morb Mortal Wkly Rep 54, 1205–1208.[Medline]
Cosgrove, D., Rodgers, K., Meehan, D., Miller, C., Bovard, K., Gilroy, A., Gardner, H., Kotelianski, V., Gotwals, P. & other authors (2000). Integrin alpha1beta1 and transforming growth factor-beta1 play distinct roles in alport glomerular pathogenesis and serve as dual targets for metabolic therapy. Am J Pathol 157, 1649–1659.
Creti, R., Fabretti, F., Orefici, G. & von Hunolstein, C. (2004). Multiplex PCR assay for direct identification of group B streptococcal alpha-protein-like protein genes. J Clin Microbiol 42, 1326–1329.
Dahl, M. S., Tessin, I. & Trollfors, B. (2003). Invasive group B streptococcal infections in Sweden: incidence, predisposing factors and prognosis. Int J Infect Dis 7, 113–119.[CrossRef][Medline]
de Fougerolles, A. R., Chi-Rosso, G., Bajardi, A., Gotwals, P., Green, C. D. & Koteliansky, V. E. (2000). Global expression analysis of extracellular matrix-integrin interactions in monocytes. Immunity 13, 749–758.[CrossRef][Medline]
Duband, J. L., Belkin, A. M., Syfrig, J., Thiery, J. P. & Koteliansky, V. E. (1992). Expression of alpha 1 integrin, a laminin-collagen receptor, during myogenesis and neurogenesis in the avian embryo. Development 116, 585–600.[Abstract]
Fabbri, M., Castellani, P., Gotwals, P. J., Kotelianski, V., Zardi, L. & Zocchi, M. R. (1996). A functional monoclonal antibody recognizing the human alpha 1-integrin I-domain. Tissue Antigens 48, 47–51.[Medline]
Farley, M. M., Harvey, R. C., Stull, T., Smith, J. D., Schuchat, A., Wenger, J. D. & Stephens, D. S. (1993). A population-based assessment of invasive disease due to group B Streptococcus in nonpregnant adults. N Engl J Med 328, 1807–1811.
Fischer, J. R., LeBlanc, K. T. & Leong, J. M. (2006). Fibronectin binding protein BBK32 of the Lyme disease spirochete promotes bacterial attachment to glycosaminoglycans. Infect Immun 74, 435–441.
Gibson, R. L., Lee, M. K., Soderland, C., Chi, E. Y. & Rubens, C. E. (1993). Group B streptococci invade endothelial cells: type III capsular polysaccharide attenuates invasion. Infect Immun 61, 478–485.
Gravekamp, C., Horensky, D. S., Michel, J. L. & Madoff, L. C. (1996). Variation in repeat number within the alpha C protein of group B streptococci alters antigenicity and protective epitopes. Infect Immun 64, 3576–3583.[Abstract]
Gullberg, D. E. & Lundgren-Akerlund, E. (2002). Collagen-binding I domain integrins – what do they do? Prog Histochem Cytochem 37, 3–54.[CrossRef][Medline]
Gutekunst, H., Eikmanns, B. J. & Reinscheid, D. J. (2004). The novel fibrinogen-binding protein FbsB promotes Streptococcus agalactiae invasion into epithelial cells. Infect Immun 72, 3495–3504.
Hamburger, Z. A., Brown, M. S., Isberg, R. R. & Bjorkman, P. J. (1999). Crystal structure of invasin: a bacterial integrin-binding protein. Science 286, 291–295.
Hemler, M. E. (1990). VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol 8, 365–400.[CrossRef][Medline]
Hemler, M. E., Glass, D., Coblyn, J. S. & Jacobson, J. G. (1986). Very late activation antigens on rheumatoid synovial fluid T lymphocytes. Association with stages of T cell activation. J Clin Invest 78, 696–702.[Medline]
Isberg, R. R., Hamburger, Z. & Dersch, P. (2000). Signaling and invasin-promoted uptake via integrin receptors. Microbes Infect 2, 793–801.[CrossRef][Medline]
Jackson, L. A., Hilsdon, R., Farley, M. M., Harrison, L. H., Reingold, A. L., Plikaytis, B. D., Wenger, J. D. & Schuchat, A. (1995). Risk factors for group B streptococcal disease in adults. Ann Intern Med 123, 415–420.
Kling, D. E., Gravekamp, C., Madoff, L. C. & Michel, J. L. (1997). Characterization of two distinct opsonic and protective epitopes within the alpha C protein of the group B Streptococcus. Infect Immun 65, 1462–1467.[Abstract]
Lachenauer, C. S., Creti, R., Michel, J. L. & Madoff, L. C. (2000). Mosaicism in the alpha-like protein genes of group B streptococci. Proc Natl Acad Sci U S A 97, 9630–9635.
Lancefield, R. C., McCarty, M. & Everly, W. N. (1975). Multiple mouse-protective antibodies directed against group B streptococci. Special reference to antibodies effective against protein antigens. J Exp Med 142, 165–179.
Levy, O., Jean-Jacques, R. M., Cywes, C., Sisson, R. B., Zarember, K. A., Godowski, P. J., Christianson, J. L., Guttormsen, H. K., Carroll, M. C. & other authors (2003). Critical role of the complement system in group B streptococcus-induced tumor necrosis factor alpha release. Infect Immun 71, 6344–6353.
Li, J., Kasper, D. L., Ausubel, F. M., Rosner, B. & Michel, J. L. (1997). Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus. Proc Natl Acad Sci U S A 94, 13251–13256.
Lindahl, G., Stalhammar-Carlemalm, M. & Areschoug, T. (2005). Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens. Clin Microbiol Rev 18, 102–127.
Loster, K., Vossmeyer, D., Hofmann, W., Reutter, W. & Danker, K. (2001). 1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins. Biochem J 356, 233–240.[CrossRef][Medline]
Madoff, L. C., Hori, S., Michel, J. L., Baker, C. J. & Kasper, D. L. (1991). Phenotypic diversity in the alpha C protein of group B streptococci. Infect Immun 59, 2638–2644.
Manning, S. D., Neighbors, K., Tallman, P. A., Gillespie, B., Marrs, C. F., Borchardt, S. M., Baker, C. J., Pearlman, M. D. & Foxman, B. (2004). Prevalence of group B streptococcus colonization and potential for transmission by casual contact in healthy young men and women. Clin Infect Dis 39, 380–388.[CrossRef][Medline]
Marcinkiewicz, C., Weinreb, P. H., Calvete, J. J., Kisiel, D. G., Mousa, S. A., Tuszynski, G. P. & Lobb, R. R. (2003). Obtustatin: a potent selective inhibitor of alpha1beta1 integrin in vitro and angiogenesis in vivo. Cancer Res 63, 2020–2023.
Meyn, L. A., Moore, D. M., Hillier, S. L. & Krohn, M. A. (2002). Association of sexual activity with colonization and vaginal acquisition of group B Streptococcus in nonpregnant women. Am J Epidemiol 155, 949–957.
Michel, J. L., Madoff, L. C., Olson, K., Kling, D. E., Kasper, D. L. & Ausubel, F. M. (1992). Large, identical, tandem repeating units in the C protein alpha antigen gene, bca, of group B streptococci. Proc Natl Acad Sci U S A 89, 10060–10064.
Miettinen, M., Castello, R., Wayner, E. & Schwarting, R. (1993). Distribution of VLA integrins in solid tumors. Emergence of tumor-type-related expression. Patterns in carcinomas and sarcomas. Am J Pathol 142, 1009–1018.[Abstract]
Picard, F. J. & Bergeron, M. G. (2004). Laboratory detection of group B Streptococcus for prevention of perinatal disease. Eur J Clin Microbiol Infect Dis 23, 665–671.[Medline]
Riikonen, T., Vihinen, P., Potila, M., Rettig, W. & Heino, J. (1995). Antibody against human alpha 1 beta 1 integrin inhibits HeLa cell adhesion to laminin and to type I, IV, and V collagens. Biochem Biophys Res Commun 209, 205–212.[CrossRef][Medline]
Rubio, M. A., Sotillos, M., Jochems, G., Alvarez, V. & Corbi, A. L. (1995). Monocyte activation: rapid induction of alpha 1/beta 1 (VLA-1) integrin expression by lipopolysaccharide and interferon-gamma. Eur J Immunol 25, 2701–2705.[Medline]
Sanz, L., Bazaa, A., Marrakchi, N., Perez, A., Chenik, M., Bel Lasfer, Z., El Ayeb, M. & Calvete, J. J. (2006). Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system. Biochem J 395, 385–392.[CrossRef][Medline]
Schrag, S. J., Zywicki, S., Farley, M. M., Reingold, A. L., Harrison, L. H., Lefkowitz, L. B., Hadler, J. L., Danila, R., Cieslak, P. R. & Schuchat, A. (2000). Group B streptococcal disease in the era of intrapartum antibiotic prophylaxis. N Engl J Med 342, 15–20.
Schubert, A., Zakikhany, K., Schreiner, M., Frank, R., Spellerberg, B., Eikmanns, B. J. & Reinscheid, D. J. (2002). A fibrinogen receptor from group B Streptococcus interacts with fibrinogen by repetitive units with novel ligand binding sites. Mol Microbiol 46, 557–569.[CrossRef][Medline]
Schuchat, A. (1999). Group B streptococcus. Lancet 353, 51–56.[CrossRef][Medline]
Senger, D. R., Claffey, K. P., Benes, J. E., Perruzzi, C. A., Sergiou, A. P. & Detmar, M. (1997). Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins. Proc Natl Acad Sci U S A 94, 13612–13617.
Shankar, V., Baghdayan, A. S., Huycke, M. M., Lindahl, G. & Gilmore, M. S. (1999). Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect Immun 67, 193–200.
Shankar, N., Lockatell, C. V., Baghdayan, A. S., Drachenberg, C., Gilmore, M. S. & Johnson, D. E. (2001). Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection. Infect Immun 69, 4366–4372.
Soriani, M., Santi, I., Taddei, A., Rappuoli, R., Grandi, G. & Telford, J. L. (2006). Group B Streptococcus crosses human epithelial cells by a paracellular route. J Infect Dis 193, 241–250.[CrossRef][Medline]
Spellerberg, B., Rozdzinski, E., Martin, S., Weber-Heynemann, J., Schnitzler, N., Lutticken, R. & Podbielski, A. (1999). Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin. Infect Immun 67, 871–878.
Stalhammar-Carlemalm, M., Areschoug, T., Larsson, C. & Lindahl, G. (1999). The R28 protein of Streptococcus pyogenes is related to several group B streptococcal surface proteins, confers protective immunity and promotes binding to human epithelial cells. Mol Microbiol 33, 208–219.[CrossRef][Medline]
Sykes, J. A., Whitescarver, J., Jernstrom, P., Nolan, J. F. & Byatt, P. (1970). Some properties of a new epithelial cell line of human origin. J Natl Cancer Inst 45, 107–122.[Medline]
Tamura, G. S., Kuypers, J. M., Smith, S., Raff, H. & Rubens, C. E. (1994). Adherence of group B streptococci to cultured epithelial cells: roles of environmental factors and bacterial surface components. Infect Immun 62, 2450–2458.
Turner, M. S., Hafner, L. M., Walsh, T. & Giffard, P. M. (2003). Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11. Appl Environ Microbiol 69, 5855–5863.
Tyrrell, G. J., Senzilet, L. D., Spika, J. S., Kertesz, D. A., Alagaratnam, M., Lovgren, M. & Talbot, J. A. (2000). Invasive disease due to group B streptococcal infection in adults: results from a Canadian, population-based, active laboratory surveillance study – 1996. Sentinel Health Unit Surveillance System Site Coordinators. J Infect Dis 182, 168–173.[CrossRef][Medline]
Valentin-Weigand, P., Jungnitz, H., Zock, A., Rohde, M. & Chhatwal, G. S. (1997). Characterization of group B streptococcal invasion in HEp-2 epithelial cells. FEMS Microbiol Lett 147, 69–74.[CrossRef][Medline]
Van Nhieu, G. T. & Isberg, R. R. (1991). The Yersinia pseudotuberculosis invasin protein and human fibronectin bind to mutually exclusive sites on the alpha 5 beta 1 integrin receptor. J Biol Chem 266, 24367–24375.
van Putten, J. P., Duensing, T. D. & Cole, R. L. (1998). Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors. Mol Microbiol 29, 369–379.[CrossRef][Medline]
Received 16 April 2007;
revised 17 June 2007;
accepted 18 August 2007.
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), NtACP D146A (
), RR' (
), D2-R (10 µM only; 
) and BSA (
) were added to 96-well plates coated with 
0.05, *P=0.05–0.1, and NS=not significant (P>0.1)]. Each assay was performed twice.