The Streptococcus agalactiae cell wall‐anchored protein PbsP mediates adhesion to and invasion of epithelial cells by exploiting the host vitronectin/αv integrin axis - De Gaetano - 2018 - Molecular Microbiology - Wiley Online Library Giuseppe Valerio De Gaetano, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalyThese authors participated equally.Search for more papers by this authorGiampiero Pietrocola, Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Pavia, ItalyThese authors participated equally.Search for more papers by this authorLetizia Romeo, IRCCS Centro Neurolesi Bonino Pulejo, Messina, ItalySearch for more papers by this authorRoberta Galbo, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorGermana Lentini, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorMiriam Giardina, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorCarmelo Biondo, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorAngelina Midiri, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorGiuseppe Mancuso, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorMario Venza, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorIsabella Venza, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorArnaud Firon, Institut Pasteur, Unite de Biologie des Bacteriés Pathogènes a Gram positif, CNRS ERL6002, 75015 Paris, FranceSearch for more papers by this authorPatrick Trieu-Cuot, Institut Pasteur, Unite de Biologie des Bacteriés Pathogènes a Gram positif, CNRS ERL6002, 75015 Paris, FranceSearch for more papers by this authorGiuseppe Teti, Corresponding Author gteti@unime.it orcid.org/0000-0002-6315-8644 Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, Italy For correspondence. E-mail gteti@unime.it; Tel. +39 090 2213310; Fax +39 090 2213312. Search for more papers by this authorPietro Speziale, Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Pavia, Italy Department of Industrial and Information Engineering, University of Pavia, Pavia, ItalySearch for more papers by this authorConcetta Beninati, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, Italy Scylla Biotech Srl, Messina, ItalySearch for more papers by this author Giuseppe Valerio De Gaetano, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalyThese authors participated equally.Search for more papers by this authorGiampiero Pietrocola, Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Pavia, ItalyThese authors participated equally.Search for more papers by this authorLetizia Romeo, IRCCS Centro Neurolesi Bonino Pulejo, Messina, ItalySearch for more papers by this authorRoberta Galbo, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorGermana Lentini, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorMiriam Giardina, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorCarmelo Biondo, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorAngelina Midiri, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorGiuseppe Mancuso, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorMario Venza, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorIsabella Venza, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, ItalySearch for more papers by this authorArnaud Firon, Institut Pasteur, Unite de Biologie des Bacteriés Pathogènes a Gram positif, CNRS ERL6002, 75015 Paris, FranceSearch for more papers by this authorPatrick Trieu-Cuot, Institut Pasteur, Unite de Biologie des Bacteriés Pathogènes a Gram positif, CNRS ERL6002, 75015 Paris, FranceSearch for more papers by this authorGiuseppe Teti, Corresponding Author gteti@unime.it orcid.org/0000-0002-6315-8644 Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, Italy For correspondence. E-mail gteti@unime.it; Tel. +39 090 2213310; Fax +39 090 2213312. Search for more papers by this authorPietro Speziale, Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Pavia, Italy Department of Industrial and Information Engineering, University of Pavia, Pavia, ItalySearch for more papers by this authorConcetta Beninati, Metchnikoff Laboratory, Departments of Human Pathology, Medicine, Biomedical Sciences and Chemical Sciences, University of Messina, Messina, Italy Scylla Biotech Srl, Messina, ItalySearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin αv subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface andhost integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS. Introduction Streptococcus agalactiae (also called group B streptococcus or GBS) is a Gram-positive bacterium that can act either as a harmless colonizer or a deadly pathogen in humans. In particular, GBS is a leading cause of sepsis and meningitis in the neonate, with a reported incidence of 0.4–0.6 per 1,000 live births (Edmond et al., 2012; Madrid et al., 2017). In addition, this organism can frequently cause spontaneous abortions and stillbirths (Nan et al., 2015). Although intrapartum antibiotic prophylaxis of colonized women has decreased the incidence of GBS disease occurring during the first week of life (traditionally defined as early-onset sepsis or EOS), it has left unchanged the incidence of neonatal late-onset disease (LOS) occurring at up to three months after birth (Van Dyke et al., 2009). In addition, GBS morbidity outside of the perinatal risk period has been steadily increasing and now accounts for more than 90% of all cases of invasive GBS disease in the U.S.A. (Centers for Disease Control and Prevention, 2015). Thus, GBS disease persists as a major health problem for which additional or alternative control measures are needed. A particularly effective strategy, which could potentially block bacterial transmission to susceptible individuals, would involve interfering with GBS colonization of mucosal surfaces. GBS can be detected in 5–40 % of asymptomatic women at any single point in time, varying with the age group considered and the number of sampled sites (Edwards M.S., 2000; Russell et al., 2017). Longitudinal studies indicate that GBS colonization might be more common than generally estimated, with up to 65% of women being colonized at least once during one year (Meyn et al., 2009). Carriage rates are higher in the rectum than in other sites and rectal colonization is a significant predictor of vaginal colonization, suggesting that the gastrointestinal tract is the main reservoir for GBS and that vaginal colonization occurs as a secondary event (Badri et al., 1977; Dillon et al., 1982; Boyer et al., 1983). Molecular mechanisms sustaining GBS colonization involve complex adaptive changes, including the regulated expression of specific adhesins that enable GBS to adhere to various receptors present on mucosal surfaces and in the extracellular matrix (ECM). Binding of GBS to ECM components, such as fibrinogen, plasminogen, fibronectin, laminin and collagen, is necessary at the initial colonization stage or during the invasive steps (Lindahl et al., 2005). Interestingly, not all ECM-binding proteins of GBS are conserved in all strains, and the set of adhesins at the surface of specific GBS clones is associated with their virulence capacities (Santi et al., 2007; Seo et al., 2012; Tazi et al., 2010; Six et al., 2015). PbsP (Plasminogen binding surface Protein) is a recently identified cell wall protein and virulence factor of GBS containing a novel plasminogen-binding domain (defined as the MK-rich region) and one or two repeated Streptococcal Surface Repeat (SSURE) domains, which are homologous to domains found in other streptococcal species (Bumbaca et al., 2004; Buscetta et al., 2016). PbsP differs from most of the ECM-binding proteins described thus far for its high degree of conservation among human GBS isolates and for being positively regulated to a remarkable extent both in vivo, during experimental infection, and in vitro, upon contact with human cells. For example, in a study which analyzed changes in the bacterial transcriptome induced by contact with human blood, none of the known or suspected virulence factors of GBS were strongly upregulated, with the exception of pbsP and a few other genes involved in plasminogen binding (Mereghetti et al., 2008). Similarly, transcriptomic analysis in a murine model of vaginal colonization indicated that pbsP was among the 10 most highly upregulated GBS genes in vivo (Cook et al., 2018). Gene deletion experiments also evidenced that pbsP was required for colonization of the murine vaginal tract, although the mechanisms involved were not investigated. In addition to its role during colonization, PbsP is also necessary for hematogenous dissemination during invasive GBS disease (Buscetta et al., 2016). Initially, the PbsP homolog in Streptococcus pneumoniae (PfbB) was identified as a dual plasminogen and fibronectin adhesin (Papasergi et al., 2010), while the GBS PbsP binds plasminogen, but not fibronectin (Buscetta et al., 2016). In the present study, we investigated whether PbsP is involved in interactions between GBS and human epithelial cells. We demonstrated that PbsP promotes GBS adherence to and invasion of epithelial cells via its ability to bind vitronectin (Vtn), a ubiquitous plasma and ECM component. The Vtn-binding ability of PbsP resides in a molecular region that encompasses its SSURE domains and differs from the part of the protein that predominantly mediates plasminogen binding. We conclude that the PbsP adhesin is able to bind at least two host ECM components to promote GBS adherence and invasion of host barriers. The lung and the gut are frequent sites of GBS entry in neonatal EOS and LOS respectively (Tazi et al., 2010). Bacterial adherence to the mucosal layer of these organs and invasion of epithelial barriers are considered as fundamental steps in the pathogenesis of GBS infections. To investigate the potential involvement of PbsP in GBS–host cell interactions, we used the human epithelial cell lines A549 (type II lung alveolar cells) and Caco-2 (colon epithelial cells). We first studied the role of PbsP in bacterial adherence to these cells using a deletion mutant (ΔpbsP) obtained in BM110, a capsular serotype III strain belonging to the hypervirulent CC17 clonal complex. Deletion of pbsP significantly decreased the ability of BM110 GBS to adhere to Caco-2 and A549 cells, as observed by microscopy (Fig. 1A–C and Suppl. Fig 1A–C) and by CFU counting of adherent bacteria (Fig. 1D). Cell invasion was also significantly impaired using both cell types (Fig. 1E). The decreased adherence and invasion phenotypes of the ΔpbsP mutant were reversed by genetic complementation (Fig. 1), demonstrating that PbsP is involved in interactions between GBS and epithelial cells. Figure 1Open in figure viewerPowerPoint PbsP is involved in interactions between GBS and epithelial cells. Adherence to and invasion of Caco-2 (colon epithelial) and A549 (alveolar epithelial) cells by GBS strain BM110 (WT), its pbsP deletion mutant (ΔpbsP) and the complemented strain (ΔpbsP+pbsP). A, B and C. Bacterial adherence to Caco-2 cells as evidenced by immunofluorescence microscopy; shown are actin filaments (red), GBS (green), cell nuclei and bacterial nucleoids (blue). D. Adherence and E. Invasion measured by CFU counting and expressed as the percentages of cell-associated bacteria relative to the total number of bacteria added to the monolayers. Shown are means + standard deviations of three independent experiments conducted in triplicate. *p 0.05; ***p 0.001; by one-way ANOVA and Bonferroni test. To test if this interaction involved direct binding of PbsP to a host component, we evaluated the ability of whole recombinant PbsP to adhere to epithelial cells. To this end, we used fluorescent beads coated with recombinant PbsP or PbsP fragments fused to glutathione-S-transferase (GST; Fig. 2B). Beads coated with the whole PbsP protein efficiently adhered to A549 cells, while the GST-coated control beads did not bind (Fig. 2A, C and F). Beads coated with a PbsP fragment encompassing both SSURE domains (SSURE1+2) adhered to A549 cells similarly as beads coated with the whole PbsP molecule, while beads coated with the MK-rich domain of the protein showed only punctuate binding to the cells (Fig. 2A, D and E). Addition of soluble SSURE1+2 domains inhibited the adherence of PbsP-coated beads in a dose-dependent manner, confirming that the SSURE1+2 domains mediate the interaction with cells (Suppl. Fig2). Figure 2Open in figure viewerPowerPoint Adherence to epithelial cells of beads coated with isolated recombinant PbsP and its fragments. A. Number of adherent beads per cell expressed as means + standard deviations of three independent experiments performed in triplicate. *p 0.05; **p 0.01; ns, non-significant by one-way ANOVA and Bonferroni test. B. Schematic representation of the PbsP, SSURE 1+2 and MK-rich recombinant proteins used in the above experiments. C, D, E and F. Representative images obtained by fluorescent microscopy showing actin filaments (red), latex microspheres (green) and cell nuclei (blue). Alveolar epithelial cells (A549) grown on coverslips were incubated with fluorescent latex microspheres (1µ in diameter) covalently bound with PbsP or PbsP fragments (SSURE 1+2, MK-rich). Latex micropheres covalently bound with glutathione-S-transferase (GST) were used as a negative control. We hypothesized that the moderate adherence to epithelial cells of GST-MK-rich coated beads might be due to the ability of the MK-rich region to efficiently bind plasminogen, a protein present on epithelial cells (Buscetta et al., 2016). Indeed, pretreatment of epithelial cells with anti-plasminogen antibodies inhibited the binding of MK-rich-coated beads to epithelial cells, but not that of PbsP- or SSURE1+2-coated beads (Suppl. Fig. 3). Collectively these data indicate that PbsP strongly binds to the surface of human epithelial cells through its SSURE domains. This property is apparently unrelated to the previously reported ability of PbsP to bind plasminogen, which resides predominantly in the MK-rich region of the protein (Buscetta et al., 2016). Figure 3Open in figure viewerPowerPoint PbsP selectively binds to immobilized human Vtn. A. binding of recombinant PbsP to immobilized extracellular matrix components using plates coated with fibronectin, vitronectin, collagen, plasminogen or thrombospondin; binding of the GST-PbsP fusion protein was revealed by ELISA using anti-GST antibodies. Shown are the results of three experiments performed in duplicate. ***p 0.001; ns, non-significant by one-way ANOVA and Bonferroni test. B. Binding of PbsP to Vtn in comparison with other recombinant cell wall proteins (FbsA and FbsC) and with PbsP fragments (SSURE 1+2 and MK-rich). Plates were coated with human Vtn or with bovine serum albumin (BSA, used as a negative control) and binding of recombinant proteins fused to GST was revealed by ELISA using anti-GST antibodies. C. Dose-dependent binding of PbsP and its SSURE1+2 region to immobilized Vtn; binding of increasing concentrations of recombinant proteins fused to GST was revealed by ELISA using anti-GST antibodies. Shown are the results of three experiments performed in duplicate. D. Dot blot analysis of PbsP-Vtn interactions; increasing concentrations of Vtn were spotted onto the nitrocellulose membranes and probed using 5 μg of recombinant proteins (PbsP, SSURE1+2 or the negative control GST), which were detected using anti-GST serum. Shown are data from one representative experiment of two producing similar results. E and F. Surface plasmon resonance analysis of interactions between PbsP (E) or SSURE1+2 (F) with Vtn. PbsP or SSURE1+2 were captured on a BIAcore sensor chip using immobilized human Vtn. Each sensorgram is representative of three independent experiments conducted in duplicate; affinity was calculated from curve fitting to a plot of the RU values against different concentrations of soluble PbsP (E) or SSURE1+2 (F) (inset graphs). Recombinant PbsP binding to 10 host components (immobilized human plasminogen, collagen, fibronectin, fibrinogen, C-reactive protein, complement components factor H, factor I, factor B, C1q and C3) has been previously tested, demonstrating preferential binding to plasminogen (Buscetta et al., 2016). In order to identify the receptor responsible for plasminogen-independent PbsP binding to the surface of epithelial cells, we tested additional host components, including thrombospondin (Binsker et al., 2015) and vitronectin (Vtn) (Singh et al., 2010) in ELISA experiments in which Plg was used as a positive control. Recombinant PbsP bound as efficiently to Vtn as to plasminogen, while no significant binding to fibronectin, thrombospondin or collagen was observed (Fig. 3A). Binding of PbsP to Vtn is specific since Vtn did not interact with two other recombinant GBS cell wall adhesins, such as FbsA and FbsC (Fig. 3B). Experiments involving recombinant PbsP fragments demonstrated that the SSURE1+2 domains of PbsP, but not the MK-rich region, bind Vtn (Fig. 3B). ELISA and dot blot experiments showed a similar dose-dependent and saturable interaction between PbsP or SSURE1+2 domains and Vtn (Fig. 3C and D). Cell pretreatment with anti-Vtn antibodies inhibited binding of PbsP or of the SSURE1+2 domain to the epithelial cells surface, while binding of the MK-rich domain involved in plasminogen binding was unaffected (Suppl. Fig. 3). Dissociation constants (KD) measured by surface plasmon resonance using immobilized Vtn on the sensor chip and different concentrations of soluble ligands were 289 and 219 nM for PbsP and SSURE1+2, respectively (Fig. 3E and F). Since Vtn-binding proteins from different bacterial species are known to preferentially target heparin-binding sites in the Vtn molecule (Liang et al., 1997; Hallstrom et al., 2016), we next investigated whether PbsP also interacts with these sites. To this end, immobilized Vtn was pretreated with heparin or chondroitin sulfate (used as a negative control) before adding PbsP. Under these conditions, heparin pretreatment prevented PbsP binding to Vtn in a dose-dependent fashion (Suppl. Fig. 4). The heparin effect was specific since chondroitin sulfate pretreatment was not inhibitory. Collectively, these data suggest that the GBS cell wall protein PbsP efficiently recognizes heparin-binding sites on the human Vtn molecule and that this property is mediated by the SSURE domains of PbsP, but not by its MK-rich region. Figure 4Open in figure viewerPowerPoint Binding of GBS to immobilized Vtn. A. Silanized coverslips were sensitized with Vtn or BSA (used as a negative control) and incubated with the indicated GBS strains (NEM316, 6313, A909, COH1, BM110 and 2603l), S.pneumoniae (unencapsulated R6 strain) or L. lactis (subsp. Cremoris, MG1363 strain). Bacterial binding was detected after Gram staining of coverslips. Results were expressed as bacteria per field of vision (FOV) at the indicated magnification and represent means + standard deviations of three independent experiments. B. Inhibition of bacterial binding to Vtn by anti-Vtn antibodies. Silanized coverslips were sensitized with Vtn and treated with the indicated IgG preparation. Bacterial binding was assessed, and results expressed, as described in A. C. The BM110 strain (WT) was compared with its pbsP deletion mutant (ΔpbsP) or with the complemented strain (ΔpbsP+pbsP) for its ability to adhere to immobilized Vtn or BSA, used as a control. D. The BM110 strain (WT), its pbsP deletion mutant (ΔpbsP) or the complemented strain (ΔpbsP+pbsP) were treated with anti-PbsP or anti-GST mouse serum, used as a control, before assessing bacterial adherence to immobilized Vtn by ELISA using an anti-GBS serum. E. Inhibition of adherence to Vtn of different GBS strains (NEM316, 6313, A909, COH1 and 2603) by anti-PbsP or anti-GST mouse serum, used as a control; bacterial binding was assessed by ELISA using an anti-GBS serum. *p 0.05; ***p 0.001, by one-way ANOVA and Bonferroni test. Clinical GBS strains do not have an identical set of adhesins, a diversity associated to the emergence of hypervirulent clones (Brochet et al., 2006, Tazi et al., 2012). Therefore, it was of interest to ascertain whether unrelated GBS clones bind Vtn. GBS strains belonging to the major pathogenic clonal complexes adhered to Vtn immobilized on glass coverslips but not to bovin ealbumin, used as a negative control (Fig. 4A and Suppl. Fig. 5). GBS binding to Vtn is similar to that of the control species Streptococcus pneumoniae, a Gram-positive pathogen known to adhere efficiently to Vtn (Voss et al., 2013). In contrast, the non-pathogenic species Lactococcus lactis was unable to bind Vtn (Fig. 4A and Suppl. Fig. 5). GBS adherence to Vtn-coated coverslips was abrogated by pre-incubating coverslips with anti-Vtn antibodies, confirming specific bacterial binding to Vtn (Fig. 4B). Comparative binding of the parental strain BM110 and its pbsP deletion mutant (ΔpbsP) to immobilized Vtn were measured using a sensitive ELISA assay in which GBS binding to Vtn-coated plates was revealed by anti-GBS polyclonal antibodies. As shown in Fig. 4C, deletion of pbsP significantly reduced, but did not completely abrogate, GBS binding to Vtn and this phenotype was reversed by genetic complementation. To confirm the role of PbsP in GBS-Vtn interactions, we next assessed whether antibodies raised against a recombinant GST-PbsP fusion protein inhibited GBS binding. Pretreatment with anti-GST-PbsP, but not anti-GST serum, significantly reduced Vtn binding of GBS strains belonging to different clonal complexes (Fig. 4D and 4E). Overall, our results demonstrate that GBS strains interact with Vtn, that this interaction is dependent on the conserved adhesion PbsP through its SSURE domain and that at least one additional adhesin is necessary for optimal GBS binding to Vtn. Figure 5Open in figure viewerPowerPoint The Vtn/αv integrin axis is involved in interactions between GBS and epithelial cells. A–D. Effects of pretreating epithelial cells with anti-Vtn IgG (A and B) or Vtn (C and D) on adhesion or invasion of GBS strain BM110 to intestinal (Caco-2) or respiratory (A549) epithelial cells. Adherence (A and C) and invasion (B and D) were measured by CFU counts and expressed as the percentages of cell-associated bacteria relative to the total number bacteria added to the monolayers. E. Monolayers of A549 respiratory cells were pretreated with Vtn in the presence of various blocking polyclonal or monoclonal anti-integrin antibodies before the addition of GBS. Bacterial invasion was assessed by CFU counts and expressed as the percentage of cell-associated bacteria relative to the total number bacteria added to the monolayers. Shown are the means+SD of results from 3 independent experiments performed in triplicate (A–D) or in duplicate (E); *p 0.05; **p 0.01;***p 0.001 by Bonferroni test and one-way ANOVA. The αvintegrin subunit promotes Vtn-dependent invasion of epithelial cells by GBS Vtn is expressed at the surface of various cell types, including A549 cells, where it can behave as an adherence or internalization substrate for pathogens (Leroy-Dudal et al., 2004; Singh et al., 2010; Su et al., 2016). In preliminary experiments, the presence of Vtn on the surface of Caco-2 cells was demonstrated by immunofluorence microscopy (Suppl. Fig. 1D). To test if Vtn plays a significant role in GBS-host cell interactions, we pretreated A549 or Caco-2 cells with anti-Vtn antibodies before infection with the BM110 GBS strain. Vtn masking by antibodies significantly decreased the number of adhering or invading bacteria (Fig. 5A and B). Conversely, pre-incubation of either epithelial cell lines with purified human Vtn (5 µ ml–1) enhanced interactions of GBS with these cells (Fig. 5C and D). The enhancing effect of Vtn pretreatment was particularly evident in the case of bacterial invasion, as evidenced by an approximately fivefold increase in the number of internalized GBS. The marked invasion phenotypes suggest that GBS might exploit Vtn to enter cells. Vtn is anchored to the cell surface through interactions with specific transmembrane proteins of the integrin family, displayed as α and β chain heterodimers. To gain entry into host cells, bacterial pathogens can use Vtn as a molecular bridge, thereby triggering integrin-dependent internalization responses (Grashoff et al., 2004). To assess whether GBS can exploit Vtn-specific integrins to invade epithelial cells, we used a panel of blocking antibodies directed againsteither integrin subunit. In these experiments, monolayers of A549 respiratory cells were pretreated with Vtn in the presence of the various anti-integrin antibodies before infection with GBS. Under these conditions, anti-αv, but not anti-α1/α2 polyclonal antibodies or anti-β1/β3/β5 monoclonal antibodies, significantly decreased cell invasion by GBS (Fig. 5E). These data suggest that the Vtn-specific αv integrin subunit might function as a receptor for GBS entry into respiratory cells. In this study, we demonstrated that GBS interacts with human Vtn through the SSURE domains of the PbsP adhesin to promote bacterial adherence and invasion of epithelial cells. Vtn is a ubiquitous glycoprotein involved in blood coagulation and inhibition of the assembly of the terminal complex of complement, among other functions (Preissner and Jenne, 1991). The Vtn function is dependent upon its conformation and multimerization, which regulate its interaction with different ligands. The folded monomeric form is largely predominant in plasma, where the protein is present at high concentrations (100–400 mg l–1), while Vtn is multimeric in the ECM. The unfolded multimeric state exposes cryptic epitopes and acts as an anchor point for integrins expressed on the cell surface, thereby promoting cell adherence to the ECM (Schvartz et al., 1999). Invasive pathogens have an independently evolved mechanism to bind Vtn in order to evade the cytolytic or pro-inflammatory activities of the terminal complement complex (Singh et al., 2010; Hallstrom et al., 2016). Moreover, Vtn is expressed at the surface of different cell types as a result of endogenous secretion or of absorption from extracellular fluids and is upregulated during inflammation and injury (Hallstrom et al., 2016; Aulakh, 2018). Consequently, microbial pathogens might target Vtn for host invasion. Extracellular bacterial pathogens, in particular, interact with Vtn to efficiently adhere to host tissues and cross cell barriers, as shown for S. pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Haemophylus influenzae (Chhatwal et al., 1987; Kostrzynska et al., 1992; Liang et al., 1995; Virkola et al., 2000; Li et al., 2001; Heilmann et al., 2003; Styriak and Ljungh, 2003; Leroy-Dudal et al., 2004; Bergmann et al., 2009; Singh et al., 2014; Hallstrom et al., 2016). Bacteria–host interactions may involve Vtn-mediated bridging with integrins, particularly αvβ3 or αvβ5 integrins, followed by intracellular signaling, activation and bacterial uptake (Dehio et al., 1998; Spreghini et al., 1999; Leroy-Dudal et al., 2004; Bergmann et al., 2009; Singh et al., 2014). Our results suggest a similar mechanism in Vtn-dependent GBS internalization by epithelial cells, which was found to involve αv subunit-containing integrins. This mechanism does not exclude the participation of other integrins/integrin receptors in cell invasion mediated by GBS. For example, GBS can adhere to surface-immobilized fibronectin (Beckmann et al., 2002) and invade lung epithelial cells (Cheng et al., 2002) through the cell wall protein C5a peptidase, which suggests the potential involvement of fibronectin-binding integrins, such as the α5-β1 integrin. Moreover, the α-C protein of GBS can directly bind to α1-β1 integrins (Bolduc and Madoff, 2007). It will be of interest to assess in future studies the relative importance of various integrins in GBS invasion of different cell types in order to better understand the tissue tropism of these bacteria. Our data show that the PbsP cell wall protein, which was previously found to mediate plasmin-dependent invasion of endothelial barriers (Buscetta et al., 2016), also promotes Vtn-dependent bacterial adhesion and invasion of epithelial cells. This indicates that PbsP is a multi-ligand protein capable of hijacking, either at the same or at different times during pathogenesis, at least two major components of the host ECM. Interactions with Vtn may contribute to the initial colonization process (Cook et al., 2018), enabling GBS to attach to the mucosal surface, whereas the uptake of plasminogen, after invasion of deeper tissues, may be indispensable to acquire surface proteolytic activity and cross the blood–brain barrier (Magalhaes et al., 2013; Buscetta et al., 2016). Interestingly, the plasminogen- and Vtn-binding abilities of PbsP apparently reside on different domains. The SSURE1+2 domains bind Vtn while the C-terminal MK-rich region of the molecule (so-called for the presence of several lysine and methionine residues) is unable to bind Vtn. On the contrary, plasminogen binding on PbsP predominantly involved the MK-rich region (Buscetta et al., 2016). The 150 amino acids long repeated domain, designated as SSURE, is conserved in a family of cell wall proteins widespread in streptococci. In pneumococci, the PfbP protein containing 6 repetitions of the SSURE domain is involved in adhesion to respiratory epithelial cells (Papasergi et al., 2010). Moreover, fragments containing single SSURE domains were shown to bind to the surface of epithelial cells, as well as to plasminogen and fibronectin immobilized on inert substrates (Papasergi et al., 2010). Binsker et al. later found that fragments of PfbP (referred to as PavB), containing multiple SSURE domains also bind thrombospondin (Binsker et al., 2015). We show here that instead of fibronectin or thrombospondin, the GBS SSURE region binds Vtn. Functional differences between the S. pneumoniae and S. agalactiae SSURE domains might be explained by structural differences since their amino acid sequences cluster apart from each other (Buscetta et al., 2016) (Suppl. Fig 6). Some bacterial pathogens, such as S. pneumoniae (Bergmann et al., 2009) or Neisseria meningitidis (Cunha et al., 2010), preferentially bind to the multimeric, unfolded form of Vtn. In our study, we measured binding to Vtn after its adsorption to a solid phase, using an initially monomeric form of the molecule. Once bound to a solid surface, monomeric Vtn can be recognized by a large array of ligands, including mAbs binding to cryptic epitopes exposed only on the unfolded, multimeric form (Seiffert and Smith, 1997; Underwood et al., 2002). Thus, it cannot be inferred from our data whether PbsP binds preferentially to any particular form of Vtn and further studies are necessary to decipher the in vivo contribution of Vtn in cellular adhesion or in the manipulation of the terminal complement complex by GBS. Elucidation of the functional role of PbsP as a multifaceted virulence factor highlights the various strategies used by GBS to interact with the host. The PbsP adhesinis conserved in GBS strains belong to different clonal complexes. Its expression was recently demonstrated to be regulated at the transcriptional level by the SaeRS two-component system in a murine model in which PbsP is necessary for vaginal colonization (Cook et al., 2018). In conclusion, PbsP is a multi-ligand adhesion playing important roles in different steps of the pathogenesis of GBS disease. Its conservation in all human isolates and its strong in vivo upregulation make it an interesting vaccine candidate. The following GBS reference strains were used: NEM316 (serotype III, CC23); 6313 (serotype III, CC23); BM110 (serotype III, CC17); COH1 (serotype III, CC17); A909 (serotype Ia, CC7); 2603V/R (serotype V, CC19) (Glaser et al., 2002; Tettelin et al., 2005; Da Cunha et al., 2014). In some experiments, we also used the pneumococcal R6 strain, an unencapsulated type 2 isolate originally obtained from Alexander Tomasz (Rockefeller Institute, New York, N.Y.) and the Lactococcus lactis subsp. Cremoris MG1363 strain (Mancuso et al., 2009). The mutant ΔpbsP strain used here was obtained from GBS strain BM110, as previously described (Buscetta et al., 2016). All GBS strains were grown at 37°C in Todd–Hewitt broth or Todd–Hewitt agar (both from Difco Laboratories). Cloning, production and purification of recombinant PbsP and of the PbsP fragments MK-rich and SSURE1+2 fused to GST have been previously described (Buscetta et al., 2016). Recombinant FbsA and FbsC were produced as previously described (Buscetta et al., 2014). Recombinant, monomeric, human Vtn was purchased from Abcam (ab 94369) and used throughout this study. Bovine serum albumin (BSA), heparin, chondroitin sulfate and thrombospondin were obtained from Sigma–Aldrich. Collagen type II was purified from bovine nasal septum as reported previously (Reese and Mayne, 1981). Human fibronectin was prepared as previously described (Pietrocola et al., 2017). Polyclonal anti-Vtn (ab20091) and anti-plasminogen (ab7336) rabbit IgG were purchased from Abcam. Polyclonal anti-GST goat IgG and normal rabbit IgG were from Sigma–Aldrich. Rabbit anti-α1, anti-α2 and anti-αv IgG and mouse monoclonal anti-β1 (BV7) and anti-β3 antibodies (B212) were a generous gift from Dr. G. Tarone (University of Turin, Italy). Rabbit anti-β5 IgG (Ab 15459) was purchased from Abcam. Rabbit anti-GBS antibody was prepared as previously described (Papasergi et al., 2013). Rabbit anti-mouse or goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from DakoCytomation (Glostrup, Denmark). The human epithelial cell lines A549 (ATCC CCL-185; lung carcinoma) and Caco-2 (ATCC HTB-37; colorectal adenocarcinoma) were used throughout this study. Cells were cultured in 24-well plates at a density of 1 × 105 cells per well in Dulbecco’s modified Eagle medium (DMEM) supplemented with fetal bovine serum to a final concentration of 10 %. At 24 h before the adherence or invasion assays, the medium was removed and replaced with serum-free medium. The adherence and invasion assays were performed as described (Buscetta et al., 2014). Briefly, bacteria were grown to the mid-log phase and added to sub-confluent monolayers at a multiplicity of infection (MOI) of 30 for 1 h. To determine bacterial adhesion, the infected cells were washed three times with Dulbecco’s phosphate buffered saline, lysed and plated on Todd–Hewitt agar for CFU counts. To enumerate internalized bacteria, the monolayers were further incubated for 1 h before cell lysis in medium supplemented with penicillin and streptomycin (200 U ml–1 and 200 μg ml–1, respectively) to kill extracellular bacteria. Bacterial adherence and invasion were calculated as: recovered CFU/initial inoculum CFU × 100. In some experiments, cells were pretreated for 30 min with various reagents, including Vtn (5μg ml–1), anti-integrin, anti-plasminogen and anti-Vtn antibodies (all at a concentration of 5 μg ml–1) before the addition of bacteria. In further experiments, bacteria were pre-incubated for 30 min with mouse serum (1:100) raised against recombinant PbsP fused to GST or against GST, as previously described (Buscetta et al., 2016). In selected experiments, adherent bacteria were visualized by immunofluorence microscopy, as described (Papasergi et al., 2010). Fluorescent beads (Fluoresbrite YG 1.00 µm microspheres, Polysciences) were conjugated with recombinant PbsP, PbsP fragments or with GST at equimolar concentrations (10 µM) by previously described procedures (Papasergi et al., 2010). The amount of protein coupled on beads was calculated by subtracting the amount of protein present in the supernatant after adsorption. Adhesion of microspheres to A549 cells was performed as previously described (Papasergi et al., 2010). Briefly, protein-coupled beads were added to cells at a concentration of 108 beads ml–1. After 30 min of incubation at 37°C (in the presence of 1 µM GST to avoid non-specific binding), monolayers were washed, fixed with 3.7 % formaldehyde and permeabilized with Triton 0.1% in phosphate buffered saline (PBS), as described (Papasergi et al., 2010). After visualizing actin and nuclei with, respectively, phalloidin-iFluor 555 (ABCAM ab 176756) and DAPI, attached beads were counted using a fluorescent microscope equipped with structured illumination (Apotome, Zeiss), as previously described (Papasergi et al., 2010). In binding inhibition assays, cell monolayers were pre-incubated with increasing concentrations of soluble SSURE1+2, MK-rich, anti-Vtn or anti-plasminogen antibodies for 30 min before performing the assay with PbsP-coated microspheres. 100µl of a 10 µg ml–1 solution of vitronectin, fibronectin, collagen or thrombospondin were coated onto microtiter wells overnight at 4°C in 0.05 M carbonate buffer (pH 9.0). After washing with PBS supplemented with 0.05% Tween 20, the wells were blocked with PBS supplemented with 0.01% Tween 20 and 1% non-fat dry milk for 2 h at 25°C. After incubation with 5 μg ml–1 of recombinant PbsP, MK-rich, SSURE1+2, FbsA or FbsC (all fused to GST) for 1 h, complex formation was detected with goat anti-GST (1:4,000; GE Healthcare), followed by the addition of alkaline phosphatase-conjugated rabbit anti-goat IgG (1:5,000; Sigma–Aldrich). For dot blot analysis, increasing concentrations of Vtn were spotted through circular templates directly onto the nitrocellulose membranes and probed using 5 μg of recombinant proteins. The latter were detected using anti-GST serum as described above. The effect of heparin and chondroitin sulfate on PbsP/Vtn interaction was examined incubating Vtn immobilized onto microtiter plates with 0.5 µg of recombinant PbsP in fusion with GST in the presence of increasing concentrations (0–2 µM) of heparin or chondroitin sulfate. PbsP bound to Vtn was detected by addition of goat anti-GST antibodies followed by HRP-conjugated anti-goat IgG. Surface plasmon resonance measurements were performed on a Biacore X-100 instrument (GE Health-care, Piscataway, NJ) as previously described (Buscetta et al., 2016). Briefly, to measure KD values of vitronectin binding to recombinant PbsP or SSURE1+2 fused to GST, Vtn (5 µg ml–1) dissolved in 10 mM sodium acetate buffer (pH 5.0) was immobilized onto a carboxy-derivatized sensor chips (CM5). PbsP or SSURE1+2 (both at 500 nM) was passed over a flow cell, whereas GST alone was passed in a reference cell at increasing concentrations at a rate of 10 µl min–1. Assay channel data were subtracted from reference flow cell data to eliminate the effects of nonspecific interactions. The response units (RU) at steady state were plotted as a function of ligand concentration, and fitted to the Langmuir equation to yield the KD of the Vtn–PbsP or Vtn-SSURE1+2 interactions. For microscopic assessment of bacterial adhesion to immobilized Vtn, silane-treated 18-mm2 glass coverslips were incubated overnight at 4°C with Vtn or BSA (both at 10 µg ml–1 in PBS), blocked with 2% casein for 1 h at 20°C and exposed to bacteria, as previously described (Papasergi et al., 2010). Briefly, bacteria were grown to the late log phase (A560 = 0.8), washed, resuspended in PBS and applied to the coverslips at a concentration of approximately 1 x 105 CFU ml–1. Slides were then Gram-stained and observed under a bright field microscope. Results were expressed as numbers of bacteria per field of vision (FOV) at the indicated magnification. At least 20 different fields per slide were counted. Adherence of bacteria to immobilized Vtn was also assessed by ELISA. After sensitizing microtiter plates overnight at 4°C with 10 µg ml–1 of Vtn or BSA in PBS, plates were blocked with 2 % casein for 1 h at 20°C. Bacteria grown to the late log phase were resuspended in PBS to a concentration of approximately 1 × 105 CFU ml–1 and incubated at 37°C for 1 h. After washing, anti-GBS rabbit serum (1:10,000) was added and incubated for 1 h at 37°C followed by the addition of alkaline phosphatase-conjugated rabbit anti-goat IgG (1:5,000; Sigma–Aldrich). One-way analysis of variance (ANOVA) followed by Bonferroni correction was used to assess statistical significance of differences between the number of adhering or invading bacteria and between ELISA test absorbance values. Work described here was supported in part by funds granted to Scylla Biotech Srl by the Ministero dell’Università e della Ricerca Scientifica of Italy (Project n.4/13 ex art. 11 D.M. n. 593). GVDG, GP, LR, RG, GL and MG performed the experiments and analyzed the data. CBi, AM, MV, IV and GM analyzed the data. PS, CBe, AF, PT and GT designed the experiments, analyzed the data and wrote the manuscript. 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