Staphylococcus aureus vWF-binding protein triggers a strong interaction between clumping factor A and host vWF.
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ABSTRACT:
SUBMITTER: Viljoen A
PROVIDER: S-EPMC8041789 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The staphylococcal adhesin clumping factor A (ClfA) has a variant amino acid sequence, generating the potential for alterations in epitope structure and immunogenicity of this vaccine candidate. We demonstrated for two recombinant ClfA(40-531) (a slightly truncated version of the fibrinogen-binding domain of ClfA containing amino acids 40 to 531) genetic variants that strain-specific epitopes are immunodominant. This work indicates that immune responses elicited by ClfA may, at least in part, be dependent on the strain of origin of the ClfA.
| S-EPMC3754511 | biostudies-literature
Project description:Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF-binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A-dependent surface protein Clumping factor A (ClfA). VWF-vWbp-ClfA anchor S. aureus to vascular endothelium under shear stress. SUMMARY:Objective When establishing endovascular infections, Staphylococcus aureus (S. aureus) overcomes shear forces of flowing blood by binding to von Willebrand factor (VWF). Staphylococcal VWF-binding protein (vWbp) interacts with VWF, but it is unknown how this secreted protein binds to the bacterial cell wall. We hypothesized that vWbp interacts with a staphylococcal surface protein, mediating the adhesion of S. aureus to VWF and vascular endothelium under shear stress. Methods We studied the binding of S. aureus to vWbp, VWF and endothelial cells in a micro-parallel flow chamber using various mutants deficient in Sortase A (SrtA) and SrtA-dependent surface proteins, and Lactococcus lactis expressing single staphylococcal surface proteins. In vivo adhesion of bacteria was evaluated in the murine mesenteric circulation using real-time intravital vascular microscopy. Results vWbp bridges the bacterial cell wall and VWF, allowing shear-resistant binding of S. aureus to inflamed or damaged endothelium. Absence of SrtA and Clumping factor A (ClfA) reduced adhesion of S. aureus to vWbp, VWF and activated endothelial cells. ADAMTS-13 and an anti-VWF A1 domain antibody, when combined, reduced S. aureus adhesion to activated endothelial cells by 90%. Selective overexpression of ClfA in the membrane of Lactococcus lactis enabled these bacteria to bind to VWF and activated endothelial cells but only in the presence of vWbp. Absence of ClfA abolished bacterial adhesion to the activated murine vessel wall. Conclusions vWbp interacts with VWF and with the SrtA-dependent staphylococcal surface protein ClfA. The complex formed by VWF, secreted vWbp and bacterial ClfA anchors S. aureus to vascular endothelium under shear stress.
| S-EPMC6232194 | biostudies-literature
Project description:We have earlier shown that clumping factor A (ClfA), a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P(336) and Y(338), were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP(336)Y(338) mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP(336)Y(338) mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP(336) and Y(338) were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP(336)Y(338) mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP(336)SY(338)A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.
| S-EPMC2374910 | biostudies-literature
Project description:UNLABELLED:Treatment of Staphylococcus aureus infections has become increasingly difficult because of the emergence of multidrug-resistant isolates. Development of a vaccine to prevent staphylococcal infections remains a priority. To determine whether clumping factor A (ClfA) is a good target protein for inclusion in a multivalent vaccine, we evaluated its efficacy in a variety of relevant staphylococcal infection models, challenging with different S. aureus strains. ClfA adsorbed to Alhydrogel and mixed with Sigma Adjuvant System was more immunogenic and stimulated a more robust Th17 response than ClfA administered with alum alone. ClfA immunization induced the production of functional antibodies in rabbits and mice that blocked S. aureus binding to fibrinogen and were opsonic for S. aureus strains that produced little or no capsular polysaccharide. Mice immunized with ClfA showed a modest reduction in the bacterial burden recovered from subcutaneous abscesses provoked by S. aureus USA300 strain LAC. In addition, the ClfA vaccine reduced lethality in a sepsis model following challenge with strain Newman, but not ST80. Vaccination with ClfA did not protect against surgical wound infection, renal abscess formation, or bacteremia. Passive immunization with antibodies to ClfA did not protect against staphylococcal bacteremia in mice or catheter-induced endocarditis in rats. Some enhancement of bacteremia was observed by ClfA immunization or passive administration of ClfA antibodies when mice were challenged by the intraperitoneal route. Although rodent models of staphylococcal infection have their limitations, our data do not support the inclusion of ClfA in an S. aureus multivalent vaccine. IMPORTANCE:Antibiotics are often ineffective in eradicating Staphylococcus aureus infections, and thus, a preventative vaccine is sorely needed. Two single-component vaccines and two immunoglobulin preparations failed to meet their designated endpoints in phase III clinical trials. Importantly, recipients of an S. aureus surface protein (iron surface determinant B) vaccine who developed a staphylococcal infection experienced a higher rate of multiorgan failure and mortality than placebo controls, raising safety concerns. Multicomponent S. aureus vaccines have now been generated, and several include surface protein clumping factor A (ClfA). We immunized mice with ClfA and generated a robust T cell response and serum antibodies that were functional in vitro. Nonetheless, ClfA was not protective in a number of relevant animal models of S. aureus infection, and high levels of ClfA antibodies enhanced bacteremia when mice were challenged with community-acquired methicillin-resistant S. aureus strains. Evidence supporting ClfA as a vaccine component is lacking.
| S-EPMC4742718 | biostudies-literature
Project description:Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg ?-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel ?-sheet complementation as observed for the clumping factor A·?-chain peptide complex. The ?-sheet complementation is shorter in the ClfB·Fg ?-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.
| S-EPMC3138276 | biostudies-literature
Project description:Staphylococcus aureus is a leading cause of contagious mastitis in dairy cattle. Internalization of S. aureus by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood. In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in S. aureus invasion into bovine mammary epithelial cell line (MAC-T). In vitro binding assays were performed to co-immunoprecipitate the binding proteins of AnxA2 in the lysates of S. aureus. AnxA2 mediated the internalization but not adherence of S. aureus. Engagement of AnxA2 stimulated an integrin-linked protein kinase (ILK)/p38 MAPK cascade to induce S. aureus invasion. One of the AnxA2-precipitated proteins was identified as S. aureus clumping factor B (ClfB) through use of mass spectrometry. Direct binding of ClfB to AnxA2 was further confirmed by using a pull-down assay. Pre-incubation with recombinant ClfB protein enhanced S. aureus internalization, an effect that was specially blocked by anti-AnxA2 antibody. Our results demonstrate that binding of ClfB to AnxA2 has a function in promoting S. aureus internalization. Targeting the interaction of ClfB and AnxA2 may confer protection against S. aureus mastitis.
| S-EPMC8538401 | biostudies-literature
Project description:Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.
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Project description:Staphylococcus aureus is one of major pathogens that can cause a series of diseases in different hosts. In the bovine, it mainly causes subclinical and contagious mastitis, but its mechanisms of infection are not fully understood. Considering the fact that virulence factors play key roles in interactions between the bacterium and host cells, this study aimed to identify if a binding partner of S. aureus clumping factor A (ClfA) exists on the bovine mammary epithelial cells. The ClfA protein was used as a bait to pull down lysates of cultured bovine mammary epithelial cells (MAC-T cells). One pull-down protein was identified through use of mass spectrometry and bioinformatics analyses as bovine AnnexinA2. The Western blot and in vitro binding assay confirmed that the full A domain of ClfA was necessary to bind to AnnexinA2. In addition, the interaction between ClfA and AnnexinA2 was validated biochemically by ELISA with a KD value of 418+/-93 nM. The confocal microscopy demonstrated that ClfA and AnnexinA2 partially co-localized in the plasma membrane and that the majority of them were transported into cytoplasm. Taken together, the results demonstrate that ClfA binds with AnnexinA2 and this interaction could mediate S. aureus invasion into bovine mammary epithelial cells.
| S-EPMC5244385 | biostudies-literature
Project description:The present study evaluated the potential of recombinant binding region A of clumping factor A (rClfA-A) to be an effective component of a vaccine against mastitis induced by Staphylococcus aureus in the mouse. rClfA-A and inactivated S. aureus were each emulsified in Freund's adjuvant, mineral oil adjuvant, and Seppic adjuvant; phosphate-buffered saline was used as a control. Seven groups of 12 mice each were immunized intraperitoneally three times at 2-week intervals. The titers of IgG and subtypes thereof (IgG1 and IgG2a) in the rClfA-A-immunized group were more than 1,000-fold higher than those in the killed-bacteria-immunized group (P < 0.01). Of the three adjuvants used, mineral oil adjuvant induced the highest antibody levels for both antigens (P < 0.001). Furthermore, the anti-rClfA-A antibody capacities for bacterial adhesion and opsonizing phagocytosis were significantly greater in the rClfA-A-immunized group than in the killed-bacteria-immunized group (P < 0.05). Lactating mice immunized with either rClfA-A or inactivated vaccine were challenged with S. aureus via the intramammary route. The numbers of bacteria recovered from the murine mammary glands 24 h after inoculation were significantly lower in the rClfA-A group than in the killed-bacteria-immunized group (P < 0.001). Histologic examination of the mammary glands showed that rClfA-A immunization effectively preserved tissue integrity. Thus, rClfA-A emulsified in an oil adjuvant provides strong immune protection against S. aureus-induced mastitis in the mouse.
| S-EPMC2976091 | biostudies-literature
Project description:Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the "dock, lock and latch" mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB?S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor?/?) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB? mutant colonised wild-type mice less efficiently than the parental ClfB? strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB? mutant in the Lor?/? mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor?/? mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.
| S-EPMC3531522 | biostudies-literature