Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood.
ABSTRACT: Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.
Project description:Staphylococcus aureus produces numerous factors that facilitate survival in the human host. S. aureus coagulase (Coa) and von Willebrand factor-binding protein (vWbp) are known to clot plasma through activation of prothrombin and conversion of fibrinogen to fibrin. In addition, S. aureus clumping factor A (ClfA) binds fibrinogen and contributes to platelet aggregation via a fibrinogen- or complement-dependent mechanism. Here, we evaluated the contribution of Coa, vWbp and ClfA to S. aureus pathogenesis in a rabbit model of skin and soft tissue infection. Compared to skin abscesses caused by the Newman wild-type strain, those caused by isogenic coa, vwb, or clfA deletion strains, or a strain deficient in coa and vwb, were significantly smaller following subcutaneous inoculation in rabbits. Unexpectedly, we found that fibrin deposition and abscess capsule formation appear to be independent of S. aureus coagulase activity in the rabbit infection model. Similarities notwithstanding, S. aureus strains deficient in coa and vwb elicited reduced levels of several proinflammatory molecules in human blood in vitro. Although a specific mechanism remains to be determined, we conclude that S. aureus Coa, vWbp and ClfA contribute to abscess formation in rabbits.
Project description:The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine.
Project description:Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman ?coa, ?vWbp, ?clfA or ?coa/?vWbp/?clfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P < 0.01). Likewise, expression of clfA increased L. lactis infectivity (incidence: 80%), which was not further affected by co-expression of vWbp. In symmetry, deletion of the coa or vWbp genes in S. aureus did not decrease infectivity (incidence: 68 and 64%, respectively) whereas deletion of clfA did decrease valve infection (incidence: 45%; P = 0.03 versus parent), which was not further affected by the triple deletion ?coa/?vWbp/?clfA (incidence: 36%; P > 0.05 versus ?clfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA.
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.
Project description:During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.
Project description:Host immunity against bacteria typically involves antibodies that recognize the microbial surface and promote phagocytic killing. Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of lethal bloodstream infection; however, vaccines and antibody therapeutics targeting staphylococcal surface molecules have thus far failed to achieve clinical efficacy. S. aureus secretes coagulase (Coa), which activates host prothrombin and generates fibrin fibrils that protect the pathogen against phagocytosis by immune cells. Because of negative selection, the coding sequence for the prothrombin-binding D1-D2 domain is highly variable and does not elicit cross-protective immune responses. The R domain, tandem repeats of a 27-residue peptide that bind fibrinogen, is conserved at the C terminus of all Coa molecules, but its functional significance is not known. We show here that the R domain enables bloodstream infections by directing fibrinogen to the staphylococcal surface, generating a protective fibrin shield that inhibits phagocytosis. The fibrin shield can be marked with R-specific antibodies, which trigger phagocytic killing of staphylococci and protect mice against lethal bloodstream infections caused by a broad spectrum of MRSA isolates. These findings emphasize the critical role of coagulase in staphylococcal escape from opsonophagocytic killing and as a protective antigen for S. aureus vaccines.
Project description:Von Willebrand factor-binding protein (vWbp), secreted by Staphylococcus aureus (S. aureus), can activate host prothrombin, convert fibrinogen to fibrin clots, induce blood clotting, and contribute to pathophysiology of S. aureus-related diseases, including infective endocarditis, staphylococcal sepsis and pneumonia. Therefore, vWbp is an promising drug target in the treatment of S. aureus-related infections. Here, we report that dryocrassin ABBA (ABBA), a natural compound derived from Dryopteris crassirhizoma, can significantly inhibit the coagulase activity of vWbp in vitro by directly interacting with vWbp without killing the bacteria or inhibiting the expression of the vWbp. Using molecular dynamics simulations, we demonstrate that ABBA binds to the "central cavity" in the elbow of vWbp by interacting with Arg-70, His-71, Ala-72, Gly-73, Tyr-74, Glu-75, Tyr-83, and Gln-87 in vWbp, thus interfering with the binding of vWbp to prothrombin. Furthermore, in vivo studies demonstrated that ABBA can attenuate injury and inflammation of mouse lung tissues caused by S. aureus and increase survival of mice. Together these findings indicate that ABBA is a promising lead drug for the treatment of S. aureus-related infections. This is the first report of potential inhibitor which inhibit the coagulase activity of vWbp by directly interacting with vWbp.
Project description:Coagulation is a critical pathogenic process in Staphylococcus aureus. Although the agglutination of S. aureus has been studied for a long time, the genes involved in this process are not completely clear. We performed tube agglutination and dynamic turbidimetry tests to identify novel genes involved in reduced plasma coagulation. A total of 15 genes were identified, including coa, clfA, vwbp, saeS, agrA, trpC, spdC, mroQ, cydA, qoxC, sucC, pyrP, menH, threonine aldolase, and truncated transposase for IS1272. The functions of these genes include bicomponent regulation, membrane transport, energy metabolism and biosynthesis, respectively. cydA, spdC, and mroQ genes were further studied by gene knockout and complementation. Results of gene knockout and complementation and real-time-qPCR proved that cydA, spdC, and mroQ genes are necessary for plasma coagulation. Furthermore, the survival ability of 7 day mice decreased significantly when cydA, spdC, and mroQ genes had been knocked out.
Project description:The two coagulases, von Willebrand factor binding protein (vWbp) and Coagulase (Coa), are critical virulence factors in several animal models of invasive Staphylococcus aureus (S. aureus) infections. These proteins are part of an intricate system of proteins that S. aureus uses to assemble a fibrinogen (Fg)/fibrin protective shield surrounding itself. This shield allows the microorganism to evade clearance by the host phagocytic cells. The coagulases can non-proteolytically activate the zymogen prothrombin to convert Fg to fibrin and promote the Fg/fibrin shield formation. The coagulases also bind directly to Fg and the interaction between Coa and Fg has been previously characterized in some detail. However, the mechanism(s) by which vWbp interacts with Fg remains unclear. Here, we show that vWbp and Coa have distinct interactions with Fg, despite being structurally similar. Coa binds with a significantly higher affinity to soluble Fg than to Fg coated on a plastic surface, whereas vWbp demonstrates no preference between the two forms of Fg. The two coagulases appear to target different sites on Fg, as they do not compete with each other in binding to Fg. Similar to Coa, both the N- and C-terminal halves of vWbp (vWbp-N, vWbp-C, respectively) harbor Fg-binding activities. The higher affinity Fg-binding activity resides in vWbp-N; whereas, the C-terminal region of Coa encompasses the major Fg-binding activity. Peptides constituting the previously identified Coa/Efb1 Fg-binding motif fail to inhibit vWbp-C from binding to Fg, indicating that vWbp-C lacks a functional homolog to this motif. Interestingly, the N-terminal prothrombin-binding domains of both coagulases recognize the Fg ?-chain, but they appear to interact with different sequence motifs in the host protein. Collectively, our data provide insight into the complex interactions between Fg and the S. aureus coagulases.
Project description:Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordonii was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deleting clfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of clfA-positive streptococci when both clfA and coa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.