Coagulases as determinants of protective immune responses against Staphylococcus aureus.
ABSTRACT: 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: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: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: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: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: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: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:Staphylococcus aureus is a prominent etiological agent of suppurative abscesses. In principle, abscess formation and purulent exudate are classical physiological features of healing and tissue repair. However, S. aureus deploys two coagulases that can usurp this classical host response and form distinct abscess lesions. Here, we establish that during coinfection with coagulase producers and non-producers, coagulases are shared public goods that contribute to staphylococcal persistence, abscess formation, and disease progression. Coagulase-negative mutants that do not produce the public goods themselves are able to exploit those cooperatively secreted by producers and thereby thrive during coinfection at the expense of others. This study shows the importance of social interactions among pathogens concerning clinical outcomes.
Project description:Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options.
Project description:Coagulation is an innate defense mechanism intended to limit blood loss and trap invading pathogens during infection. However, <i>Staphylococcus aureus</i> has the ability to hijack the coagulation cascade and generate clots via secretion of coagulases. Although many <i>S. aureus</i> have this characteristic, some do not. The population dynamics regarding this defining trait have yet to be explored. We report here that coagulases are public goods that confer protection against antimicrobials and immune factors within a local population or community, thus promoting growth and virulence. By utilizing variants of a methicillin-resistant <i>S. aureus</i> we infer that the secretion of coagulases is a cooperative trait, which is subject to exploitation by invading mutants that do not produce the public goods themselves. However, overexploitation, "tragedy of the commons," does not occur at clinically relevant conditions. Our micrographs indicate this is due to spatial segregation and population viscosity. These findings emphasize the critical role of coagulases in a social evolution context and provide a possible explanation as to why the secretion of these public goods is maintained in mixed <i>S. aureus</i> communities.
Project description:Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.