Disrupting the Fibrin Shield to Combat Staphylococcus aureus Bloodstream Infections - ABSTRACT Staphylococcus aureus (S. aureus) is a leading causative agent of life-threatening bloodstream infections. The increasing prevalence of antibiotic-resistant strains (e.g., methicillin-resistant S. aureus), as well as understudied mechanisms of antibiotic tolerance, highlights the necessity of identifying strategies to better combat S. aureus virulence. Fibrinogen is a monomeric plasma glycoprotein which polymerizes to form a fibrin matrix following proteolytic cleavage by the coagulation protease thrombin. The physiological role of fibrin(ogen) is in hemostasis and vascular repair following blood vessel injury. However, S. aureus has evolved an extensive repertoire of virulence factors that engage host fibrin(ogen), including the bacterial coagulases ‘staphylocoagulase’ and ‘von Willebrand factor-binding protein’ that hijack host prothrombin to catalyze fibrin polymerization. The coagulases, along with key bacterial fibrin(ogen)-binding proteins (FBPs; e.g., clumping factor A) support the formation of a ‘fibrin shield’ around the pathogen to promote protection from the host and antibiotic therapies in the bloodstream. The central hypotheses of this proposal are that (i) S. aureus fibrin shield formation and function is driven by bacterial binding of circulating fibrinogen followed by coagulase-mediated fibrin polymerization, and that (ii) disruption of fibrin shield formation with novel fibrinogen variants (e.g., nonpolymerizable or non-S. aureus- binding variants) will result in increased bacterial susceptibility to antibiotic treatment during the course of a bloodstream infection. Preliminary in vitro studies suggest collaborative roles for each coagulase and other FBPs in fibrin shield formation. Moreover, a major mechanism of protection induced by the fibrin shield is induction of an antibiotic-tolerant state. In vivo studies support critical roles for coagulases and FBPs in S. aureus virulence in a murine model of intravenous infection and demonstrate the capacity for fibrinogen variants to disrupt fibrin(ogen)-dependent S. aureus virulence and improve host infection outcomes. The studies proposed will 1) determine the contribution of S. aureus-mediated fibrin(ogen) binding and fibrin polymerization to pathogen virulence in the bloodstream and 2) evaluate the efficacy of fibrinogen variants, which lack key S. aureus binding motifs, in disrupting S. aureus fibrin shield formation in vivo as a therapeutic strategy to increase bacterial susceptibility to antibiotic therapies. In vitro analyses of fibrin shield formation and function, mechanisms of fibrin(ogen)-mediated S. aureus antibiotic tolerance, and in vivo murine models of bloodstream infection will build upon preliminary results to rigorously investigate the role of fibrin shield formation in S. aureus pathogenicity and antibiotic tolerance as well as examine the potential for novel fibrinogen-directed therapeutics in improving infection outcomes. The UNC Blood Research Center, Department of Biochemistry and Biophysics, and Integrative Vascular Biology Training Program (NIH T32 fellowship) provide an outstanding environment to successfully complete the proposed work. The completion of this F31 fellowship will play an instrumental role in reaching my goal of becoming an independent researcher.
