ABSTRACT
BACKGROUND: Staphylococcus aureus is a common gram-positive bacterium that is the causative agent for several human diseases, including sepsis. A key virulence mechanism is pathogen binding to host fibrinogen through the C-terminal region of the γ-chain. Previous work demonstrated that FggΔ5 mice expressing mutant fibrinogen γΔ5 lacking a S. aureus binding motif had significantly improved survival following S. aureus septicemia. Fibrinogen γ' is a human splice variant that represents about 10% to 15% of the total fibrinogen in plasma and circulates as a fibrinogen γ'-γ heterodimer (phFibγ'-γ). The fibrinogen γ'-chain is also expected to lack S. aureus binding function. OBJECTIVE: Determine if human fibrinogen γ'-γ confers host protection during S. aureus septicemia. METHODS: Analyses of survival and the host response following S. aureus septicemia challenge in FggΔ5 mice and mice reconstituted with purified phFibγ'-γ or phFibγ-γ. RESULTS: Reconstitution of fibrinogen-deficient or wildtype mice with purified phFibγ'-γ prior to infection provided a significant prolongation in host survival relative to mice reconstituted with purified phFibγ-γ, which was superior to that observed with heterozygous FggΔ5 mice. Improved survival could not be accounted for by quantitative differences in fibrinogen-dependent adhesion or clumping, but phFibγ'-γ-containing mixtures generated notably smaller bacterial aggregates. Importantly, administration of phFibγ'-γ after infection also provided a therapeutic benefit by prolonging host survival relative to administration of phFibγ-γ. CONCLUSION: These findings provide the proof-of-concept that changing the ratio of naturally occurring fibrinogen variants in blood could offer significant therapeutic potential against bacterial infection and potentially other diseases.
Subject(s)
Bacteremia , Fibrinogens, Abnormal , Sepsis , Staphylococcal Infections , Humans , Mice , Animals , Staphylococcus aureus/metabolism , Fibrinogen/metabolismABSTRACT
The blood-clotting protein fibrin(ogen) plays a critical role in host defense against invading pathogens, particularly against peritoneal infection by the Gram-positive microbe Staphylococcus aureus. Here, we tested the hypothesis that direct binding between fibrin(ogen) and S. aureus is a component of the primary host antimicrobial response mechanism and prevention of secondary microbe dissemination from the peritoneal cavity. To establish a model system, we showed that fibrinogen isolated from FibγΔ5 mice, which express a mutant form lacking the final 5 amino acids of the fibrinogen γ chain (termed fibrinogenγΔ5), did not support S. aureus adherence when immobilized and clumping when in suspension. In contrast, purified wildtype fibrinogen supported robust adhesion and clumping that was largely dependent on S. aureus expression of the receptor clumping factor A (ClfA). Following peritoneal infection with S. aureus USA300, FibγΔ5 mice displayed worse survival compared to WT mice coupled to reduced bacterial killing within the peritoneal cavity and increased dissemination of the microbes into circulation and distant organs. The failure of acute bacterial killing, but not enhanced dissemination, was partially recapitulated by mice infected with S. aureus USA300 lacking ClfA. Fibrin polymer formation and coagulation transglutaminase Factor XIII each contributed to killing of the microbes within the peritoneal cavity, but only elimination of polymer formation enhanced systemic dissemination. Host macrophage depletion or selective elimination of the fibrin(ogen) ß2-integrin binding motif both compromised local bacterial killing and enhanced S. aureus systemic dissemination, suggesting fibrin polymer formation in and of itself was not sufficient to retain S. aureus within the peritoneal cavity. Collectively, these findings suggest that following peritoneal infection, the binding of S. aureus to stabilized fibrin matrices promotes a local, macrophage-mediated antimicrobial response essential for prevention of microbe dissemination and downstream host mortality.
Subject(s)
Fibrinogen/immunology , Peritonitis/immunology , Staphylococcal Infections/immunology , Animals , Coagulase/immunology , Coagulase/metabolism , Fibrin/metabolism , Mice , Peritonitis/metabolism , Staphylococcal Infections/metabolism , Staphylococcus aureus/immunology , Staphylococcus aureus/metabolismABSTRACT
Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with â¼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite â¼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was â¼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to â¼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.
Subject(s)
Afibrinogenemia , Blood Platelets/metabolism , Fibrinogen , Hemostasis/genetics , Mutation , Platelet Aggregation/genetics , Thrombosis , Afibrinogenemia/genetics , Afibrinogenemia/metabolism , Animals , Fibrinogen/genetics , Fibrinogen/metabolism , Mice , Mice, Knockout , Thrombosis/genetics , Thrombosis/metabolismABSTRACT
The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of â¼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.
Subject(s)
Fibrinogen/metabolism , Peritonitis/metabolism , Prothrombin/metabolism , Animals , Anti-Bacterial Agents/metabolism , Anti-Infective Agents/metabolism , Blood Coagulation , Coagulase/metabolism , Female , Fibrin/metabolism , Fibrinogen/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Staphylococcal Infections/microbiology , Staphylococcus aureus/metabolism , Staphylococcus aureus/pathogenicity , ThromboplastinABSTRACT
PURPOSE OF REVIEW: Fibrin(ogen) is a multifunctional clotting protein that not only has critical roles in hemostasis but is also important in inflammatory processes that control bacterial infection. As a provisional extracellular matrix protein, fibrin(ogen) functions as a physical barrier, a scaffold for immune cell migration, or as a spatially-defined cue to drive inflammatory cell activation. These mechanisms contribute to overall host antimicrobial defense against infection. However, numerous bacterial species have evolved mechanisms to manipulate host fibrin(ogen) to promote microbial virulence and survival. Staphylococcal species, in particular, express numerous virulence factors capable of engaging fibrin(ogen), promoting fibrin formation, and driving the dissolution of fibrin matrices. RECENT FINDINGS: Recent studies have highlighted both new insights into the molecular mechanisms involved in fibrin(ogen)-mediated host defense and pathogen-driven virulence. Of particular interest is the role of fibrin(ogen) in forming host protective biofilms versus pathogen protective barriers and biofilms as well as the role of fibrin(ogen) in mediating direct host antimicrobial responses. SUMMARY: Current data suggest that the role of fibrin(ogen) in staphylococcal infection is highly context-dependent and that better defining the precise cellular and molecular pathways activated will provide unique opportunities of therapeutic intervention to better treat Staphylococcal disease.