Studies on fibronectin binding proteins, proteases, and virulence in Staphylococcus aureus

Abstract: Staphylococcus aureus produces several surface proteins, many of which bind specifically to different host extracellular matrix (ECM) proteins and plasma proteins, such as fibronectin, collagen and fibrinogen. Two highly homologous fibronectin binding proteins (FnBPA and FnBPB), enconded by fnbA and fnbB, have been characterized. Both FnBPs are produced by most S. aureus and contribute to the fibronectin binding phenotype. Very little was known about the regulation of fnbA and fnbB expression except that mutants defective in the global virulence regulator agr (accessory gene regulator) bound fibronectin (Fn) better that the corresponding parent strain. This study was initiated to find out whether the increased Fn binding of agr mutant cells was due to increased transcription of fnb genes or to reduced degradation of FnBPs due to decreased production of extracellular proteases. We found that transcription of the fnb genes is negatively regulated by agr and also by an agr-independent mechanism that restricts synthesis of FnBPs to early exponential phase of growth. Fn binding assays confirmed agr mutant strains have an increased capacity to bind soluble fibronectin compared to the wild type. However, while the concentration of cell wall associated FnBPs was 16-fold higher in the agr mutants than in the parent strain, Fn binding was only two-fold higher in the agr mutant, suggesting that other cell surface proteins that are up-regulated in the agr mutant had a negative effect on Fn binding. We also found that the amount of FnBPs at the bacteria] surface is affected by the extracellular staphylococcal serine protease, SspA. In fact, release of FnBPs from the bacterial surface due to extracellular proteases explained the apparent down-regulation of fnb expression in the sarA mutant derived from a clinical S. aureus strain with high fibronectin binding capacity. Thus, it seems that depending on the regulation of agr and sarA expression, the amount of FnBPs on the bacterial surface can be modulated to meet specific requirements during the course of infection. Local infection and abscess formation caused by S. aureus is a common complication found in patients bitten by the viperid snake Bothrops asper. We found that inoculation of venom significantly potentiated S. aureus infection in mouse gastrocnemius muscle. Acute muscle damage caused by the myotoxic phospholipase A2 MTIII and ECM degradation by endogenous matrix metalloproteinases (MMP-2 and MMP-9) seemed to be the most important factors that promoted infection. While activation of proMMP-9 has been attributed to mediators of the inflammatory response, we showed that proMMP-2 could be directly activated by serine proteinases contained in the snake venom. Comparing the infectivity of S. aureus DB strain to that of its sarA and agr mutants indicated that the adhesive phenotype of DB, i.e., high ability to bind fibronectin and fibrinogen, was important for survival. Taken all results together, we speculated that degradation of ECM proteins and possibly other proteins promoted infection by providing soluble fragments that can bind to the surface of the bacteria so that they are masked from the host immune system.