The identification of two M20B family peptidases required for full virulence in Staphylococcus aureus.

We have previously demonstrated that deletion of an intracellular leucine aminopeptidase results in attenuated virulence of S. aureus. Herein we explore the role of 10 other aminopeptidases in S. aureus pathogenesis. Using a human blood survival assay we identified mutations in two enzymes from the M20B family (PepT1 and PepT2) as having markedly decreased survival compared to the parent. We further reveal that pepT1, pepT2 and pepT1/2 mutant strains are impaired in their ability to resist phagocytosis by, and engender survival within, human macrophages. Using a co-infection model of murine sepsis, we demonstrate impairment of dissemination and survival for both single mutants that is even more pronounced in the double mutant. We show that these enzymes are localized to the cytosol and membrane but are not necessary for peptide-based nutrition, a hallmark of cell-associated aminopeptidases. Furthermore, none of the survival defects appear to be the result of altered virulence factor production. An exploration of their regulation reveals that both are controlled by known regulators of the S. aureus virulence process, including Agr, Rot and/or SarA, and that this cascade may be mediated by FarR. Structural modeling of PepT1 reveals it bears all the hallmarks of a tripeptidase, whilst PepT2 differs significantly in its catalytic pocket, suggesting a broader substrate preference. In sum, we have identified two M20B aminopeptidases that are integral to S. aureus pathogenesis. The future identification of protein and/or peptide targets for these proteases will be critical to understanding their important virulence impacting functions.

Unraveling the Impact of Secreted Proteases on Hypervirulence in Staphylococcus aureus.

Staphylococcus aureus controls the progression of infection through the coordinated production of extracellular proteases, which selectively modulate virulence determinant stability. This is evidenced by our previous finding that a protease-null strain has a hypervirulent phenotype in a murine model of sepsis, resulting from the unchecked accumulation of virulence factors. Here, we dissect the individual roles of these proteases by constructing and assessing the pathogenic potential of a combinatorial protease mutant library. When strains were constructed bearing increasing numbers of secreted proteases, we observed a variable impact on infectious capacity, where some exhibited hypervirulence, while others phenocopied the wild-type. The common thread for hypervirulent strains was that each lacked both aureolysin and staphopain A. Upon assessment, we found that the combined loss of these two enzymes alone was necessary and sufficient to engender hypervirulence. Using proteomics, we identified a number of important secreted factors, including SPIN, LukA, Sbi, SEK, and PSMα4, as well as an uncharacterized chitinase-related protein (SAUSA300_0964), to be overrepresented in both the aur scpA and the protease-null mutants. When assessing the virulence of aur scpA SAUSA300_0964 and aur scpA lukA mutants, we found that hypervirulence was completely eliminated, whereas aur scpA spn and aur scpA sek strains elicited aggressive infections akin to the protease double mutant. Collectively, our findings shed light on the influence of extracellular proteases in controlling the infectious process and identifies SAUSA300_0964 as an important new component of the S. aureus virulence factor arsenal.IMPORTANCE A key feature of the pathogenic success of S. aureus is the myriad virulence factors encoded within its genome. These are subject to multifactorial control, ensuring their timely production only within an intended infectious niche. A key node in this network of control is the secreted proteases of S. aureus, who specifically and selectively modulate virulence factor stability. In our previous work we demonstrated that deletion of all 10 secreted proteases results in hypervirulence, since virulence factors exist unchecked, leading to overly aggressive infections. Here, using a combinatorial collection of protease mutants, we reveal that deletion of aureolysin and staphopain A is necessary and sufficient to elicit hypervirulence. Using proteomic techniques, we identify the collection of virulence factors that accumulate in hypervirulent protease mutants, and demonstrate that a well-known toxin (LukA) and an entirely novel secreted element (SAUSA300_0964) are the leading contributors to deadly infections observed in protease-lacking strains.