Rot is a key regulator of Staphylococcus aureus biofilm formation.

Staphylococcus aureus is a significant cause of chronic biofilm infections on medical implants. We investigated the biofilm regulatory cascade and discovered that the repressor of toxins (Rot) is part of this pathway. A USA300 community-associated methicillin-resistant S. aureus strain deficient in Rot was unable to form a biofilm using multiple different assays, and we found rot mutants in other strain lineages were also biofilm deficient. By performing a global analysis of transcripts and protein production controlled by Rot, we observed that all the secreted protease genes were up-regulated in a rot mutant, and we hypothesized that this regulation could be responsible for the biofilm phenotype. To investigate this question, we determined that Rot bound to the protease promoters, and we observed that activity levels of these enzymes, in particular the cysteine proteases, were increased in a rot mutant. By inactivating these proteases, biofilm capacity was restored to the mutant, demonstrating they are responsible for the biofilm negative phenotype. Finally, we tested the rot mutant in a mouse catheter model of biofilm infection and observed a significant reduction in biofilm burden. Thus S. aureus uses the transcription factor Rot to repress secreted protease levels in order to build a biofilm.

Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis.

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N-terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin-resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in-vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N-terminal domain of human CXCR2.

Staphopains modulate Staphylococcus aureus biofilm integrity.

Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ΔsigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture.

Extracellular proteases are key mediators of Staphylococcus aureus virulence via the global modulation of virulence-determinant stability.

Staphylococcus aureus is a highly virulent and successful pathogen that causes a diverse array of diseases. Recently, an increase of severe infections in healthy subjects has been observed, caused by community-associated methicillin-resistant S. aureus (CA-MRSA). The reason for enhanced CA-MRSA virulence is unclear; however, work suggests that it results from hypersecretion of agr-regulated toxins, including secreted proteases. In this study, we explore the contribution of exo-proteases to CA-MRSA pathogenesis using a mutant lacking all 10 enzymes. We show that they are required for growth in peptide-rich environments, serum, in the presence of antimicrobial peptides (AMPs), and in human blood. We also reveal that extracellular proteases are important for resisting phagocytosis by human leukocytes. Using murine infection models, we reveal contrasting roles for the proteases in morbidity and mortality. Upon exo-protease deletion, we observed decreases in abscess formation, and impairment during organ invasion. In contrast, we observed hypervirulence of the protease-null strain in the context of mortality. This dichotomy is explained by proteomic analyses, which demonstrates exo-proteases to be key mediators of virulence-determinant stability. Specifically, increased abundance of both secreted (e.g. α-toxin, Psms, LukAB, LukE, PVL, Sbi, γ-hemolysin) and surface-associated (e.g. ClfA+B, FnbA+B, IsdA, Spa) proteins was observed upon protease deletion. Collectively, our findings provide a unique insight into the progression of CA-MRSA infections, and the role of secreted proteolytic enzymes.

Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation.

Regulation of sialic acid transport and catabolism in Haemophilus influenzae.

Virulence of nontypeable Haemophilus influenzae (NTHi) is dependent on the decoration of lipooligosaccharide with sialic acid. This sugar must be derived from the host, as NTHi cannot synthesize sialic acids. NTHi can also use sialic acid as a carbon source. The genes encoding the sialic acid transporter and the genes encoding the catabolic activities are localized to two divergently transcribed operons, the siaPT operon and the nan operon respectively. In this study, we identified SiaR as a repressor of sialic acid transport and catabolism in NTHi. Inactivation of siaR resulted in the unregulated expression of the genes in both operons. Unregulated catabolism of sialic acid in the siaR mutant resulted in the reduction of surface sialylation and an increase in serum sensitivity. In addition to SiaR-mediated repression, CRP, the cAMP receptor protein, was shown to activate expression of the siaPT operon but not the nan operon. We describe a model in which SiaR and CRP work to modulate intracellular sialic acid levels. Our results demonstrate the importance of SiaR-mediated regulation to balance the requirement of surface sialylation and the toxic accumulation of intracellular sialic acid.