Gram-positive bacteria process and release small peptides, or pheromones, that act as signals for the induction of adaptive traits, including those involved in pathogenesis. One class of small signaling pheromones is the cyclic autoinducing peptides (AIPs), which regulate expression of genes that orchestrate virulence and persistence in a range of microbes, including staphylococci, listeriae, clostridia, and enterococci. In a genetic screen for Staphylococcus aureus secreted virulence factors, we identified an S. aureus mutant containing an insertion in the gene SAUSA300_1984 (mroQ), which encodes a putative membrane-embedded metalloprotease. A ΔmroQ mutant exhibited impaired induction of Toll-like receptor 2-dependent inflammatory responses from macrophages but elicited greater production of the inflammatory cytokine interleukin-1␤ and was attenuated in a murine skin and soft tissue infection model. The ΔmroQ mutant phenocopies an S. aureus mutant containing a deletion of the accessory gene regulatory system (Agr), wherein both strains have significantly reduced production of secreted toxins and virulence factors but increased surface protein A abundance. The Agr system controls virulence factor gene expression in S. aureus by sensing the accumulation of AIP via the histidine kinase AgrC and the response regulator AgrA. We provide evidence to suggest that MroQ acts within the Agr pathway to facilitate the optimal processing or export of AIP for signal amplification through AgrC/A and induction of virulence factor gene expression. Mutation of MroQ active-site residues significantly reduces AIP signaling and attenuates virulence. Altogether, this work identifies a new component of the Agr quorum-sensing circuit that is critical for the production of S. aureus virulence factors.

26Gram-positive bacteria process and release small peptides or "pheromones" that act as signals 27 for the induction of adaptive traits including those involved in pathogenesis. One class of small 28 signaling pheromones is the cyclic auto-inducing peptides (AIPs), which regulate expression of 29 genes that orchestrate virulence and persistence in a range of microbes including 30Staphylococci, Listeria, Clostridia, and Enterococci. In a genetic screen for Staphylococcus 31 aureus secreted virulence factors, we identified a S. aureus mutant containing an insertion in 32 gene SAUSA300_1984 (mroQ), which encodes a putative membrane-embedded 33 metalloprotease. A ΔmroQ mutant exhibits impaired induction of TLR2-dependent 34 inflammatory responses from macrophages, but elicits greater production of the inflammatory 35 cytokine IL-1β and is attenuated in a murine skin and soft tissue infection model. The ΔmroQ 36 mutant phenocopies a S. aureus mutant containing a deletion of the accessory gene regulatory 37 system (Agr), wherein both strains have significantly reduced production of secreted toxins and 38 virulence factors, but increased surface Protein A abundance. The Agr system controls 39 virulence factor gene expression in S. aureus through sensing accumulation of AIP via the 40 histidine kinase AgrC and response regulator AgrA. We provide evidence to suggest that MroQ 41 acts within the Agr pathway to facilitate optimal processing or export of AIP for signal 42 amplification through AgrC/A and induction of virulence factor gene expression. Mutation of 43MroQ active site residues significantly reduces AIP signaling and attenuates virulence. 44Altogether, this work identifies a new component of the Agr quorum sensing circuit that is 45 critical for the production of S. aureus virulence factors. 46 47 48 from the bacterial cell. While many signaling molecules of Gram-negative bacteria are freely 56 diffusible, the peptides of Gram-positives generally must transit the membrane via a dedicated 57 transporter (4, 5). After processing and transport, the peptide is either imported back into the 58 bacterial cell or transmits signal from the extracellular environment by binding to membrane-59 embedded sensor histidine kinases (6). Peptide signaling culminates in a change in gene 60 expression mediated by transcription factors that respond to the peptide. Many Gram-positive 61 bacterial pathogens use these "quorum-sensing" peptides to induce gene expression programs 62 that promote virulence adaptations such as competence, toxin production, biofilm formation, 63 and establishment of persistence traits. Because of its importance in activating virulence 64 programs in S. aureus, quorum-sensing inhibition has been the focus of many therapeutic 65 initiatives (7)(8)(9)(10)(11)(12)(13)(14). 66The ability of S. aureus to infect host tissues and cause acute and chronic disease is 67 partially due to its use of complex gene regulatory systems that control virulence factor gene 68 expression (15-17). S. aureus employs 16 two-compone...

Gram-positive bacteria produce small autoinducing peptides (AIPs), which act to regulate expression of genes that promote adaptive traits, including virulence. The Gram-positive pathogen Staphylococcus aureus generates a cyclic AIP that controls expression of virulence factors via the accessory gene regulatory (Agr) system. S. aureus strains belong to one of four Agr groups (Agr-I, -II, -III, and -IV); each group harbors allelic variants of AgrD, the precursor of AIP.