Determinants of maturation of the Staphylococcus aureus autoinducing peptide.

The accessory gene regulatory (Agr) system is required for virulence factor gene expression and pathogenesis of Staphylococcus aureus. The Agr system is activated in response to the accumulation of a cyclic autoinducing peptide (AIP), which is matured and secreted by the bacterium. The precursor of AIP, AgrD, consists of the AIP flanked by an N-terminal [Formula: see text]-helical Leader and a charged C-terminal tail. AgrD is matured to AIP by the action of two proteases, AgrB and MroQ. AgrB cleaves the C-terminal tail and promotes the formation of a thiolactone ring, whereas MroQ cleaves the N-terminal Leader in a manner that depends on the four-amino acid linker immediately following a conserved IG helix breaker motif. However, the attributes of AgrD that dictate the sequence of events in peptide maturation are not fully defined. Here, we used engineered AgrD peptide intermediates to ascertain the sufficiency of MroQ for N-terminal peptide cleavage, peptide export, and generation of mature AIP. We found that MroQ promotes the removal of the N-terminal Leader peptide from both linear and cyclic peptide intermediates, while peptide cyclization remained essential for signaling. The expression of the Leader peptide in isolation was sufficient for MroQ-dependent cleavage proximal to the four-amino-acid linker. In addition, active site mutations within AgrB destabilized full-length AgrD and thiolactone-containing intermediates and prevented the release of the Leader peptide. Altogether, our data support a tandem peptide maturation event involving both MroQ and AgrB that appears to couple protease activity and export of bioactive AIP.IMPORTANCEThe accessory gene regulatory (Agr) system is important for S. aureus pathogenesis. Activation of the Agr system requires recognition of a cyclic peptide pheromone, which must be fully matured to exert its biological activity. The complete events in cyclic peptide maturation and export from the bacterial cell remain to be fully defined. We and others recently discovered that the membrane peptidase MroQ is required for pheromone maturation. This study builds off the identification of MroQ and considers the attributes of the pheromone pro-peptide that are required for MroQ-mediated processing as well as uncovers features important for peptide stability and export. Overall, the findings in this study have implications for understanding bacterial pheromone maturation and virulence.

Characterization of MroQ-Dependent Maturation and Export of the Staphylococcus aureus Accessory Gene Regulatory System Autoinducing Peptide.

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. In a prior screen for S. aureus virulence factors, we identified MroQ, a putative peptidase. A ΔmroQ mutant closely resembled a Δagr mutant and had significant defects in AIP production in an Agr-I strain. Here, we show that expression of AgrD-I in a ΔmroQ mutant leads to accumulation of an AIP processing intermediate at the membrane that coincides with a loss of secreted mature AIP, indicating that MroQ promotes maturation of AgrD-I. MroQ is conserved in all Agr sequence variants, suggesting either identical function among all Agr types or activity specific to Agr-I strains. Our data indicate that MroQ is required for AIP maturation and activity in Agr-I, -II, and -IV strains irrespective of background. However, MroQ is not required for Agr-III activity despite an identifiable role in peptide maturation. Isogenic Δagr and Δagr ΔmroQ strains complemented with Agr-I to -IV validated the critical role of MroQ in the generation of active AIP-I, -II, and -IV but not AIP-III. These findings were reinforced by skin infection studies with mice. Our data substantiate the prevailing model that MroQ is a mediator of cyclic peptide maturation.

Control of Staphylococcus aureus Quorum Sensing by a Membrane-Embedded Peptidase.

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-1ß 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.