Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis

Abstract

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator ( agr ) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1–32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Critical Assessment of the Prospects of Quorum-Quenching Therapy for Staphylococcus aureus Infection

“…Four different subgroups of Agr exist with considerable differences in the amino acid sequence of, and to some extent, length of the mature autoinducing peptide (AIP) [ 21 ]. The AIP can be a hepta- to nonapeptide which is exported likely also by AgrB and trimmed at the N-terminus with the help of the non-Agr-encoded membrane-located protease MroQ, at least for the AIPs of groups 1, 2, and likely 4, while the proteolytic maturation of the product of the AgrB thiolactone-introducing step remains unknown for subgroup 3 [ 24 , 25 ]. The AIP activates the AgrC-AgrA two-component system by binding to the membrane histidine kinase AgrC, which in turn phosphorylates AgrA, a DNA-binding response regulator that when phosphorylated binds to and activates the promoters driving transcription of agrBDCA and RNAIII [ 18 ].…”

Proteases Involved in Leader Peptide Removal during RiPP Biosynthesis

“…The necessity of MroQ in AIP production was demonstrated via generation of an mroQ knockout; deletion of the protease eliminated or severely reduced group I and II AIP production, which was restored with mroQ complementation. 156 In vitro studies further showed that substrate specificity of MroQ is dependent on the linker peptide connecting the N-terminal αhelix and the C-terminal thiolactone of its substrate; AIPs containing the helix and C-terminal macrocycle of one AIP group and the linker of another AIP group were efficiently processed. 156 However, whereas MroQ was shown to be active with groups I and II, and by homology group IV, AIP substrates, maturation of group III AIPs remains unresolved.…”

“…The second step of AIP biosynthesis requires cleavage of the LP by another integral membrane metalloprotease (M79) MroQ. The necessity of MroQ in AIP production was demonstrated via generation of an mroQ knockout; deletion of the protease eliminated or severely reduced group I and II AIP production, which was restored with mroQ complementation . In vitro studies further showed that substrate specificity of MroQ is dependent on the linker peptide connecting the N-terminal α-helix and the C-terminal thiolactone of its substrate; AIPs containing the helix and C-terminal macrocycle of one AIP group and the linker of another AIP group were efficiently processed .…”

Characterization of an Autoinducing Peptide Signal Reveals Highly Efficacious Synthetic Inhibitors and Activators of Quorum Sensing and Biofilm Formation in Listeria monocytogenes

“…Recent work implicates another membrane-associated peptidase in this process (MroQ), at least in certain S. aureus strains. , As the bacterial population grows, the extracellular AIP concentration likewise increases to eventually reach a local threshold that can activate the two-component signaling system formed by AgrC and AgrA. AgrC is a transmembrane receptor histidine kinase that, upon binding of AIP to its extracellular domain, trans -autophosphorylates its cytosolic domain and then transfers the phosphoryl group to its partner intracellular response regulator, AgrA .…”