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

Abstract

In <i>Staphylococcus aureus,</i> virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (<i>agr</i>) 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 <i>agr</i> QS (MroQ), an integral membrane protease recently implicated in the <i>agr</i> response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in <i>agr</i> 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 <i>agr</i> specificity group identity. Collectively, our study adds to the molecular understanding of the <i>agr</i> response and <i>Staphylococcus aureus</i> virulence.