Membrane Anchoring of the AgrD N-terminal Amphipathic Region Is Required for Its Processing to Produce a Quorum-sensing Pheromone in Staphylococcus aureus

Abstract

Quorum-sensing pheromones are signal molecules that are secreted from Gram-positive bacteria and utilized by these bacteria to communicate among individual cells to regulate their activities as a group through a cell density-sensing mechanism. Typically, these pheromones are processed from precursor polypeptides. The mechanisms of trafficking, processing, and modification of the precursor to generate a mature pheromone are unclear. In Staphylococcus aureus, AgrD is the propeptide for an autoinducing peptide (AIP) pheromone that triggers the Agr cell density-sensing system upon reaching a threshold and subsequently regulates expression of virulence factor genes. The transmembrane protein AgrB, encoded in the agr locus, is necessary for the processing of AgrD to produce mature AIP; however, it is not clear how AgrD interacts with AgrB and how this interaction results in the generation of mature AIP. In this study, we found that the AgrD propeptide was integrated into the cytoplasmic membrane by a conserved ␣-helical amphipathic motif in its N-terminal region. We demonstrated that membrane targeting of AgrD by this motif was required for the stabilization of AgrD and the production of mature AIP, although this region was not specifically involved in the interaction with AgrB. An artificial amphipathic peptide replacing the N-terminal amphipathic motif of AgrD directed the protein to the cytoplasmic membrane and enabled the production of AIP. Analysis of Bacillus ComX precursor protein sequences suggested that the amphipathic membrane-targeting motif might also exist in pheromone precursors of other Gram-positive bacteria.

The AgrD N-Terminal Leader Peptide of Staphylococcus aureus Has Cytolytic and Amyloidogenic Properties

“…1D). N-AgrD has also been predicted to adopt an amphipathic ␣-helical structure that is similar to those of the PSMs (12,38) (Fig. 1E).…”

“…The N-terminal region of AgrD is essential for directing the propeptide to the cell membrane (35), where the integral membrane endopeptidase AgrB removes the C-terminal tail, catalyzes thiolactone ring formation, and transports the AgrD AIP intermediate across the cell membrane (34,36). Once this intermediate peptide is secreted from the cell, the SpsB signal peptidase cleaves the N-terminal peptide to release the active form of AIP, leaving the leader peptide associated with the cytoplasmic membrane (37,38). Thus, the current model of AIP processing suggests that the N-terminal leader fragment is essential for targeting the propeptide to the cell membrane, but the fate of the peptide after AIP processing is unknown.…”

Quorum sensing in bacterial virulence

“…The autoinducer in the agr system is an oligopeptide that has been termed the autoinducing peptide (AIP), encoded by agrD. AIP is trimmed and secreted by AgrB, a membrane-bound protein (Ji et al, 1995;Saenz et al, 2000;Zhang et al, 2004). The active AIP is 7-9 aa, with a 5-membered thiolactone ring (Roux et al, 2009).…”

Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence

“…1A, black arrows), the peptide is ribosomally translated as a precursor, AgrD, in which the mature AIP sequence is flanked by an N-terminal leader peptide and a C-terminal recognition sequence (6). The N-terminal leader forms an amphipathic helix, which attaches the precursor to the inner leaflet of the cell membrane (7). In the first step, AgrD is processed by a membrane-integrated peptidase, AgrB, such that the C-terminal recognition sequence (AgrD C ) is removed with concomitant installation of the thiolactone (Fig.…”