Transmembrane Topology of AgrB, the Protein Involved in the Post-translational Modification of AgrD in Staphylococcus aureus

Abstract

The accessory gene regulator (agr) of Staphylococcus aureus is the central regulatory system that controls the gene expression for a large set of virulence factors. This global regulatory locus consists of two transcripts: RNAII and RNAIII. RNAII encodes four genes (agrA, B, C, and D) whose gene products assemble a quorum sensing system. RNAIII is the effector of the Agr response. Both the agrB and agrD genes are essential for the production of the autoinducing peptide, which functions as a signal for the quorum sensing system. In this study, we demonstrated the transmembrane nature of AgrB protein in S. aureus. A transmembrane topology model of AgrB was proposed based on AgrB-PhoA fusion analyses in Escherichia coli. Two hydrophilic regions with several highly conserved positively charged amino acid residues among various AgrBs were found to be located in the cytoplasmic membrane as suggested by PhoA-AgrB fusion studies. However, this finding is inconsistent with the putative transmembrane profile of AgrB by computer analysis. Furthermore, we detected an intermediate peptide of processed AgrD from S. aureus cells expressing AgrB and a 6 histidine-tagged AgrD. These results provide direct evidence that AgrB is involved in the proteolytic processing of AgrD. We speculate that AgrB is a novel protein with proteolytic enzyme activity and a transporter facilitating the export of the processed AgrD peptide.

Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

“…AIP is processed from the precursor peptide AgrD by AgrB and other proteases, and the AIP is secreted 26,27 . Extracellular AIP is detected by a cognate transmembrane-bound receptor histidine kinase, AgrC, that upon AIP binding, autophosphorylates and subsequently funnels a phosphoryl group to the partner response regulator, AgrA 28,29 (Fig.…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…aureus AIPs are generated from the precursor ArgD following two proteolytic cleavage events that remove the leader and tail segments of the propeptide. The protein AgrB removes the C-terminal tail of the propeptide (161,162), while removal of the Nterminal leader segment is catalyzed by the type I signal peptidase SpsB (Fig. 1D) (163).…”

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

“…Plasmid pLZ4005 contains an NdeIEcoRI DNA fragment (encoding the His 6 -T7 tag-Xpress TM epitope and multiple cloning sites sequence) of pRSET-A (Invitrogen) ligated to the 5Ј-end of agrD (16). Plasmid pLZ4011 was constructed as follows.…”

“…AgrB is required for the production of AIP (13) and functions as a protease that is involved in the proteolytic cleavage of AgrD (16). It has also been proposed that AgrB functions as an oligopeptide transporter that facilitates the secretion of mature AIP (10,16). However, the interaction between AgrD and AgrB and the mechanisms of the processing of AgrD and the secretion of mature AIP by AgrB remain unclear.…”

“…AgrD is the precursor of AIP, a peptide pheromone that is secreted from the bacteria into the culture medium (12,13) and that is the ligand of AgrC, a membrane protein with its N-terminal half, which has been proposed to contain the AIP-binding site, anchored in the cytoplasmic membrane via five transmembrane helices (14,15) and with its C-terminal half, which composes a histidine kinase domain, located in the cytoplasm (11). AgrB is required for the production of AIP (13) and functions as a protease that is involved in the proteolytic cleavage of AgrD (16). It has also been proposed that AgrB functions as an oligopeptide transporter that facilitates the secretion of mature AIP (10,16).…”