Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone.

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.

Identification of the putative staphylococcal AgrB catalytic residues involving the proteolytic cleavage of AgrD to generate autoinducing peptide.

The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic alpha-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria.

Identification of a staphylococcal AgrB segment(s) responsible for group-specific processing of AgrD by gene swapping.

The four gene products of the accessory gene regulator (agr) P2 operon of Staphylococcus aureus assemble a quorum-sensing system: AgrA and AgrC resemble a two-component signal transduction system, and AgrB and AgrD are required to produce an autoinducing peptide. Upon activation, this quorum-sensing system positively regulates the transcription of the P2 operon as well as the P3 operon, whose transcript, RNAIII, regulates the expression of virulence genes. Four groups of S. aureus have been identified based on the agr sequences and the group-specific interaction between the autoinducing peptide and AgrC. AgrB is a transmembrane protein involved in the processing of AgrD propeptide, and its interaction with AgrD is also group specific. In this study, a series of chimeric AgrBs were constructed by swapping between group I and group II AgrBs, and these mutants were used to analyze the group-specific segment(s) in AgrB that was responsible for AgrD processing. Our results revealed that the first transmembrane alpha-helix and the extracellular loop 1 of group I AgrB were decisive in the specific processing of group I AgrD. In contrast, two hydrophilic segments of group II AgrB played a crucial role in the group-specific processing of group II AgrD. We also found that several chimeric AgrBs were capable of processing AgrD from both groups, suggesting that all AgrB homologues may utilize the same or a similar mechanism in the processing of AgrDs.

Membrane anchoring of the AgrD N-terminal amphipathic region is required for its processing to produce a quorum-sensing pheromone in Staphylococcus aureus.

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 alpha-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.

Transmembrane topology of AgrB, the protein involved in the post-translational modification of AgrD in Staphylococcus aureus.

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.