Staphylococcus aureus is a gram-positive bacterium that is part of the normal healthy flora but that can become virulent and cause infections by producing biofilms and toxins. The production of virulence factors is regulated by cell-cell communication (quorum sensing) through the histidine phosphorylation of target of RNAIII-activating protein (TRAP), which is a 21-kDa protein that is highly conserved among staphylococci. Using microarray analysis, we show here that the expression and phosphorylation of TRAP upregulate the expression of most, if not all, toxins known to date, as well as their global regulator agr. In addition, we show here that the expression and phosphorylation of TRAP are also necessary for the expression of genes known to be necessary for the survival of the bacteria in a biofilm, like arc, pyr, and ure. TRAP is thus demonstrated to be a master regulator of staphylococcal pathogenesis.Staphylococcus aureus is a gram-positive bacterium that is part of the normal flora of the skin, but it can become pathogenic and cause fatal diseases once it forms a biofilm and/or produces toxins (18,25). Biofilm formation and toxin production are regulated by a quorum-sensing mechanism, where molecules produced and secreted by the bacteria (autoinducers) reach a threshold concentration and activate signal transduction pathways, leading to activation of the genes that encode virulence factors (22,27,32,35,37).To date, two staphylococcal quorum-sensing systems (SQS) have been described. SQS 1 consists of the autoinducer RNAIII-activating protein (RAP) and its target molecule, target of RNAIII-activating protein (TRAP) (4,5,20,21). SQS 2 consists of the molecules encoded by agr (28, 29). The bacteria secrete RAP, a 33-kDa protein, as they multiply (23); when RAP reaches a threshold concentration (in the mid-exponential phase of growth), RAP induces the histidine phosphorylation of its target molecule TRAP (5, 20). The phosphorylation of TRAP leads, in an as-yet-unknown mechanism, to the synthesis of SQS 2, which is composed of the products of the agr system. agr encodes two divergently transcribed transcripts, RNAII and RNAIII (28,29). RNAII encodes AgrA, AgrC, AgrD, and AgrB, where AgrD is a propeptide that yields an autoinducing peptide (AIP) that is processed and secreted with the aid of AgrB. Once agr is activated and AIP is secreted, AIP induces the phosphorylation of its receptor AgrC, leading to the production of the regulatory RNA molecule termed RNAIII (28). RNAIII upregulates the production of numerous secreted toxins (28). SQS 1 and SQS 2 interact with one another because once AIP is made in the mid-exponential phase of growth, it indirectly downregulates the phosphorylation of TRAP (5). The interplay between the phosphorylation of TRAP and AgrC by their respective autoinducers, RAP or AIP, regulates the expression of adhesion molecules or toxins Like typical sensors of classical two component systems, TRAP is histidine phosphorylated in the presence of RAP (5, 20); immunoelectron microscopy and West...

Staphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP ؊ strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP ؊ strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers.

Staphylococcus aureus are Gram-positive bacteria and cause diverse serious diseases in humans and animals through the production of toxins. The production of toxins is regulated by quorum sensing mechanisms, where proteins such as RNAIII activating protein (RAP) are secreted by the bacteria and induce virulence. Antibodies to RAP have been shown to protect mice from infection, but the molecular structure of RAP was not known and hindered vaccine development. To characterize RAP, recombinant protein was made and tested for its ability to induce genes important for pathogenesis (agr). In addition, monoclonal antibodies were produced to identify its cellular localization. Results shown here indicate that RAP is a 277-aa protein that is an ortholog of the ribosomal protein L2. Like the native molecule, recombinant RAP activates the production of RNAIII (encoded by agr). Using RAP specific monoclonal antibodies we demonstrate that RAP is continuously secreted and while RAP is expressed also in other bacteria (like Staphylococcus epidermidis, Staphylococcus xylosus and Escherichia coli), it is secreted to the culture medium only by S. aureus. Our results show that the ribosomal protein L2 has an extraribosomal function and that when secreted RAP acts as an autoinducer of virulence to regulate S. aureus pathogenesis.

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