Characterization of RAP, a quorum sensing activator of Staphylococcus aureus1

Abstract

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.

A Nonsense Mutation in agrA Accounts for the Defect in agr Expression and the Avirulence of Staphylococcus aureus 8325-4 traP :: kan

“…Rap-Trap is thus considered to represent a second agr activation pathway, designated "SQS1" (8), which is superficially similar to the second activating pathway in the competenceregulating system of Bacillus subtilis, which uses the CSF peptide (10).…”

Mutation of traP in Staphylococcus aureus Has No Impact on Expression of agr or Biofilm Formation

“…The traP gene reportedly encodes a response regulator (TraP) that is triply phosphorylated as a result of the accumulation of a protein designated RAP (RNAIII-activating peptide), resulting in activation of the agr regulatory system and induction of toxin synthesis (21). A number of studies have also concluded that clinical isolates of S. aureus are responsive to RAP and that a modified peptide derivative designated RIP (RNAIIIinhibiting peptide) can be used to limit phosphorylation of TraP and thereby limit induction of agr, the capacity to form a biofilm, and virulence (1,3,12,14,24).To the extent that both RAP and RIP reportedly function by modulating the activity of TraP, studies indicating that clinical isolates are responsive to RAP and RIP would suggest that the traP regulatory system functions in the same fashion in most if not all S. aureus strains. However, the primary focus to date, particularly in terms of direct mutagenesis studies, has been on derivatives of the prototype 8325-4 laboratory strain such as RN6390, and recent studies from our laboratory have confirmed that regulatory circuits in RN6390 are different than those observed in at least some clinical isolates (9, 11).…”

Transcriptional Profiling of Target of RNAIII-Activating Protein, a Master Regulator of Staphylococcal Virulence

“…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.…”