The Primary Study On A Virulence Regulon-RAP And TRAP From Staphylococcus Aureus

Staphylococcus aureus is a common Gram positive pathogen that cause pathologies from minor skin infections to life-threatening toxic shock. It accounts for more than 80% of purulent cases. Because of survival pressure, S. aureus become resistance to most of the traditional antibiotics, a question in dire need of resolve in clinic. The pathogenesis of S. aureus is essentially due to the expression of numerous virulence factors, including exotoxins, proteases, and surface adhesin et al. They are expressed in a fashion of growth phase dependent manner, which is also called quorum sensing phenomena. The blockade of quorum sensing offers a novel strategy for attenuating infection, and it need to gain a comprehensive understand toward quorum sensing mechanism. In S. aureus, many global regulator systems are responsible for controlling virulence gene expression via quorum sensing, among which agr (accessory gene regulator) system plays a central role. It is also a typical two-component regulatory system that is self-activated. The key molecule in agr is RNAIII, an effector that cause cell-density dependant phenotype changes.Balaban's research group reported a protein that could activate agr system, which is encoded outside of agr system, with molecular weight of about 38kD, and named RAP (RNAIII-activating protein) according its activity. They elucidated that RAP was continuously secreted by S. aureus, and once the concentration of extracellular RAP reached a threshold, it phosphorylated a protein of about 21kD(accordingly, the protein is named TRAP in terms of target of RAP). Thereafter, phosphorylated TRAP activated transcription of RNAIII, and regulated expression of virulent factors. In strains of trap minus mutant, RAP couldn't increase RNAIII level. But due to the controversy about RAP, there is still a long way to go to clarify RAP-TRAP pathway at large. .According to the method reported by Balaban, we purified the components that could increase RNAIII transcription from the culture medium of S. aureus clinicalisolate strain 04018 (we termed the active components as RAP like proteins). We screened the binding peptides of the active components by phage display, and found that the screened R15 peptide could inhibit RNAIII transcription in S. aureus, as well as protect hairless mouse from S. aureus infection on the skin model. We also identified ~38kD proteins from the active components by bio-mass spectrum. The purpose of this research is to gain novel insights into RAP and TRAP on the basis of the above studies.Firstly, to confirm whether or not there exists RAP, and to realize more about RAP, we expressed the two proteins of RNAIII activating components (beta-hemolysin, and panton-Valentine leucocidin S) which we identified before in prokaryotic expression system. Northern Blot results suggested that neither protein could activate RNAIII transcription in S. aureus. But we found that the concentrated >10kD component from S. aureus cultured medium by ultrafiltration still accounted for about 30% of total RNAIII activation activity. It is suggested that there might be another RNAIII activating substance besides AIP (auto-inducing peptide, a peptide of 7 to 9 amino acids in length). We concluded that some special characteristics of RAP bottlenecked the study on itself.By using anti-TRAP polyclonal antibodies, we confirmed the location of TRAP in S. aureus cells through immunoelectronmicroscopy and Western blot, and fished out its extracellular binding protein through different co-immunopurification or co-immuno-precipitation. We found that TRAP might be a surface protein or a secreted protein, which was different from the predicted result by bio-informatics. We also found that TRAP could bind extracellular autolysin protein. The polyclonal antibody to TRAP could increase the expression level of autolysin (ATL) in S. aureus culture medium, but had no effect on the level of ATL non-covalently linked to the cell wall, so we speculated that TRAP and ATL might form a new quorum sensing pathway. Further researc...