The Research On The Anti-S. Aureus. Infection

The Gram-positive bacteria Staphylococcus aureus (S. aureus) is an important pathogen, causing a variety of diseases, such as endocarditis, septic arthritis, and toxic shock syndrome. In America, hospital infections by S. aureus alone surpass 500,000 person-times every year.Clinically, application of combined different antibiotics is a major way to cure S. aureus infection, but the effect is often unideal because most of Staphylococcus aureus have the character of drug-resistance, which results from abuse of antibiotics ironically.Nowadays, how to control S. aureus infection is a question to answer desperately. It was demonstrated that Staphylococcal pathogenesis primarily involves the production and secretion of toxins that damage or lyse host cells or interfere with the immune system, enzymes that degrade tissue components, and cell wall-associated proteins that may be involved in adhesion and protection against host defenses. The expression of most of these virulence factors is regulated by at least one well-characterized global regulator, agr. As cells enter the postexponentical phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The agr locus consists of two divergent transcription units, RNAâ…¢ and RNAâ…¢. RNAâ…¢ acts primarily at the level of target gene transcription and independently regulated the transcription of exoprotein.RAP(RNAâ…¢ activating protein) is a -38KD S. aureus secretory protein which could activate the transcription of RNAâ…¢ in S. aureus. Now it was found that RAP activated the toxin production via its sensor TRAP (Target of RAP), a membrane-associated -21 kD protein. As the cell multiplies and the colony grows, the cells secrete RAP. When RAP reaches a threshold concentration, it induces the histidine phosphorylation of its target molecular TRAP and then the agr is activated. It was shown that the antibodies of RAP and TRAP could decrease the secretion of exotoxin. It was suggested that inhibitor of RAP and TRAP may be the useful way to cure the Staphylococcus aureus infecion.We purified RAP from a wild type S. aureus as the method of Naomi Balaban. It was tested that RAP purified by us could activated RNAâ…¢ transcription by Northern blot. The polyclonal antibodies against to RAP could deduce the level of RNAâ…¢ in S.aureus. The binding peptides of RAP were selected by phage display, which were checked if they could inhibit RAP activity by testing the level of RNAâ…¢. It was found that phage clone 15 has the highest activity. The GST fusion of this peptide(GST-15) was expressed in E.coli which could specifically bind to RAP. We also chemically synthesize the peptide 15. The results of Northern blot showed that both GST-15 and synthesized peptide 15 could reduce the activity of RAP. Our results showed that GST-15 could suppress infection in vivo in the murine cutaneous S. aureus infection model. The mean size of lesions in the mice treated with GST- 15 was smaller than that in control animals or non-related GST-fusion peptide group. The difference between experimental group and control groups was significant. The Chinese patent on binding peptides of RAP was already applied and the application number of the patent is 01119975.x.The trap gene was obtained by PCR from the genome of RN6390B strains. The trap gene was sequenced and cloned into the pET-28a vector. The soluble TRAP protein was expressed in E.coli, which could be specifically bind to antibodies provided by Dr. Naomi Balaban, the trap and rap discover. It was shown that the polyclonal antibodies of the TRAP prepared by us could specifically bind to TRAP protein existing in four different S. aureus strains. The binding peptides of TRAP were screened by phage display. The peptide T2 could specifically deduce the phosphorylation of TRAP in strain RN6390B. The level of exotoxins in RN6390B was decreased by peptide T2 by tested in MDBK cell model.We also identified the epitope and mimotope of TRAP by phage display. The binding peptides of polyclonal antibodies ag...