TraP Types Of Staphylococcus Aureus And Interaction Of TraP And Rap In Vitro

Target of RNAIII-activiting protein (TraP) is a 167-amino acid residue membrane-associated protein of Staphylococcus aureus. Multiple sequence alignment of traP indicates that the traP gene sequence is highly conserved. However, individual traP nucleotide and amino acid sequences can be divided into five major groups based on MseⅠrestriction fragment length polymorphisms (RFLP). There is no report on traP of S.aureus isolates from China in previous studies. RNAIII-activating protein (Rap) is a 33 kDa protein, which is secreted as an autoinducer. Rap could induce TraP phosphorylated on three conserved histidine residues, and regulate S.aureus pathogens. But there is no detail report about the direct interaction of TraP and Rap.In order to know the traP type of isolated S.aureus in China, traP gene of 26 dairy mastitis isolates and 4 representative strains were amplified by polymerase chain reaction and cloned into pMD18-T vector to be sequenced for further homologous analysis of sequence. The results showed that the homology of traP gene of 30 S.aureus strains could reach 85.5%. 30 S.aureus strains could be typed into 4 traP types based on MseⅠRFLP, 13 strains in type traPⅠ, 2 strains in type traPⅡ, 14 strains in type traPⅢ, and 1 strain in type traPⅤ, no type traPⅣwas found.In order to know whether TraP can directly interact with Rap, recombinant TraP and Rap were expressed and purified. Detection of TraP location by indirect Enzyme-linked Immunosorbent Assay (ELISA) with mice anti-TraP antibody prepared with recombined TraP protein showed that TraP exposes on the bacterium surface. To verify TraP binding with Rap in vitro, double-antibody sandwich ELISA with mice anti-TraP polyclonal antibody, tandem affinity purification, and agar diffusion were performed. The results showed that TraP could directly bind Rap in vitro. So as to identify interaction site of TraP binding with Rap, the secondary structure of TraP was analyzed by Protean of DNAStar and SWISS-PORT, and the tertiary structure of TraP and Rap were analyzed by SWISS-MODEL, peptides that could bind with TraP or Rap were showed by phage display. Furthermore truncated mutation and site-directed mutation of TraP were used to locate the detail site of TraP binding with Rap. The interaction site of TraP binding with Rap was localized on TraP Ser-75 and Thr-76. These results are useful for researches of the signal transduction pathway mediated by TraP and Rap, and vaccine anti S.aureus infection and targeting drug treating S.aureus infection.