Screening And Identifying The Proteins Interacting With PBP2a In The Methicillin-resistant Staphylococcus Aureus

With the widespread use of the conventional antibiotics in clinic, bacterial resistance to antibiotics has become a challenge to the anti-infection treatments for clinical infectious disease. Staphylococcus aureus is one of the important opportunistic pathogens. With the development of drug-resistance, the methicillin-resistant Staphylococcus aureus (MRSA) has become the most important pathogens of nosocomial infection. Because the multi-resistant strains of MRSA are emerging, treatments have become more difficult. With the extensive use of vancomycin,the vancomycin-resistant Staphylococcus aureus (VRSA) infection have been reported.Thus, finding a new reagent to MRSA infection is necessary. The resistance mechanism of MRSA is complex, including drug-target changes, producing passivation enzymes as well as active effluxing,and so on. Among them, the change of target sites for antibiotics is a major mechanism. Typically, the bacterial cell wall is built by Penicillin-binding proteins (PBPs), a family of enzymes which provide transglycosylase (TGase) and transpeptidase (TPase).Theβ-lactam antibiotics can selectively bind to TPase domain of PBPs,make it acetylated and inactivated, block the synthesis of bacterial peptidoglycan, and then kill the sensitive bacteria. MRSA obtains a mecA gene from an unknown host, which encodes a new PBP,called PBP2a,whose TPase domain has low affinity with theβ-lactam antibiotics.When the normal PBPs is combined with theβ-lactam antibiotics and losses its TPase activity, PBP2a can provide a TPase function for the synthesis of cell peptidoglycan and maintain the growth and survival of Staphylococcus aureus in the existance ofβ-lactam antibiotics.That is, the expression of PBP2a in MRSA is involved in the drug resistance. Many studies have showed that, in MRSA, PBP2a is required toβ-lactam resistance. However, how PBP2a participates in the drug resistance of MRSA? Are there some activation or inhibition regulatory factors? In this study,we cloned the TPase domain of MRSA PBP2a into pRBR vector to construct a bait plasmid to screen a MRSA genomic library constructed in pRAC vector using bacterial two-hybrid system (B2H), and nine candidates were screened out that might interact with PBP2a. One of the candidates (P2) was confirmed to have the interaction with PBP2a by the pull-down assay.The main contents and results are as followings:1. Screen the proteins that interacts with PBP2a using a bacterial two-hybrid system:①Using the pMD-PBP2a plasmid (carrying the coding sequence of TPase domain of MRSA PBP2a) as template, the fragment encoded PBP2a TPase domain of MRSA strain N315 was amplified by PCR,digested with BamHI / XhoI and cloned into pRBR plasmid vector to obtain a bait plasmid pBR-PBP2a. The bait plasmid was identified by PCR, restriction enzyme digestion and sequencing.②The genomic DNA of MRSA strain N315 was extracted and partially digested with Sau3A I,then the fragments between 500~1500bp were recovered, and ligated into pRAC vector to get a genomic DNA library. 50 clones were selected randomly and the colony PCR amplification was used to detect the distribution of library inserts with pRAC sequencing primers. The constructed MRSA N315 strains genomic library constructed were 9 times of coverage,which meeted the needs for subsequent library screening.③The bait plasmid pBR-PBP2a and genomic library co- transformed KS1 cells (a reporter strain with a genomic integrated lacZ gene under the control ofλoperator) were cultured in agar plates with 50mg/ml X-gal,100μmol/L IPTG,100μg/ml AMP,50μg/ml Kan,34μg/ml Cl and the blue colonies were picked up for LacZ assay. The empty vector pRAC transformed into pBR-PBP2a /KS1 served as a negative control and the KS1 carrying pACλCI-βflap and pBRL28 (with the confirmed interaction between chlamydialβflap and CT663 expressed by pACλCI-βflap and pBRL28 respectively) used as positive control.④The colony with LacZ activities significantly increased after induction of IPTG was selected as the a prey. Then the pRAC plasmid with a candidate insertion was isolated and sequenced. After performing LacZ assay upon 200 colonies, 9 prey plasmids were obtained, named as pAC-p1 ~ pAC-p9.⑤The 9 inserted sequences were subjected to BLAST analysis and we found that all of them came from the MRSA N315 genome.The longest insert was 649bp and the shortest one was 335bp. There were 14 genes related with the 9 inserts and 10 of them encoded proteins with unknow functions. Among the 4 familiar genes, one encoded dihydrodipicolinate synthase,one encoded dihydrodipicolinate reductase and the others encoded chromosome segregation SMC proteins. The peptides that might interact with PBP2a encoded by the 9 prey plasmids were composed of 14~46 amino acids.2. Identifying the interaction between PBP2a and prey peptide:①According to evaluation ofβ-galactosidase activity and sequence analysis, the peptide encoded by p2 was selected for further in vitro Pull-down experiments.The insert of p2 was amplificated by PCR and cloned into pGEX-6p-1 vector to construct the pGEX-p2 plasmid,then pGEX-p2 was transformed into E. coli BL21 competent cells to screen the recombinants.②The pGEX-p2/BL21 recombinant bacteria was induced for the expression of GST-P2 fusion proteins, and the optimal expression condition was explored. We find that the optimal expression condition of pGEX-p2/BL21 were 6 hours post induction by 500μmol/L IPTG at 28℃.③The recombinant GST-P2 fusion protein was expressed using pGEX-p2/BL21 engineered bacteria, purified by GST-Sepharose4B immunoaffinity chromatography and characterized by Western blotting with the polyclone sera against GST tag. As for negative control, the GST protein was expressed by pGEX-6p-1 and purified with GST-Sepharose4B resin.④The purified GST or GST-P2 proteins were imobilized by GST-Sepharose4B resin and subjected to capture PBP2-2a fusion proteins in a pull down assay. The results showed that the GST-P2 could pull down PBP2-2a determined by Western blot using the polyclone mouse sera against PBP2-2a.In the same condition, the purified GST protein could not pull down PBP2-2a.In summary, we successfully screened out the proteins that interact with PBP2a from MRSA genomic library using a bacterial two-hybrid system and the interaction between the two proteins was verified by in vitro Pull-down experiments. Our results facilitated further understanding the mechanism of PBP2a mediated the drug-resistance in MRSA.