Design And Expression Of A Chimeric Drug Target Against MRSA And The Analysis Of Its Bioactivity In Vivo

Staphylococcus aureus is a major cause of hospital- and community-acquired infections worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) strains account for >80 % of S. aureus clinical isolates in China, >60 % in western countries. The bacterial cell wall is built by Penicillin-binding proteins (PBPs), a family of enzymes which provide transglycosylase (TGase) and transpeptidase (TPase) to be responsible for the construction of cross-linked peptidoglycan mesh that gives the bacterium its strength and shape. These enzymes are excellent drug targets because they are essential among bacteria, are accessible from the periplasm, and have no equivalent in mammalian cells. TPase is the well known target of theβ-lactam antibiotics. Unfortunately, MRSA can encode a new penicillin binding protein, called PBP2a, which shares lower sequence identity (<21 %) with the normal PBPs produced by S. aureus and shows unusually lowβ-lactam affinity and remains active to allow cell wall synthesis at normally lethalβ-lactam concentrations. In recent years, some researchers indicated that PBP2a only provid transpeptidase activity in the process of cell wall synthesis and PBP2 provid the transglycosylase activity. That's to say, the multi-drug resistance of MRSA is contributed by both PBP2 and PBP2a. If a new chimeric molecule carrying the PBP2 and PBP2a functional domains was designed and expressed, that will facilitate in the screening of inhibitors anainst TGase or TPase as an anti-MRSA drugs and make great sense to prevent and control the MRSA infection.Under the fully analysis of MRSA PBP2 and PBP2a functional domains, the PBP2 N-terminal transglycosylase domain and PBP2a C-terminal transpeptidase domain were cloned by PCR, respectively. TG-TPase chimeric gene was constructed by general molecular techniques and subcloned into prokaryotic, eukaryotic expression vectors. We expressed and initially purified the chimeric protein in prokaryon and eucaryou, and confirm the bioactivity of TG-TPase chimeric drug target in MSSA. These results established the foundation of screening anti-MRSA inhibitors by the enzymologic activities of the chimeric protein. The contents and results of this dissertation are as follows:1.The design of chimeric drug target:①The multi-aligment analysis of MRSA PBP2 sequence;②The structurial features and activity performance of MRSA PBP2 TGase domain;③The structure and drug-resistant mechanism of MRSA PBP2a TPase domain;④The design of the TG-TPase chimeric molecule.2.Expression of MRSA TG-TPase chimeric protein in E. coli:①Two gene fragments encoding PBP2a TPase domain and PBP2 TGase domain were amplified by PCR, respectively, and cloned into pMD18-T to generate pMD-TG and pMD-TP;②The pMD-TG-TP carrying TG-TPase chimeric gene was constructed by ligating the large fragment of pMD-TG/Xho I-Not I and the small insert of pMD-TP/Xho I-Not I. Then the interest TG-TPase chimeric gene was subcloned into pET22b+ and pET30b+ and transformed into Rosetta(DE3) plysS;③Fusion protein was expressed by engineering bacteria harboring the chimeric gene and about 90 % of the fusion protein was expressed as inclusion-body;④Fusion protein was purified to 90 % homogenicity by Ni-NTA affinity chromatography and evaluated by mass spectrogram and Western blotting analysis;⑤The purified fusion protein was renatured through dialysis and the concentration of the protein was determined by Bradford method;⑥In order to obtain the antibody against the fusion protein, Balb/c mice were immuned and the anti sera were prepared.3.Expression of TG-TPase chimeric gene in Pichia pastoris:①The TG-TPase chimeric gene was digested from pMD-TG-TP and inserted into pPIC9K vector to produce pPIC9K-TG-TP;②The recombinant plasmid was digested with Sal I and electrotransformated into Pichia pastoris strain GS115. The multicopy transformants were screened by adding gradually increasing the G418 concentration;③Then the recombinants was identified by performing PCR to test the chimeric gene in the genome of recombinant Pichia pastoris, doing RT-PCR to probe the gene of interest expression and SDS-PAGE analysis to see the band of interest. The results showed that a 1 923 bp fragment could be amplified by PCR from the chromosome DNA of recombinant Pichia pastoris. After induced by methanol, a 1 923 bp fragment could also be amplified by RT-PCR, but we failed in detection of the recombinant protein by SDS-PAGE analysis, that may due to the low expression of chimeric gene in Pichia pastoris.4.Expression and identification the biological activity of TG-TPase chimeric drug target in MSSA:①Two DNA fragments encoding PBP2a TP'ase domain and PBP2 TG'ase domain, respectively, were amplified by PCR with 2 pairs of new primers by adding the putative ribosome binding site to the 5'of PBP2 domain and cloned into pMD18-T to generate pMD-TG'and pMD-TP';②The pMD-TG-TP'carrying TG-TP'ase chimeric gene was constructed by ligating the large fragment of pMD-TG'/Xho I-Not I and the small fragment from pMD-TP'/Xho I-Not I;③T he recombinant plasmid pYT3-TG-TP'was constructed by subcloning the interest TG-TP'ase chimeric gene into pYT3 and transformed into DH5α. After identifying the recombinant plasmids by PCR, enzyme digestion and nucleotide sequencing, the recombinant plasmid was extracted and electrotransformated into Staphylococcus aureus strain N315;④The recombinant plasmid was extracted and electrotransformated into Staphylococcus aureus strain MSSA;⑤The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of several antibiotics, including oxacillin, imipenem, gatifloxacin,cefalexin,nafcillin,vancomycin hydrochloride,against the recombinant bacteria pYT3-TG-TP'/ MSSA as well as pYT3/MSSA and MSSA only were evaluated by well dilution assay. The results show that the values of MIC and MBC of the recombinant engineering bacteria had been changed in different degrees. Among these antibiotics, the values of MIC and MBC to oxacillin, cefalexin, gatifloxacin, nafcillin had been changed more obviously; meantime, the curve of growth inhibiting in vitro of pYT3-TG-TP'/MSSA is determined, showing the relationship between the different concentrations of oxacillin, cefalexin, gatifloxacin, nafcillin and the bacteria densities of pYT3-TG-TP'/MSSA, pYT3/MSSA and MSSA and the sterilizing effect in vitro of these antibiotics. The result of sterilizing in effect was identical to the value of MIC, proving the recombinant engineering bacteria pYT3-TG-TP'/MSSA had the multi-drug resistant activity.In conclusion, the TG-TPase chimeric drug target molecule was designed based on the structure and functions of MRSA PBP2 and PBP2a. The TG-TPase chimeric gene and the engineering bacteria pET22b-TG-TP/Rosetta and pPIC9k-TG-TP/GS115 were successfully constructed, the chimeric protein was expressed and purified from prokaryotic cells. The acquired recombinant chimeric protein may facilitate the analysis of TGase and TPase activities in vitro. Using a S. aureus shuttle vector pYT3, we performed the expression of TG-TP'ase chimeric gene in MSSA and the biological activity of this drug target was also evaluated by determining MIC and MBC. The result shows that the chimeric gene can introduce the drug resistance of MSSA to antibiotics, this observation may lay down great foundation for later screening of inhibitors against MRSA.