Clone And Expression Of PBP2a C-terminal Fragment In E.coli, And Screening Anti-Methicillin Resistant Staphylococcus Aureus (MRSA) Components From Traditional Chinese Herbs

Objective Methicillin Resistant Staphylococcus aureus (MRSA) infection, with high mortality, is a common complication in patients with extensive deep burns and severe trauma. The mechanism of MRSA resistance is associated with the abundant PBP2a, a special penicillin binding protein that expressed by mecA gene. PBP2a is composed with 668 amino acids, and its molecular weight is 76.1 kDa. PBP2a has low affinity to all antibiotics. The soluble PBP 2a (644 amino acids, 74 kDa) has three domains. They are an N-terminal lobe, a centralized non-penicillin binding domain of unknown function and a C-terminal transpeptidase domain. Of particular interest is the C-terminal transpeptidase domain, which has a folding pattern that is typical of the PBP transpeptidases and the serineβ-lactamases. PBP2a provides complementary transpeptidase activity for the cells in order that cell wall synthesize although other PBPs were inhibited byβ-lactams. MRSA strains account for >60% of S. aureus clinical isolates in Japan, Singapore and Taiwan, >50% in Italy and Portugal. These isolates are typically multidrug resistant and often are treated only by glycopeptides such as vancomycin. However, vancomycin-resistant MRSA strain had come truth in Japan in 1996 (followed by similar strains isolated in the United States in 2002) , and no effective drug can cure this kind of infection caused by MRSA. Therfore, it is the need to discover new antibiotics or drugs to decrease resistance of MRSA. Among literateurs, there are a lot of reports that traditional Chinese herbs possessed anti-MRSA effects. So it is possible to get new drugs to kill MRSA or decrease resistance of MRSA.With these considerations in mind, we undertook the current study to construct a recombinant vector containing C-terminatio of mecA gene and express it in E.coli M15.Using the biosensor technology to screen anti-PBP2a components from traditional Chinese herbs, and to sort them according to the content of anti-MRSA effective materials.Methods①the sensitivities of MRSA to antibiotics were tested by microdilution method.②mecA gene of MRSA was identified by using PCR method.③C-terminatio of mecA DNA was amplified by PCR method and inserted into expression vector pQE30. The recombinant was identified by PCR method and enzyme digestions. E.coli M15 was transformed with the positive reconstructed expression vector pQE30-mecA and induced with 1 mmol/L IPTG. The induced protein PBP2a was identified by SDS-PAGE and Western blot .The soluble protein was purified with Ni-NTA agarose.⑤Dissoluble PBP2a was immobilized onto the surface of biotin cuvette for establishing target. The tannins were removed from aqueous extracts of 115 traditional Chinese herbs, and then the remainders were tested by affinity biosensor. Aqueous extractions of the TCM with high affinity for PBP2a were incubated with PBP2a(50 ng/ml)at 37℃for 30 min, subsequently the mixtures were tested by affinity biosensor, too.⑦The activites of the fractions were isolated by chromatography from Coptis chinensis. After the activites of the fractions were evaluated, the most active fraction of Coptis chinensis was confirmed.Results①We found that MRSA was resistant to all of oxacillin, gatifloxacin, benzylpenicillin, sulbactam sodium-ampicillin sodium, tienam, but it was sensitive to vancomycin.②Among 26 MRSA clinical isolates, there were 22 isolates which mecA gene was positive.③Positive recombinant plasmid pQE30-mecA was successfully constructed. The target protein of 39.7 kDa with 6-His tag was confirmed by SDS-PAGE, Western blot and PMF. And the target PBP2a had transpeptidase activity.④Dissoluble PBP2a was immobilized onto the surface of biotin cuvette.⑤Among the 115 traditional Chinese herbs, twenty-five ones such as radix ilecis pubescens, wild buckwheat, Tripterygium wilfordii possessed the higher binding activity for PBP2a, and seven ones such as airpotato yam, Chinese gall and scutellariae barbatae, herba contained more specific binding-PBP2a components.⑥MICs ofβ-lactams against MRSA could be significantly decreased when components from above seven herbs were added.⑦Among components from above seven herbs, the fraction from Coptis chinensis had most effective ability against MRSA.Conclusions The soluble PBP2a from MRSA was successfully expressed in E.coli M15 and purified. Biosensor technology was a fast, highly effective, objective and feasible method. Seven traditional Chinese herbs possessed components with specific affinity to PBP2a, and the fraction from Coptis chinensis had most effective ability against MRSA.