Search For Anti-methicillin Resistant Staphylococcus Aureus (MRSA) Potentiator From Traditional Chinese Herbs Targeting Multidrug-resistant Protein PBP2a

Methicillin-resistant Staphylococcus aureus (MRSA) is a very important pathogen in nosocomial infection, and one common cause of deep burns infections and severe trauma infections. Nosocomial infection caused by MRSA, among Staphylococcus aureus, become more and more popular, especially in intensive care units and burn wards. The detective rate of MRSA is increasing gradually, even up to over 90%. This issue brings great challenges to the clinical treatmant and nursing. Actually, the senile, the severe, the immunological- deficient and the neonatal, are high-risk groups with MRSA infection. The iatrogenic factors such as longtime administration of antibiotics, invading manipulation and sterilisation and isolation, can result in the growth of MRSA infection.The mechanism of MRSA resistance is mainly associated with the abundant PBP2a, a special penicillin-binding protein that encoded by mecA gene. PBP2a is composed of 668 amino acids, and its molecular weight is 76.1 kDa. PBP2a has low affinity for all beta-lactam antibiotics. The soluble PBP2a (644 amino acids, 74 kDa) has two domains. One is an N-terminal lobe, a centralized non-penicillin binding domain of unknown function, the other is 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 serine beta-lactamases. PBP2a provides complementary transpeptidase activity for the cells so that cell walls can be synthesized although other PBPs are inhibited by beta-lactams, thus result in drug resistance. MRSA clinical isolates are typically multidrug resistant and could only be treated with glycopeptides such as vancomycin. However, vancomycin-resistant MRSA strain had emerged 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. Therefore, it is very important and significant to search for new agents that inhibit PBP2a in order to restore the susceptibility of antibiotics in MRSA or decrease MRSA resistance.ObjectiveConsidering that PBP2a plays an important role in MRSA resistance, we undertake the current study to express the soluble PBP2a from clinical MRSA isolate. Using the biosensor technology to screen traditional Chinese herbs with high affinity for PBP2a, and then search for anti-MRSA potentiator to restore antibiotics susceptibility in order to provide a new treatment for MRSA.Methods1. The expression, purification and identification of the soluble PBP2a.The mecA gene fragments including bases 76 to 2007, encoding soluble PBP2a, were amplified by PCR to construct prokaryotic expression vector pQE30-mecA. After the positive recombinant identified by enzyme digestion was sequenced correctly, the recombinant was transformed into E.coli M15. The bacteria were induced with 1 mmol/L IPTG, and the soluble PBP2a was expressed. After the protein was purified with Ni-NTA agarose, its purity, amino acid sequences and molecular wieght were identified. Subsequently, biological activities of the PBP2a such as the transpeptidase and beta-lactamase activities were respectively detected by spectrophotometric method, and binding activities to antibiotics in vitro were observed through double micropore dilution method and confocal technology.2. Screening of anti-MRSA traditional Chinese herbs targeting PBP2a.The soluble PBP2a was immobilized onto the surface of carboxymethyl dextran cuvette as a target. According to the affinity capacities of 26 traditional Chinese herbs for PBP2a, 10 herbs were selected to test their antibacterial efficiency, then the specific anti-MRSA herb was chosen out.3. Isolation of the anti-MRSA components from Spica Prunellae and evaluation of their activities.The affinities of aqueous extractions for PBP2a from different parts of Spica Prunellae were firstly measured by biosensor. Their antibacterial effects were evaluated through synergetic antibacterial experiment in order to confirm the active components for the next study. The anti-MRSA components from entire plants of Spica Prunellae were isolated through macroporous adsorptive resins AB-8, followed by affinity tests, antibacterial and synergetic antibacterial experiments. At the same time, the material attribute was identified. Then the growth curves of MRSA and synergetic antibacterial effects of the active component combined with beta-lactams were observed respectively. Finally, the possible mechanism of the component, as anti-MRSA potentiator, was approached by observing the morphology of MRSA through transmission electron microscope.Results1. After the mecA gene fragment including bases 76 to 2007 had been amplified by PCR, the recombinant pQE30-mecA was successfully constructed. By gene sequencing it showed that the mecA fragments consisted of 1932 bp as expected, with 9 site-mutations. The recombinant protein was 96% homology with the one in GenBank. Then, soluble PBP2a was efficiently expressed in E.coli M15. The purity of recombinant PBP2a was up to 90.6%. The N-terminal amino acid sequences were MRGSH HHHHH GSKDK as expectated. Its molecular wieght was identified as 74 kDa by mass spectrography analysis. The recombinant PBP2a had transpeptidase and beta-lactamase activities, and possessed some binding effects to beta-lactam antibiotics in vitro.2. The soluble PBP2a was successfully immobilized onto the surface of carboxymethyl dextran cuvette. 10 traditional Chinese herbs with high affinity for PBP2a were selected out from 26 herbs. Scutellaria, Spica Prunellae and Coptis exerted better anti-MRSA effects, while Spica Prunellae showed the best synergetic antibacterial effect with oxacillin among 10 herbs.3. The active components of Spica Prunellae were mainly from the ear of plants, but there was no difference from the entire plants in anti-MRSA. Therefore, SP2 fraction was isolated from entire plants of Spica Prunellae through macroporous adsorptive resins AB-8. This fraction not only had high affinity for PBP2a, but also could decrease the MICs of oxacillin, penicilin, ampicillin and sulbactam sodium-ampicillin sodium on MRSA by 256, 8, 16 and 4 times respectively. Their FIC were 0.007, 0.151, 0.064 and 0.276. SP2 fraction was water-soluble and consisted of some phenolic hydroxyl groups, with the molecular wieght range of 3~5 kDa. SP2 could intensify antibacterial effects of antibiotics on MRSA by destroying their cell walls.ConclusionsThe soluble PBP2a from clinical MRSA isolates has been successfully expressed by gene recombination technique. It is feasible to sreen anti-MRSA traditional Chinese herbs targeting the multidrug-resistant protein PBP2a through biosensor technology. Using this screening technique, Spica Prunellae, with high affinity for PBP2a and the best synergetic anti-MRSA effect with oxacillin, is selected out of 26 traditional Chinese herbs. Furthermore, water-soluble active component SP2 has been derived from entire plants of Spica Prunellae, which could enhance the antibacterial effects of beta-lactams on MRSA. Molecular weight of the active component in SP2 ranges from 3 to 5 kDa. The compound as an anti-MRSA potentiator may be discovered by further isolation and purification in the future.