In Vitro Antibacterial Activity And Its Mechanism Of Recombination Protein PGRP Of Staphylococcal Aureus

Objective Staphylococcus aureus is a kind of invasive gram-positive pathogen,which can cause the community acquired and hospital acquired infections.As one of the most common pathogenic bacteria in the human purulent infection,it can cause skin purulent infection,pneumonia,pseudo membranous enteritis,pericarditis,even systemic infection,such as sepsis.The frequent use of antibiotics has led to drug resistance of Staphylococcus aureus,especially methicillin-resistant Staphylococcus aureus(MRSA),and the infection rate has been maintained at a high level.The emergence of multidrug resistance in S.aureus is generating an urgent need for alternative therapeutic methods for infections caused by this multi-drug resistant bacteria.Peptidoglycan recognition protein(PGRPs)is a kind of pattern recognition receptors.Peptidoglycan recognition protein of Staphylococcus aureus is a function fragment of Staphylococcus aureusautolysin,which is located in the amidase function domain.The aim of this study was to obtain recombinant protein PGRP of Staphylococcal aureus through prokaryotic expression system,to investigate its antibacterial activity in vitro and preliminary antibacterial mechanism,which can lay the foundation for peptidoglycan recognition protein as a new type of antimicrobial agents.Methods1.pgrp functional fragment forecast and conservative,specificity analysis:pgrp function domain of Staphylococcus aureusautolysin was predicted by NCBI Conserved Domain Search.Conservative analysis of functional fragment pgrp was finished by NCBI Blast.The specific primers of pgrp were designed according to Staphylococcal aureus autolysin gene sequences in GenBank(AC:D17366).In order to determine the specificity of this gene,pgrp was amplified by PCR from genome DNA ofStaphylococcal aureus(ATCC25923),Klebsiella pneumoniae(ATCC700603),Escherichia coli(ATCC25922),Pseudomonas aeruginosa(ATCC27853),Enterococcus faecalis(ATCC29212)and Streptococcus pneumoniae(ATCC49619),respectively.2.Prokaryotic expression and purification of recombinant protein PGRP:The specific primers of pgrpwere designed according toatlA gene sequence of Staphylococcal aureus recorded in GenBank,thenthe purpose gene fragment pgrp was amplified by PCR from the Staphylococcal aureus genome.The recombinant expression plasmid pET-32?(+)/pgrpwas constructed and transformed into BL21(DE3)to express PGRP.After induced by IPTG,the expressed protein PGRPwas separated and purified by SDS-polyacrylamide gel electrophoresis and electroeluting of bag filter.3.The bioinformatics analysis of recombinant protein PGRP:The bioinformatics of PGRP protein derived from pgrp were analysised by bioinformatics software(ProtParam?ProtScale?SignalP 4.1 server?Signal-3L?TMpred?DAS?NetPhos 2.0Server),such as the physical and chemical properties,signal peptide,transmembrane domain and phosphorylation sites.4.In vitro bactericidal experiment:The minimum bactericidal concentration(MIC)of rPGRP to S.aureus(ATCC25923)and 10 strainsclinical MRSA were determined by the broth microdilution method.To compare the bactericidal effect and differences,suspension of 30 clinical MRSA(final concentration of 5×10~4 CFU/ml)were cultivated wth recombinant protein PGRP(final drug concentrations 32?g/ml),and counted colonies at 1h,3h,5h and 12h,respectively.The results were statistically analyzed.5.Real-time fluorescent quantitative PCR:The specific fluorescence quantitative PCR primers were designed according to Staphylococcal aureus autolysin gene sequences in GenBank(AC:D17366).16S RNA was the housekeeping gene.To explore the preliminary bactericidal mechanism of rPGRP,cDNA of 16 strains clinical MRSA were used as PCR templates to compare pgrp gene expression with and without rPGRP.Results1.Functional domains pgrp was the research object,predicted through NCBI Conserved Domain Search.NCBI Blast results showed that pgrp exists in Staphylococcus aureus conservatively.Amplified stripe of pgrp was about 480 bp,while other strains amplified nothing.2.The coincidence rate was 95.7%between recombinant plasmid sequencing results and gene sequences in GenBank(AC:D17366),which showed that cloning plasmid pEASY-T1/pgrp and expression plasmid pET-32?(+)/pgrp were successfully built.rPGRP determined by SDS-PAGE were 20kDa,met the expected results.The concentration of rPGRP was 456?g/ml.3.The biological information analysis results showed that:PGRP,composed of155 amino acids,was a stable hydrophilic protein.Its relative molecular mass was17440.95 and theory pI was 5.45.There was no signal peptide in PGRP.PGRP is not a transmembrane protein.4.The MICs of rPGRP to Staphylococcus aureus(ATCC25923)was 64?g/ml;and to 10 strains clinical MRSA were 64?g/ml(4 cases),128?g/ml(1 case)and>128?g/ml and<256?g/ml(5 cases),respectively.Antibacterial test in vitro showed that rPGRP had certain inhibitory effects to Staphylococcus aureus standard strain and 30strains MRSA(bactericidal effect is very obvious and lasting to 12 h,all P values=0.000<0.01).5.The fluorescence quantitative PCR results of experimental group(with drug)and control group(without drug)showed that compared with the control group,pgrp gene expression of experimental group increased(p<0.05).The mechanism of bactericidal effect of rPGRP might be increasing the gene expression of itself to play a role of antibacterial.Conclusion1.pgrp gene is highly conserved and specific in Staphylococcus aureus,and recombinant protein PGRP can obtain from pET-32?(+)/pgrp through genetic engineering technology.2.On account of the bactericidal effects of recombinant protein PGRP toward Staphylococcus aureus standard strain and clinical drug resistant strains MRSA,rPGRP may be a new type of antibacterial drugs,and its mechanism may be stimulating pgrp gene expression of itself to inhibit bacteria.