Functional Study Of AmpC ?-lactamase Expression-regulating Gene In Yersinia Enterocolitica

Objective:Yersinia entercolitica is a Gram-negative budless bacillus,which belongs to Yersinia genus, Enterobacteriaceae. It is one of the three pathogenic bacteria of Yersinia genus, mainly infects human to cause gastrointestinal disease by contaminated food or water. Like other Enterobacteriaceae, Y. enterocolitica chromosomally encodes ampR-ampC system to produce AmpC ?-lactamase, making it intrinsic resistante to penicillins and cephalosporins. Although the regulation mechanisms of AmpC ?-lactamase in Enterobacteriaceae are well studied, the feature of ampC regulation in Y. enterocolitica is still a mistiness. In this study, we explore the regulation mechanisms of AmpC P-lactamase by using molecular and microbiological techniques, to elucidate the mechanism of Y. enterocolitica antibiotics resistance.Method:1. Predict the ampC promoter region by using the bioinformatic techniques,amplify the ampC promoter sequence by PCR and ligate the amplicon with a promoterless luxCDABE reporter plasimid pBBRLux to construct pLUXampC which could monitor the level of ampC transcription.2. Use the BLAST program to predict the presumed gene of Y. enterocolitica AmpD, Low-Molecular-Mass Penicillin-Binding Proteins and N-acetyl-?-glucosaminidase. Ligation the 5' and 3' flanking homologous recombination arms ??1000bp? with suicide plasmid pDS 132, and use it to construct the relevant mutation strains.3. Using the ampC promoter monitor plasmid pLUXampC to evaluate the ampC expressiong level of ampDl, ampD2, ampD3, pbp4, pbp5a, pbp5b, pbp7, ampR and nagZ relevant mutation strains.4. Use the nitrocefin and 4-nitrophenyl N-acetyl-?-D-glucosaminide as substrate to evaluate the activity of ?-lactamase and N-acetyl-?-glucosaminidase.5. Use the microdilution method to measure the minimal inhibitory concentrations ?MICs? of all mutation strains.Results:1. The luminescent ampC promoter monitor plasimid pLUXampC has been successfully constructed.2. According to the results of amino acid sequence homology analysis, three Y.enterocolitica proteins WP₀05156822, WP₀05164953 and WP₀13649890 which we named AmpD1, AmpD2 and AmpD3 have the same conserved domain of N-acetyl- anhydromuramyl-L-alanine amidase. To elucidate the role of three AmpD homologues in ampC regulation, we construct seven ampDl, ampD2 and ampD3 relevant single, double and triple gene mutation strains. The experimental results demonstrate that all three AmpD homologues participate in the regulation of AmpC?-lactamase, the ?-lactamase of tested strains were elevated after the ampD gene deletion. Finally, triple mutaion strain YEAD123 has the highest ?-lactamase activity which acquire the fully derepression phenotype.3. We also constructed fifteen kinds of four LMM PBPs namely PBP4?PBP5a?PBP5b and PBP7 relevant single, double, triple and quadruple mutation strains. We found all LMM PBPs headed by PBP5b plays a key role in ampC regulation, deletion of LMM PBPs gene causes varying degrees of elevation in ampC promoter activity.Quadruple deletion strain YEA4A5aA5bA7 have the highest ?-lactamase activity and also acquire the fully derepression phenotype.4. We further deleted the nagZ gene to compare the difference of two derepression strain YEAD123 and YE?4?5a?5b?7, and found deletion of nagZ have few effect in YE?D123?Z, but greatly influence YE?4?5a?5b?7?Z, which shares the same ampC expression level with wild-type strain 105.5R?r?.Conclusion:1. Through the construction of pL UXampC, we established an ampC promoter activity monitoring system, which is base on the luminescent technology. Use this method, we can survey the ampC expression level continuously.2. Three AmpD homologues participate in the ampC regulation of Y.enterocolitica. ?mpD1 is the most potent one whereas the role of AmpD2 and AmpD3 only appeared in ?ampD1 background. This is the first report of ampC multi-step upregulation mechanism driven by three AmpD homologues in Y. enterocolitica.3. Four LMM PBPs of Y. enterocolitica participate in the AmpC ?-lactamase regulation. Mutation of LMM PBPs, especially PBP5b gene, elevate the ampC expression level obviously, the function of single PBP4, PBP5a or PBP7 is relatively low, which work in coordination with PBP5b.4. Y. enterocolitica have both NagZ-depend and NagZ-independent mechanisms for ?-lactamase expression, which is the first report of such a complicate ampC regulation model in Enterobacteriaceae.