ABC转运蛋白介导副溶血性弧菌耐药性产生的分子机制

Vibrio parahaemolyticus is the primary cause of foodborne outbreaks of bacterial pathogens in the China's coastal areas, and its antibiotic resistance is growing. The studies on antibiotic resistance mechanisms of this bacterium focused on a small number of proteins such as RND efflux pump and MATE efflux pumps, etc. Our previous genomics analysis revealed that at least five genes encoding ABC transporter proteins are present in the genome of V. parahaemolyticus, among which, vpa1715 has been heterologous expressed in E. coli, and we found that it conferred efflux of multiple drugs. Therefore, this study will focus on the genes encoding for the ABC transporter proteins, which will be expressed in recombinant E. coli, and the candidate antibiotic resistance genes will be obtained by drug susceptibility testing, subcellular location assay and energy-dependent assay. Gene deletion mutants and complementary mutants will be constructed and gene function will be verified. The key functional sites in protein encoded by resistance gene will be determined through site-directed mutagenesis technology. This finding on antibiotic resistance-related ABC transporter has not been reported, which will not only contribute the new mechanisms on antibiotic resistance formation in V. parahaemolyticus, but also provide a theoretical basis for the mining of drug targets in this bacterium.

副溶血性弧菌是导致我国沿海地区食源性疾病暴发的首要致病菌，它对抗生素的耐药性日趋严重，目前对其耐药机制的研究主要集中在RND家族和MATE家族外排泵等少数蛋白。我们通过基因组序列分析发现，副溶血性弧菌中存在至少5个假定ABC转运蛋白，对其中的Vpa1715进行了异源表达，发现它能外排多种药物。本项目将以副溶血性弧菌中这5个假定ABC转运蛋白为研究对象，实现它们在大肠杆菌中的表达，通过药敏实验、表达产物的亚细胞定位和能量依赖实验，筛选出耐药候选基因；构建耐药候选基因缺失突变菌株和互补菌株，通过药敏实验、Triton X-100诱导裂解实验、溶菌活性酶谱分析和存活率实验来验证耐药候选基因的功能；采用定点突变技术构建耐药基因突变体，识别所编码蛋白的关键功能位点。副溶血性弧菌中这些ABC转运蛋白同耐药相关性的研究还未见报道，它将有助于揭示这一细菌耐药产生的新机制，并且为药物靶标的研发提供理论依据。

本研究采用环丙沙星、氨苄青霉素、四环素，分别对副溶血弧菌始发菌株F7进行体外多步诱导，最终获得三个系列耐药菌株。对诱导前后菌株MIC值进行比较，发现单一抗生素类药物诱导副溶血弧菌产生多重耐药。采用反转录荧光定量PCR，测定了ABC转运蛋白编码基因vpa1715在出发菌株和诱导耐药菌株中的表达量。实现了ABC转运蛋白编码基因vpa1715在药物高度敏感大肠杆菌中表达，通过MIC测定和能量依赖实验证实了vpa1715对大肠杆菌宿主细胞耐药的贡献。构建了vpa1715和vmeAB 的双基因敲除株、vmeAB 的单基因敲除株，发现双基因敲除株对5种抗生素的MICs相比单基因敲除株有明显的下降，从而验证了ABC转运蛋白编码基因vpa1715在副溶血弧菌耐药中的作用。本研究发表和接收期刊论文共9篇，其中SCI论文6篇、EI论文3篇，以第一作者或通讯作者发表SCI论文2篇、EI论文2篇。培养研究生5名。