沙门菌转录激活子介导的氟喹诺酮类耐药机理研究

Salmonella is an important Zoonotic pathogen. At present fluoroquinolones(FQs) resistance in Salmonella is becoming increasingly serious. People usually use susceptible strains of FQs for in vitro induction to study the mechanisms of resistance,however,the mechanisms of FQs resistance mediated by transcription activators under in vivo induction pressure is unclear. In this study, we intend to choose Caenorhabditis elegans as model for in vivo induction and induce FQs resistance in Salmonella typhimurium by colonizing in the intestine of Caenorhabditis elegans.Firstly, we ascertain which transcription activators activated under induction pressure of FQs and clarify relationships between mutations in transcription inhibitors and promoter(region) and expression of transcription activators. Then, we plan to construct the deletion strain and complementary strain of activated transcription activators and to screen active efflux pump genes using gene expression profile chip. In the end,the activated efflux pump genes will be konckout, and the changes of resistance phenotype, invasion to epithelial cell and survival ability in epithelial cell and macrophage before and after efflux pump genes knockout will be analyzed, thus the mechanisms of FQs resistance mediated by transcription activators will be elucidated. This research would provide effective methods and means for in vivo resistance induction and study on the chromosome mediated multi-resistant mechanisms, and would supply important theoretical foundation and experimental basis for the control of FQs resistance in Salmonella.

沙门菌是重要的人畜共患病原菌，目前沙门菌对氟喹诺酮类药物(FQs)的耐药性日趋严重。人们通常使用FQs对敏感菌进行体外诱导来研究耐药机理，而对于沙门菌在FQs体内诱导压力下转录激活子介导的FQs耐药机理尚不明确。本课题拟选用模式生物秀丽隐杆线虫作为体内诱导模型，将鼠伤寒沙门菌定植于秀丽隐杆线虫肠道内进行FQs耐药性诱导，探明FQs诱导压力下激活的转录激活子，明确转录抑制子和启动子(区)突变与转录激活子表达的关系，然后构建转录激活子缺失株和互补株，利用基因表达谱芯片筛选出激活的外排泵基因，最后敲除激活的外排泵基因，比较外排泵基因敲除前后菌株耐药表型及对上皮细胞侵袭力、在上皮细胞和巨噬细胞生存能力的变化，阐明沙门菌转录激活子介导的FQs耐药机理，从而为耐药性体内诱导及染色体介导的多重耐药机理研究提供有效的方法和手段，为控制沙门菌的FQs耐药性提供重要的理论依据和实验基础。

沙门菌是重要的人兽共患病原菌，目前沙门菌对氟喹诺酮类药物（fluoroquinolones, FQs）的耐药性日趋严重。人们通常使用FQs对敏感菌进行体外诱导来研究耐药机理，而对于沙门菌在FQs体内诱导压力下转录激活子介导的FQs耐药机理尚不明确。本研究建立了鼠伤寒沙门菌-秀丽隐杆线虫体内耐药性诱导模型，比较了体内诱导和体外诱导耐药菌的基因突变差异。利用转录组测序技术（RNA-seq）对鼠伤寒沙门菌和其体内、体外诱导耐药菌进行研究，筛选出了FQs体内和体外诱导压力下激活的转录激活子和转录激活子激活的外排泵基因，揭示了鼠伤寒沙门菌耐药性诱导前后差异表达的基因和显著变化的代谢途径，明确了转录抑制子和启动子(区)突变与转录激活子表达的关系。然后敲除了转录激活子激活的外排泵基因，明确了外排泵基因对沙门菌的耐药表型、上皮细胞侵袭力及在上皮细胞和巨噬细胞内生存能力的影响，阐明了沙门菌转录激活子介导的FQs耐药机理，为耐药性体内诱导及染色体介导的多重耐药机理研究提供了有效的方法和手段，为控制沙门菌的FQs耐药性提供了重要的理论依据和实验基础。