空肠弯曲杆菌CRISPR-Cas 系统对多重耐药性和毒力的双重调控机理研究

The Campylobacter jejuni with multidrug resistance and high virulence, could pose serious threat to food safety and human public health. CRISPR-Cas，as bacterial immunity/defense system may play an essential role on antimicrobial resistance and virulence in C. jejuni. .Following our previous study, this project will firstly investigate the genetic characteristic of CRISPR-Cas in C. jejuni isolates with different level of drug resistance and virulence using whole genome sequencing and bioinformation methods, and analyze the association of CRISPR-Cas with antimicrobial resistant genes and virulence genes. Then the target genes (cas1, cas2, cas9, csn and spacer) of CRISPR-Cas will be inactivated by insertional mutagenesis and the supplement mutants will be constructed by molecular cloning. The antimicrobial resistance phenotype and virulence characteristic of these constructs will be compared with the their parent strains. The ability of acquisition of antimicrobial resistance genes and virulence factors will also evaluated to explore the function of CRISPR-Cas system on antimicrobial resistance and virulence. Additionally, new generation of RNA sequencing technique will be carried out to analyze the regulatory mechanism of CRISPR-Cas system on antibiotic resistance and virulence in C. jejuni. The function of the significant gene found in RNA-seq on antimicrobial resistance and virulence will be also confirmed to reveal the indirect regulation mechanism of CRISPR-Cas on resistance and virulence. .Conclusively, this project will systematic reveal the direct and indirect regulation mechanism of CRISPR-Cas system on antimicrobial resistance and virulence in C. jejuni. Consequently, our study will provide scientific reference and basic information for designation of new drug and development of new strategy to control the C. jejuni with multidrug resistance and high virulence.

空肠弯曲杆菌(空弯)的多重耐药性和毒力耦联，必对动物健康和食品安全构成严重威胁。CRISPR-Cas作为细菌免疫防御系统，可能对空弯的耐药性和毒力至关重要。本项目将在前期研究基础上，应用全基因组测序等方法，分析不同程度耐药和毒力型空弯的CRISPR-Cas基因特征，研究其与耐药和毒力因子的并存关系；通过基因重构等技术，构建CRISPR-Cas关键成分（cas1、cas2、cas9、csn 和spacer）的缺失和互补突变体，分析该系统对耐药性和毒力的直接作用；通过RNA-seq等技术，分析CRISPR-Cas对耐药性和毒力的直接调控作用；进一步构建特殊差异基因突变体，分析CRISPR-Cas对耐药性和毒力的间接调控机制。本研究将系统揭示空弯CRISPR-Cas对耐药性和毒力的双重作用和调节机理，为对抗多重耐药毒力型空弯提供新药设计靶标，为控制多重耐药毒力型空弯的产生和传播奠定理论基础。

弯曲杆菌是全球胃肠炎的主要原因,对公共卫生构成严重威胁。该菌常含有CRISPR–cas9系统，在不同生物体中被用作基因编辑技术。然而，其CRISPR-Cas系统对耐药性和毒力耦联调控机制却尚待研究。本项目旨在探究CRISPR-Cas对弯曲杆菌耐药性和毒力的直接和间接调控机理。.本研究对临床分离菌的耐药表型和CRISPR-Cas系统特征进行了关联分析，发现耐药菌中特殊的CRISPR spacer序列特征。不同抗菌药作用压力下，标准菌株中CRISPR相关基因（cas1, cas2和cas9）的表达量显著差异，表明了CRISPR-cas系统在弯曲杆菌应对抗菌药压力中发挥着重要作用。在成功构建cas缺失、过表达/互补突变体的基础上，研究了突变体和野生菌在生物膜的形成、迁移力、粘附和侵袭、细胞内生存能力和细胞毒素的产生方面的差异，发现CRISPR-cas系统可以在一定程度上增强弯曲菌毒力。同时比较了突变菌和野生菌的药物敏感性、耐药应答能力、耐药转移特征等，发现CRISPR-cas系统可以介导弯曲菌耐药性的产生过程，并可能参与耐药基因的转移。最后进行转录组学测序分析，结果发现cas9基因可能通过调控多个通路和多个基因，间接介导弯曲杆菌毒力增强、耐药应答和耐药转移。总之，弯曲杆菌中CRISPR-cas系统可能参与其毒力和耐药性产生转移过程，且Cas9基因表达与毒力和耐药性成正相关。本研究提供了cas9参与参与毒力和耐药性的关键信息，为弯曲杆菌耐药性防控提供了理论支撑。