CRISPR-Cas调控大肠杆菌SOS反应影响志贺毒素表达的分子机制

产志贺毒素大肠杆菌（STEC）是重要的人兽共患病原菌，其产生的毒素能引起出血性肠炎和溶血性尿毒综合征，导致人和动物死亡，但其毒素产生的具体调控机制尚不完全清楚。项目组前期研究发现，大肠杆菌CRISPR-Cas系统的激活可靶向基因组中Stx2噬菌体，抑制Stx毒素表达及噬菌体释放。为阐明其分子调控机制，本项目将基于基因缺失、转录组、蛋白组等技术，重点聚焦大肠杆菌CRISPR-Cas对SOS反应关键调控因子RecA和LexA，以及对Stx噬菌体溶原转换调节因子多核苷酸磷酸化酶（PNPase）和poly(A)聚合酶（PAP I）的调控作用和网络；通过噬菌体溶原揭示CRISPR-Cas影响SOS反应从而调控Stx毒素表达的分子路径，最终阐明CRISPR-Cas调控STEC噬菌体释放及毒素表达的分子机制，为抑制Stx噬菌体溶原菌的噬菌体释放、毒素表达及基因水平转移，有效防控STEC提供理论依据。

Shiga toxin-producing Escherichia coli (STEC) strains are important zoonosis pathogens, which generate toxins that cause severe human diseases, such as haemorrhagic colitis and haemolytic–uraemic syndrome (HUS), resulting in deaths of humans and animals. However, many questions remain unclear regarding the exact regulatory mechanism of toxin production. Our preliminary data showed that the E. coli CRISPR-Cas system represses toxin expression and phage release by targeting the Stx2 phage in the bacteria genome. To elucidate the molecular network and regulation mechanism, based on the gene deletion and transcriptome techniques, as well as proteome analysis, the regulating effect of CRISPR-Cas system on SOS response regulator RecA, LexA and Stx2-converting phage regulator, polynucleotide phosphorylase (PNPase), and poly(A) polymerase (PAP I) will be mainly explored in this proposal. Furthermore, the molecular signaling pathway of activated CRISPR-Cas system affecting the SOS response will also be addressed. Taken together, this study will elucidate the molecular mechanism of how the CRISPR-Cas system of STEC strains targeting phage release and toxin expression, which are virulence markers of STEC isolates. This investigation will be conducive to inhibit the phage release, toxin production, and horizontal gene transfer, in order to provide theoretical foundation for effective control of STEC.