sRNA调控禽致病性大肠杆菌在巨噬细胞内存活的分子机制

Avian Pathogenic Escherichia coli (APEC) is an important bacterial pathogen of poultry illness , and these APEC-associated systemic infections have been proven economically devastating to global poultry industries, and APEC might hold zoonotic potential. During APEC colonizing the host extraintestinally in the early stages of avian colibacillosis, intracellular macrophage survival is a key part of APEC pathogenicity. Research currently available is not sufficient to reveal the molecular mechanisms for the expression of APEC intracellular macrophage survival. Based on the present research, the applicant proposes to explore the mechanism for small RNA (sRNA) post-transcriptionally regulating APEC intracellular survival factors. APEC transcriptional expression profiling for two different conditions (cultured in LB and infecting with HD11 macrophage cells) will be compared by transcriptome analysis, and characteristic changes for APEC interacting with HD11 cells will be identify. This comparative transcriptome analysis will reveal and screen the intracellular survival factors and correlative regulatory sRNAs, and the identification will focus on the specific coding small RNAs in APEC pathogenicity islands. Then we plan to construct mutants and complemented strains of putative sRNAs and intracellular survival factors to evaluate virulence by cell infection assay and animal experiments. At the same time, gene deletion, Northern Blot, EMSA, construction of binding site mutants, and others will be used to reveal the interaction and regulation mechanisms of sRNAs with intracellular survival factors in APEC pathogenesis. These findings will illuminate the molecular mechanisms of APEC intracellular macrophage survival during the early infection phases and pave the way to development of strategies in their prevention and treatment, increasing awareness for definitive roles in bacterial pathogenesis.

禽致病性大肠杆菌(APEC)是引起家禽致病的重要细菌病原，严重危害养禽业，甚至具有人畜共患潜力。APEC感染宿主的早期阶段，在巨噬细胞内存活是其致病的一个关键环节，目前研究成果不足以阐明APEC在胞内存活的分子机制。申请者在已有研究的基础上, 拟以小RNA(sRNA)转录后调控胞内存活因子的表达作为研究主线，通过RNA-seq转录组比较APEC在体外培养和感染HD11巨噬细胞时转录表达谱差异，分析转录组数据，筛选与APEC胞内存活相关的sRNA和毒力因子，重点研究在APEC特有毒力岛的sRNA的调控机制。通过基因敲除、Northern Blot、EMSA和定点突变等方法揭示sRNA调控APEC胞内存活因子表达的分子机制。

本研究采用转录组测序、蛋白组技术、生物信息学分析、分子生物学、、激光共聚焦、Northern Blot和电镜等手段等方法，以 鉴定APEC sRNA和胞内存活因子为研究主线，揭示APEC 感染禽巨噬细胞和在巨噬细胞内存活的分子机制。首次发现：(1) 通过dRNA-seq测序技术测定APEC O2:K1强毒株FY26的sRNA序列，鉴别出 242个新的APEC sRNA, 其长度大小在120nt至500nt之间，其中32 个sRNA编码于APEC/ExPEC特有的毒力岛中；(2) 转录因子DksA被鉴定为APEC关键的毒力调控因子，DksA通过sRNA转录后调控途径介导APEC感染巨噬细胞，APEC DksA缺失株可作为候选禽大肠杆菌病弱毒疫苗开发应用；(3) 转录因子CpxR介导APEC在巨噬细胞内存活，揭示CpxR可以调控sRNA转录水平，被调控sRNA可能影响APEC胞内存活因子的表达；(4)通过比较APEC感染HD11巨噬细胞时转录表达谱差异，鉴定出ColV毒力质粒编码的胞内存活因子(HlyF、Mig-14p和OmpTp)；(5) 研究发现APEC通过募集胞内乙酸促进巨噬细胞炎性紊乱，乙酸同化系统被认作新的胞内存活因子，该系统直接影响APEC致病表型;(6)结合对APEC感染的认识，对当今研究发现当今mcr-1阳性APEC流行菌株类群进行耐药性、致病力和人畜共患潜力评估，明确了携带mcr-1抗性基因的禽致病性大肠杆菌(APEC)具有高致病力和广泛耐药性，具有人畜共患风险。上述结果揭示APEC感染巨噬细胞的重要分子机制，为APEC新药物靶标的筛选和弱毒疫苗的开发提供理论依据。