SOS应答对大肠杆菌O157抗药性与毒力的调控机制研究

Pathogens will coordinate a variety of resistance and virulence mechanisms to resist the killing effect of antibiotics and immune biochemical system of animal body administrated with antibiotics, but its regulation mechanism has not been clarified up to now. SOS response is a stress response induced in critical condition when DNA synthesis is damaged, and it is a low-fidelity DNA repair way so as to improve the mutation rate, survival and adaptability of the bacteria. 81171621 project research shows that inhibition of the E. coli SOS response can reduce both their resistance level, mutation rate to fluoroquinolone and the virulence to mice..Accordingly in this project, the pharmacological technology platform to simulate infection status of clinical pathogens is established that take into account both pathogen virulence and resistance, immune function and pharmacology of antibiotics, and promise sufficient gene exchange between the target pathogenic bacteria and intestinal flora..E. coli O157, ATCC25922 and the strains with promoter genes recA of SOS response knocked out are marked by inserting fluorescent protein gene gfp, and orally infect SPF chicken with the corresponding route of antibiotics administration, respectively. Then, the differences and evolution of virulence and resistance of E.coli isolated from feces are detected together with genome chip, fluorescence quantitative PCR and corresponding gene sequencing, so as to clarify the regulatory mechanism of SOS response of E.coli to resistance and virulence, and evaluate the clinic curative effect of SOS response inhibitor and its combination administration with antibiotics.

施药动物体内的病原菌会运用多种抗药性、耐药性机制和毒力机制抵抗抗生素和免疫生化系统的杀灭作用，但其调控机制一直未予阐明。SOS应答（细菌应激反应）是在DNA合成受损的危急状态时所诱导的一种低保真度DNA修复方式，能提高细菌的突变率、生存率和适应性。81171621项目研究表明，抑制大肠杆菌SOS应答能同时降低其对喹诺酮的抗药性程度、突变率和对小鼠致病力。.据此，本项目拟建立整合病原菌毒力与抗药性、免疫与抗生素作用、保证肠道菌群基因充分交换的药理学技术平台，模拟临床病原菌感染状态；构建敲除SOS应答的启动蛋白基因recA的O157、ATCC25922菌株，与初始菌株均标记荧光蛋白基因gfp，分别口服感染SPF鸡并抗生素治疗，用基因组芯片和荧光定量PCR、相关基因测序检测粪样中标记菌毒力与抗药性的差异和进化，厘清SOS应答对抗药性与毒力的调控机制，并评价SOS应答抑制剂及其与抗生素联用的疗效。

SOS应答是在细菌DNA合成受损的危急状态时所诱导的一种低保真度DNA修复方式，能提高细菌的突变率、生存率和适应性。本项目以此建立整合病原菌毒力与抗药性的平台，构建敲除SOS应答的启动蛋白基因recA的E. coliO157、ATCC25922菌株，用转录组芯片和荧光定量PCR技术，探索SOS应答对抗药性与毒力的调控机制，并评价SOS应答抑制剂及其与抗生素联用的疗效。.研究结果表明，recA缺失使菌株对恩诺沙星的抗药性和抗药率均明显降低，recA缺失对O157:H7菌株中毒力基因stx2表达量下调，eaeA表达量上调。通过O157:H7及其recA缺失菌株的转录组分析，共发现20个可能受SOS调控系统的主要基因，在恩诺沙星处理下表现为表达显著地上调或下调，推测有6个转录子与细菌的耐药性、抗药性有关，2个转录子与与毒力调控相关，3个与核苷酸代谢有关，3个在信号通路的上游。其中粘附素基因eaeA，在转录组分析、荧光定量验证和体外细胞验证中，同时得到印证，此基因由SOS应答系统调控，SOS系统启动导致O157:H7中粘附素基因的下调表达，使细菌对细胞的粘附力减弱。.在此研究基础上，合成了RecA蛋白抑制剂，与恩诺沙星联合使用对E. coli生长具有相加抑制作用，RecA抑制剂能够一定程度上抑制恩诺沙星诱导的recA、umuC、eaeA、stx2的表达。.此外，研究了SOS应答对肠道菌群的微生物组、抗药性、噬菌体组的影响，以及对原噬菌体的释放及其与抗药性和毒力的调控关系。