携带usp毒力岛的尿道致病性大肠杆菌毒力基因累加效应研究

Urinary tract infections are common infectious diseases, it is easy to be recurrent and become chronic diseases after infection. The most common pathogen was Escherichia coli, often referred to as urinary tract pathogenic E. coli (UPEC). We screened strong virulent UPEC strains which carry usp and other virulence genes by detection of virulence genes and virulence phenotype experiments, and the strain can induce Hela cells to be early apoptosis. With the prolonged co-incubation time, a lot of Hela cells were damaged. At present, the pathogenic mechanism of single virulence gene or pathogenicity island was detailed studied, but the cumulative effects of multiple virulence genes are less. We presumed that the formation of recurrence and chronic diseases may be related with the the cumulative effect of multiple virulence genes. We will use gene knockout technology to build mutant strains of more than one candidate virulence genes on the basis of usp mutant strain. Changes in virulence of the original strain and mutant strains or different mutant strains will be compared through virulence phenotypic experimental and mouse infection model, revealing the cumulative effects of virulence genes and the main virulence genes, immuno-transmission electron microscope was used to locate th eUPEC, activated apoptotic signals were detected by immunoblotting, ELISA and other methods, and preliminarily elucidating pathogenic mechanisms of recurrent and chronic urinary tract infection caused by UPEC.

尿路细菌感染是常见的感染性疾病，感染后短期内易复发和形成慢性疾病。病原菌以大肠杆菌最常见，通常称为尿道致病性大肠杆菌(UPEC)。申请者通过毒力基因检测和毒力表型实验筛选到携带usp等多种毒力基因、强毒力的UPEC，有较强诱导Hela细胞凋亡的能力，随共同孵育时间延长，可大量破坏Hela细胞。目前对单毒力基因或毒力岛的致病机制研究较为深入，但多个毒力基因的累加效应研究较少。我们推测其复发和慢性疾病的形成可能与多个毒力基因累加作用有关。我们拟在usp基因突变基础上，运用基因敲除技术构建多个候选毒力基因共同突变菌株。通过毒力表型实验和小鼠感染模型比较原始菌株和突变菌株、不同突变株之间的毒力变化，揭示毒力基因间累加作用，确定在致病过程中的主毒力基因；采用免疫透射电镜对UPEC进行定位，免疫印迹、ELISA等方法检测细胞激活的凋亡信号，初步阐明由UPEC引起的尿路感染复发和形成慢性疾病的致病机理。

尿路细菌感染是常见的感染性疾病，感染后短期内易复发和形成慢性疾病。病原菌以大肠杆菌最常见，通常称为尿道致病性大肠杆菌(UPEC)。我们采用Red敲除系统成功构建了低毒力菌株的usp和fimH基因共同突变的菌株，而高毒力菌株由于特殊的耐药模式未能获得预期的突变体。课题组改变实验方案，对高毒力菌株代表株（27N和6N）以及低毒力代表菌株（30N）进行了高通量基因组测序，以了解它们的遗传背景。测序结果分析显示，强毒力菌株与经典的尿道致病性大肠埃希菌（UPEC_CFT073和_UTI189）亲缘关系相近，而30N则较远。毒力基因和耐药基因分析结果显示强毒力菌株（27N和6N）比低毒力菌株（30N）含有更多的毒力基因和耐药基因。这为治疗由高毒力合并高耐药UPEC引起的尿路感染提供了理论基础，并且提供了详细的UPEC分子流行病学资料。通过尿道滴注法成功构建了小鼠尿路感染模型，为后续研究奠定了实验基础。我们仍在筛选合适的高毒力菌株来构建多基因突变的突变菌株，以延续后续的研究。由于多粘菌素新型耐药基因的报道，我们拓展研究了多粘菌素新型耐药基因mcr-1的分子流行病学情况。我们对UPEC、肠道标本分离的大肠杆菌和水环境分离的大肠杆菌进行了mcr-1的检测和定位，未在UPEC中发现mcr-1耐药基因，而在粪便标本和水环境分离的大肠杆菌中发现了mcr-1，同时研究结果显示mcr-1不仅位于可接合的质粒上，而且存在于染色体DNA上。这一研究结果提示水环境中的微生物可以作为耐药基因的储存库，通过可接合质粒将耐药基因进行传播。