控制致病性大肠杆菌DegP表达的调控蛋白及其调控机制研究

Pathogenic Escherichia coli are one of the main causes of many diseases of human being, livestock and poultry, which threaten the human health and lead to serious economic damage in Livestock and poultry industry. In the last decades, the function of regulaotry proteins in bacterial pathogenesis is attracting the focus of researchers. However, the regulatory proteins of DegP have not been further explored in the last 30 years and no report has appeared about the high-throughput screening of them. In this project, we first constructed the ΔphoAdegP::miniTn5phoAkm mutant which is derived from the urinary tract pathogenic model strain UTI89. Then a transposon randomly inserted mutant library will be constructed with UTI89ΔphoAdegP::miniTn5phoAkm as a parent strain. Employing the bacterial alkaline phosphatase PhoA fused into DegP as a reporter, the library will be screened in a high-throughput way according to the color change quantitively and quantitatively. The insertion of the interesting mutants will be localized and interrupted genes will be as candidates of regulatory proteins of DegP. Afterwards, the regulatory gene mutants will be constructed respectively. Furthermore, the expression level of DegP will be tested by qRT-PCR and Western-blot. The binding of the regulatory proteins and the promoter region of DegP will be confirmed by EMSA (Electrophoretic Mobility Shift Assay). After analyzing the data obtained above, the regulatory mechanism of DegP will be deciphered. The achievements of this project will expectedly help to further discover novel virulence factors and better understand the pathogenesis mechanism of pathogenic E. coli in human being, livestock and poultry infection.

致病性大肠杆菌在人畜禽引起多种疾病，对人类健康是潜在的严重威胁，在畜禽养殖业则引起严重的经济损失。近年来，调控蛋白在细菌致病中的作用得到了重视。然而控制DegP的调控因子却近30年内没有更新，也没有高通量筛选的报道。本研究首先构建了模式菌株UTI89的ΔphoAdegP::miniTn5phoAKm突变体。并将对突变体进行随机插入突变，以细菌碱性磷酸酶基因phoA作为报告基因，高通量筛选得到颜色差异突变体，并鉴定插入突变基因，发现控制致病性大肠杆菌DegP表达的调控蛋白。然后在UTI89菌株分别将这些调控蛋白基因缺失，在这些突变体应用qRT-PCR和Western-blot的方法检测DegP的表达水平，以凝胶滞后实验验证调控蛋白是否和DegP的启动子区域结合。综合分析上述数据得到调控蛋白对DegP的调控机制。本项目将为鉴定新的毒力因子，揭示大肠杆菌在人畜禽感染中的作用机理奠定基础。

致病性大肠杆菌可在人和动物消化道内外引起多种类型的疾病。其中致尿路感染大肠杆菌（Uropathogenic Escherichia coli，UPEC）和产肠毒素大肠杆菌（Enterotoxigenic Escherichia coli，ETEC）是引起肠道内外感染的最常见的大肠杆菌。DegP是细菌蛋白质量控制蛋白，又称htrA；而parB基因编码细菌质粒配对蛋白。本项目分别对UPEC和ETEC开展了如下三个方面的研究。首先，DegP蛋白在膀胱炎分离株和肾盂肾炎分离株中对细菌致病力的作用不同：在感染后6小时，肾盂肾炎分离株的DegP突变体在膀胱中的定殖能力开始下降，而膀胱炎分离株的degP突变体则直到24小时，与野生菌株相比较，定殖能力才开始出现显著变化；而对DegP可影响的下游基因分析则发现在膀胱炎分离株和肾盂肾炎分离株都有各自特有的蛋白出现。其次，我们发现parB基因在细菌内生质粒的稳定遗传中发挥了重要作用。而质粒稳定性在细菌耐药、细菌致病机制等方面都具有重要意义；通过构建系列基因缺失突变株，parB基因的缺失可以影响细菌生物被膜的形成，并且该基因在急性感染阶段可能发挥了一定作用。我们通过对parB突变体与野生株的转录水平的深度测序，即RNA-seq，测序结果比较发现parB基因的缺失，导致质粒上的多数基因表达量下调，而在基因组上亦影响到200个基因的表达量发生变化。最后，我们对四株曾作为疫苗株的猪产肠毒素大肠杆菌的耐药性、12种毒力因子进行了分析，对2株菌株进行了测序，菌株及其所含有的质粒在Genbank的序列登录号分别为CP025752,CP025751，CP025753和CP024978,CP024975,CP024976，CP024977，由此我们掌握了细菌刺激宿主产生较好免疫反应的病原遗传学基础。总之，本项目的资助为揭示病原致病性大肠杆菌的致病机制、疫苗研制以及疾病预防等后续研究做了较好的探索。