致病性大肠杆菌胞外蛋白酶Prt1在破坏肠黏膜屏障功能中的作用机制研究

致病性大肠杆菌感染严重威胁人类健康。免疫功能低下人群易受致病菌感染，但致病菌与此类宿主相互作用的机制仍不明确，迫切需求寻找新的毒力因子并以此为靶点进行干预治疗。我们以条件致病菌鼠柠檬酸杆菌作为体内模型，前期研究中构建了一系列鼠柠檬酸杆菌胞外蛋白酶的缺失突变株，将其逐个感染免疫低下小鼠，从中筛选到低毒力的突变株Δprt1。prt1基因在细菌感染中的作用仍不清楚。我们发现：感染野生型鼠柠檬酸杆菌造成免疫低下小鼠肠黏膜通透性增加，肠上皮紧密连接蛋白Claudin-3的分布改变，且其蛋白总量减少，而接种Δprt1不会造成上述变化。我们推测：Prt1通过作用宿主肠上皮细胞间紧密连接而破坏肠黏膜屏障，导致致病菌感染。本研究拟探讨Prt1与鼠柠檬酸杆菌致病力的关系，分析Prt1破坏肠黏膜屏障功能的作用机制，并寻找Prt1的作用对象。本研究鉴定了致病性大肠杆菌新的毒力因子，为临床防治致病菌感染提供新靶点。

The infection of pathogenic Escherichia coli is an important public health issue and causes an enormous economic burden worldwide. However, the pathogenesis has not been fully elucidated. So far, only <1% of the total genes encoded by pathogenic E. coli and their murine equivalent Citrobacter rodentium have been identified as crucial virulence factors. Certain human populations with decreased serum levels of interleukin-22 (IL-22) exhibit much higher susceptibilities to bacterial infections, but the effective treatment is still lacking. It is, therefore, urgently needed in both fundamental research and clinical treatment to discover unknown novel bacterial virulence factors that are crucial for pathogenic E. coli infection with the defect of IL-22 signaling in the immunocompromised populations. . Of note, C. rodentium shares 66.7% of its genes and most pathogenic mechanisms with human pathogenic E. coli. C. rodentium infection leads to dramatically increased mortality and morbidity in IL-22 knockout mice, which makes it an excellent model to investigate the critical host-pathogen interactions during foodborne pathogen infections under the IL-22 compromised condition. In our study, we identified a mutant C. rodentium strain Δprt1, in which the putative extracellular metalloprotease Prt1 was deleted. We found that deletion of Prt1 dramatically attenuated the morbidity and mortality in IL-22 knockout mice, without affecting bacterial replication and colonization in the host. In addition, we found that wild-type C. rodentium infection caused disruption of Claudin-3 protein by forming those punctate and distributed throughout the cells, whereas Δprt1 C. rodentium infection, similar as PBS control, barely disrupted the Claudin-3 localization in the lateral membrane of colonocytes. Moreover, C. rodentium also caused the increase of epithelial barrier permeability and the decrease of Claudin-3 protein level in the infected mouse colon and colon carcinoma cell line, whereas Δprt1 infection did not cause such changes. Since Claudin-3 is one of tight junction associated proteins and is essential for colon epithelial integrity, we thus propose that Prt1 could be essential for C. rodentium infection-caused tight junction damage in IL-22 knockout mice. In this project, we will assess the impact of Prt1 on C. rodentium-induced inflammatory response in IL-22 knockout mice and to elucidate the molecular mechanisms through which Prt1 affects the pathogenesis of C. rodentium infection. The findings will provide novel insights into the pathogenic mechanisms for foodborne diseases and potential therapeutic targets for bacterial infections in the immune-compromised susceptible populations with low IL-22 levels.