肠致病性大肠杆菌六型分泌系统及其真核来源效应蛋白介导的上皮细胞黏附的作用机制

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative pathogen which leads multiple infections in human beings. The infection is mainly caused by the adhesion and invasion of the pathogens to the epithelial cells on intestines. Based on previous bioinformatic analysis, a widespread Type six secretion system (T6SS) gene cluster is encoded in the genome of EPEC O55:H7, suggesting a common mechanism for promoting cell adhesion and infection in pathogenic E. coli. Our results indicate that the locus plays a role in bacterial growth competition and epithelial cell adhesion. Meanwhile, one potential T6SS secreted effector, an Ankyrin repeat protein with eukaryotic origin, is found to be closely encoded downstream of T6SS cluster. Preliminary data show that, besides its bacterial toxicity, it could be a non-structural protein of T6SS which target cytoskeleton as it can be translocated into host cells by T6SS and interacts with cytoskeleton directly. This project was aimed to comprehensively elucidate the biological functions of T6SS and Ankyrin repeat protein in EPEC O55:H7. Furthermore, the molecular mechanism of the unique trans-kingdom virulence device in the adhesion of epithelial cells will also be investigated, in order to provide better understandings regarding the pathogenic T6SS of E. coli and subsequent antibacterial treatments.

肠致病性大肠杆菌（EPEC）是能导致人类多系统感染的革兰氏阴性病原菌，其感染主要通过对肠道上皮细胞的黏附侵染达成。前期分析发现EPEC O55：H7基因组中编码了一个六型分泌系统（T6SS）毒力岛，其类似结构存在于多种致病性大肠杆菌中，提示该系统是一种通用的促进细胞黏附的机制。预实验显示它参与了细菌的竞争性生长及对上皮细胞的黏附等活动。该毒力岛还编码了一个含有真核Ankyrin repeat来源的基因，初步结果表明其产物Ank不仅具有细菌毒性，还能被T6SS转运至真核细胞中与细胞骨架直接结合。因此我们提出一个假说：EPEC的T6SS参与了针对原核细菌及肠道上皮细胞的作用，且Ank为全新的T6SS作用于细胞骨架的非结构蛋白类效应蛋白。本项目拟以EPEC O55：H7为研究对象，探究此菌T6SS及其Ank效应蛋白针对原核细菌和真核上皮细胞作用的分子机制，为治疗致病性大肠杆菌感染提供新的思路。

包括肠致病性大肠杆菌在内的多种病原菌分泌系统密切参与其致病性及和环境宿主共生。以六型分泌系统(T6SS)为代表的被称为Contractile Injection System(CIS)的纳米穿刺装置分布广泛，它们主要锚定在细菌膜上发挥作用；另一类可分泌到胞外(extracellular CIS，eCIS)，以Photorhabdus virulence cassette(PVC)为代表。申请人在前期解析了一个大于10MDa的PVC结构，初步揭示了eCIS的组装过程和作用机理（Cell，2019）。随后又发现了若干该分泌系统的真核相关效应蛋白的作用分子机制和装载原理，为深入改造开发相关装置奠定了基础。其中RRSP蛋白能够通过与细胞因子CDK1的结合并降低其磷酸化水平从而抑制细胞增殖和诱导细胞凋亡（Front Microbiol, 2020）。Pdp1蛋白具有调控胞内dNTP水平的焦磷酸酶活性，Pnf则是能引起Rho GTPases的脱氨基作用最终导致细胞骨架破坏（Sci China Life Sci, 2022）。最后鉴定一类特异性信号肽，能够引导多种不同来源蛋白被以“豌豆-豆荚”的方式装配进PVC内并递送至真核细胞发挥各自生物学功能（Sci Adv，2022）。这为最终将六型分泌系统相关装置开发成生物医学治疗领域的工具提供了更多的理论和实践依据。