肠出血型大肠埃希菌VI型分泌系统调控机制研究

Bacterial type VI secretion system (T6SS) is a specific nanomachine which plays a critical role in bacterial pathogenicity. The post-transcriptional regulator of bacteria CsrA is involved in the regulation of many cellular processes including pathogenesis. Previous study from our laboratory showed T6SS is essential for Enterohemorrhagic Escherichia coli virulence (PLoS Pathog. 2017, 13(3):e1006246), but the regulation of T6SS in EHEC is unknown. Our preliminary data showed that CsrA regulates the expression of the T6SS at both transcriptional and post-transcriptional levels. Specifically, EMSA assay revealed that CsrA could bind to multiple mRNAs of T6SS. The binding sites are located in the vicinity of Shine-Dalgarno (SD) sequences, indicating CsrA may affect translation by blocking ribosome binding, which is confirmed by β-galactosidase reporter assay. Interestingly, CsrA could affect T6SS transcription. Therefore, we propose that CsrA may control the expression of T6SS in a fine-tuning manner via the different binding affinities to mRNAs of T6SS and T6SS’s regulators. To test this hypothesis, various molecular techniques will be employed to investigate the molecular mechanism of CsrA in regulating expression of T6SS in EHEC, and its consequence outcome in EHEC virulence will be examined as well. This project aims to find out how Csr system regulates the expression of T6SS in vivo and in vitro, and information derived from this application will provide a new perspective for understanding the complex regulatory networks of T6SS in EHEC.

课题组前期工作表明VI型分泌系统（T6SS）对肠出血型大肠埃希菌（EHEC）致病性至关重要，但其调控机制还不明晰。作为转录后调控因子，CsrA广泛参与病原菌的生理代谢和致病过程。初步数据显示CsrA可结合在T6SS基因簇多个基因mRNA的SD序列附近，从而调节mRNA的翻译；另外，CsrA缺失也会影响T6SS相关基因的转录。基于以上结果，我们推测CsrA可能通过直接作用于T6SS基因mRNA，或者间接地靶向T6SS转录调节因子mRNA来精细调节T6SS分泌装置和效应蛋白的表达，并最终影响EHEC的毒力。因此，本项目拟利用分子生物学技术，在细菌、细胞模型和动物模型层面分别研究CsrA调控T6SS表达的分子机制，探究该机制在EHEC致病过程中的作用。研究结果将有助于深入理解T6SS调控的机制，并为其他病原菌T6SS的研究提供可借鉴的范例。

肠出血型大肠埃希菌（EHEC）是广泛分布的条件致病菌，具有发病率和致死率双高的特点，但其致病机制还不清晰。细菌VI型分泌系统（T6SS）对病原细菌的侵染致病、菌间竞争和逆境生存具有至关重要的作用。研究发现EHEC的T6SS存在功能退化现象，可导致T6SS分泌活性降低和部分效应蛋白失活。进一步研究表明T6SS突变可显著提升产志贺毒素（Stx2）原噬菌体的裂解和III型分泌系统（T3SS）的活性，从而增强EHEC对肠上皮细胞的毒性。此外，本项目发现EHEC T6SS的核心结构蛋白编码基因tssM发生了突变，其5’端插入了28 bp的重复序列导致翻译起始密码子由ATG突变为TTG，极大地降低了翻译效率。该突变还导致TssM的N端缺失10个氨基酸，推测会对该蛋白的活性有显著的影响。生物进化分析表明TssM的突变发生在其祖先非典型肠致病性大肠埃希菌（aEPEC）获得Stx2原噬菌体之后，推测原噬菌体的整入会促进EHEC T6SS突变，从而增强Stx2-T3SS来源的细胞毒性。本研究发现福氏志贺菌也可能存在类似的T6SS退化现象。.除了全局调控因子CsrA对T6SS的功能调控，CsrA还可调控EHEC两种溶血素的活性。溶血素是EHEC重要的毒力因子。CsrA可显著抑制肠溶血素（EhxA）的分泌活性，增强溶血素E（HlyE）来源的溶血活性，表现出双向调控作用。进一步研究表明，CsrA通过结合在EhxA的结构蛋白编码基因ehxB的SD序列附近，阻碍核糖体与mRNA结合，促进RNA降解并抑制其翻译，从而抑制EhxA的分泌活性。同时CsrA可结合在hlyE 的5’-UTR端稳定其mRNA，保护mRNA不受降解，增强HlyE来源的溶血活性。本项目还鉴定了CsrA调控EHEC溶血活性的靶结合位点，阐明了CsrA作为其分子开关的调控机制。本项目有助于深入认识EHEC的致病机理，为相关药物的开发提供理论基础。