副溶血弧菌黏附因子VpadF转录调控的分子机制研究

Vibrio parahaemolyticus is the major foodborne pathogen in China. Its attachment to host cells is not only the first important step, but also the prerequisite for the infection. This pathogen expresses several adhesins to attach to host cells, but only mam7 and vpadF, which are constitutively expressed and induced expressed, respectively, were shown to be required for virulence in vivo. Moreover, the expressions of virulence factors are tightly regulated in V. parahaemolyticus. However, how the expression of vpadF is regulated remains enigmatic. To address this issue, we first use gene deletion, gene complement, β-galactosidase assay and RNA-Seq to identify regulators that affect vpadF expression. Further，site-directed mutagenesis, ChIP, EMSA and Western blot assays will be employed to study the regulation mode for the expression of vpadF. This study will uncover not only the molecular mechanism by which vpadF is regulated, but also provide new insight for the prevention of V. parahaemolyticus infection.

副溶血弧菌是我国最主要的食源性病原菌。黏附是其接触和感染宿主的第一步，也是导致感染的首要条件。已知的副溶血弧菌黏附因子有多个，但只有组成型表达的MAM7和诱导型表达的VpadF是其感染宿主必需的。在副溶血弧菌感染宿主的过程中，毒力因子的表达被紧密调控，但我们对vpadF基因表达调控的分子机制知之甚少。本项目拟首先通过基因敲除和回补、β-半乳糖苷酶活性实验和转录组测序，筛选和确定vpadF基因的调控因子。再利用定点突变、染色质免疫共沉淀(ChIP)、凝胶阻滞(EMSA)和Western blot技术，揭示这些调控调控元件的调控模式。研究结果将阐明副溶血弧菌黏附因子vpadF基因转录调控的分子机制，为预防其感染提供理论依据。

副溶血弧菌是是一种广泛存在于海水和海底沉积物的嗜盐细菌，易感染海洋鱼类、虾和贝类等海产生物。被污染的海产品如处理不当，人食用后会引起急性胃肠炎，产生发热、腹泻、呕吐等症状，严重者可引起脱水、休克，甚至死亡。黏附作用是副溶血弧菌感染宿主的第一步，也是导致感染的首要条件。已知的副溶血弧菌黏附因子有多个，其中诱导型表达的黏附因子VpadF是其感染宿主必需的，然而vpadF基因表达调控的分子机制以前并不清楚。本项目发现副溶血弧菌主要通过表达VpadF来结合I型胶原，从而激活I型胶原-α1β1整合素-磷脂酰肌醇-3激酶信号途径，从而介导其侵袭宿主细胞，这种侵袭机制与其他病原菌侵袭宿主细胞的分子机制都不相同。因此，我们不仅揭示了副溶血弧菌通过结合I型胶原蛋白感染宿主细胞，而且发现了副溶血弧菌侵袭细胞的独特方式，提出该菌黏附宿主细胞是一个多步骤的过程。其次，我们通过比较转录组、基因敲除并结合β-半乳糖苷酶活性实验，还初步发现了ExsA和VadK这2个调控基因能促进vpadF的转录，进一步的生化、细胞、EMSA、比较转录组测序和感染小鼠等实验也正在进行中。总之，本项目的研究结果对深入理解副溶血弧菌的感染机理具有重要科学意义，同时也为有效防治其感染提供了理论依据。