Regulatory mechanism of host cell contact-dependent T3SS gene expression in Vibrio parahaemolyticus

Many pathogenic bacteria have mechanisms that regulate gene expression in response to the surrounding environment to establish an infection. One such mechanism is the regulation of gene expression in response to contact with host cells. Here, we show that Vibrio parahaemolyticus, a causative agent of foodborne gastroenteritis, has a host cell contact-dependent regulatory mechanism for virulence gene expression. T3SS2, an essential virulence determinant for acute gastroenteritis encoded by V. parahaemolyticus pathogenicity island (Vp-PAI), recognizes host cell contact by sensing high intracellular K+ levels and switches its secretory substrates. This switching of secretory substrates is regulated by proteins called gatekeepers. Mutants deficient in the gatekeeper gene lose the ability to switch secretory substrates and lock their secretory state into contact with the host cell. Transcriptome analysis of T3SS2 gatekeeper gene-deficient strains showed that the genes encoded by Vp-PAI were specifically upregulated, and this upregulation was dependent on T3SS2 secretory activity, implying the presence of a negative regulator secreted by T3SS2. Comparative proteomic analyses identified a previously unidentified T3SS2 secretory substrate, VtrN, that negatively regulates Vp-PAI gene transcription. Secretion of VtrN was promoted under conditions that mimic host cell contact as well as other T3SS2 effectors. vtrN gene deletion specifically upregulated Vp-PAI gene expression, but unlike gatekeeper gene deletion, it was independent of T3SS2 secretory activity. Furthermore, VtrN interacted with VtrB, a transcription factor essential for Vp-PAI-encoded gene expression and repressed its transcriptional activity. Thus, V. parahaemolyticus has a mechanism to upregulate virulence gene expression in response to host-cell contact by utilizing the host-cell recognition mechanism of T3SS2. Overall design: To investigate the effect of gatekeeper dysfunction (vpa1360 and vpa1359) on T3SS2 gene expression, the gene transcription profiles of the gatekeeper deletion mutants and parental strain were examined under a general culture condition (RIMD2210633_vpa1360 deletion mutant and RIMD2210633_vpa1359 deletion mutant vs RIMD2210633_WT). The experiment was repeated 3 times. To investigate the function of a T3SS2 regulatory gene (vpa1369), the gene transcription profiles of the deletion mutant and parental strain were compared when culture in a general condition (RIMD2210633_vpa1369 deletion mutant vs RIMD2210633). The experiment was repeated 3 times.

诸多致病菌具备响应周遭环境以调控基因表达、进而建立感染的机制。其中一类机制为响应宿主细胞接触而调控基因表达。本研究表明，食源性胃肠炎的致病原——副溶血性弧菌（Vibrio parahaemolyticus），存在依赖宿主细胞接触的毒力基因表达调控机制。Ⅲ型分泌系统2（T3SS2）是副溶血性弧菌致病岛（Vp-PAI）所编码的急性胃肠炎关键毒力决定因子，其可通过感知胞内高钾离子浓度识别宿主细胞接触，并切换其分泌底物。这类分泌底物的切换过程受一类被称为“门卫蛋白”（gatekeepers）的调控蛋白所支配。缺失门卫蛋白编码基因的突变株会丧失分泌底物切换能力，并将分泌状态锁定在与宿主细胞接触后的状态。对T3SS2门卫蛋白基因缺失菌株的转录组分析结果显示，Vp-PAI所编码的基因出现特异性上调，且该上调依赖于T3SS2的分泌活性，这暗示存在一种由T3SS2分泌的负调控因子。比较蛋白质组学分析鉴定出一种此前未被发现的T3SS2分泌底物VtrN，其可负调控Vp-PAI基因的转录过程。VtrN与其他T3SS2效应蛋白一样，在模拟宿主细胞接触的培养条件下其分泌过程会被促进。缺失vtrN基因可特异性上调Vp-PAI基因的表达，但与门卫蛋白基因缺失不同的是，该上调过程不依赖于T3SS2的分泌活性。此外，VtrN可与Vp-PAI编码基因表达所必需的转录因子VtrB发生相互作用，并抑制其转录活性。综上，副溶血性弧菌可通过利用T3SS2的宿主细胞识别机制，响应宿主细胞接触而上调毒力基因的表达。
实验设计：为探究门卫蛋白功能异常（vpa1360与vpa1359基因缺失）对T3SS2基因表达的影响，本研究在常规培养条件下检测了门卫蛋白基因缺失突变株与亲本菌株的基因转录谱（RIMD2210633_vpa1360缺失突变株、RIMD2210633_vpa1359缺失突变株 与 RIMD2210633野生型菌株的对比），实验重复3次。为探究T3SS2调控基因vpa1369的功能，本研究在常规培养条件下对比了vpa1369缺失突变株与亲本菌株的基因转录谱（RIMD2210633_vpa1369缺失突变株 与 RIMD2210633野生型菌株的对比），实验重复3次。