T6SS2效应蛋白VP-GntR调控副溶血弧菌致病性的分子机制研究

GntR family transcriptional regulators are widespread in bacteria, and have been found to control many fundamental cellular processes such as bacterial metabolism, motility, biofilm formation, antibiotic production and virulence. Vibrio parahaemolyticus is considered as an important foodborne pathogen, which poses a threat to human public health. Type VI secretion system (T6SS) plays important roles in the survival and pathogenicity of bacteria by secreting many effector proteins to achieve its biological functions. In our previous study, we found that T6SS2 were involved in numerous pathogenic processes of V.parahaemolyticus, including biofilm formation, motility and virulence. The effectors of T6SS2 were screened and identified by comparative proteomics method. An important effector (encoded by VP1277 gene) was selected and predicted to a GntR family transcriptional regulator, and subsequently named as VP-GntR.In our preliminary study, the results revealed that VP-GntR plays important roles in the biological characteristics and pathogenicity of V.parahaemolyticus. However, the exact regulation mechanism of VP-GntR is still unknown. This study includes:（1）Determination of biological characteristics and pathogenicity of gntR mutant. (2) Identification of the target genes regulated by VP-GntR, and analysis of the transcriptional regulation mechanism of VP-GntR. (3) Clarify the roles of the important target genes regulated by VP-GntR on the molecular pathogenesis of V. parahaemolyticus. The purpose of this study is to elucidate the molecular mechanism of V.parahaemolyticus pathogenicity regulated by VP-GntR, which make for further understand V. parahaemolyticus pathogenesis and benefit for the future prevention and control strategies against the V. parahaemolyticus infection.

GntR家族蛋白是细菌中广泛存在的一类多功能调控子，参与调控细菌代谢、生物被膜形成、运动性、耐药性和毒力等。副溶血弧菌（VP）是一种重要的食源性病原菌，威胁人类公共卫生安全。VI型分泌系统（T6SS)是细菌一种重要的蛋白分泌系统，借由分泌效应蛋白实现生物学功能，与细菌环境适应性及致病力密切相关。前期研究发现T6SS2与VP生物被膜形成、运动性和毒力相关。通过比较蛋白组学技术筛选并鉴定到一个T6SS2分泌蛋白，是GntR家族成员，命名为VP-GntR。前期研究发现VP-GntR与VP致病力相关，但对其具体的调控机制尚不清楚。本研究主要内容包括：（1）测定VP-GntR的基因缺失株的生物学特性和致病性；（2）鉴定和分析VP-GntR蛋白调控的靶基因；（3）研究VP-GntR调控的靶基因在VP致病过程中的作用。最终阐明VP-GntR调控VP致病性的分子机制，研究结果为VP预防和控制理论基础。

GntR家族蛋白是细菌中广泛存在的一类多功能调控子,参与调控细菌的代谢和毒力等。副溶血弧菌（VP）是一种重要的食源性病原菌，威胁人类公共卫生安全。前期研究中通过比较蛋白组学技术筛选并鉴定到一个T6SS2分泌蛋白，是GntR家族成员，命名为VP-GntR，进一步发现其与VP致病力相关，但具体的调控机制尚不清楚。通过本项目研究发现：（1）VP-GntR对VP在环境生存和致病性中发挥重要作用。缺失VP-GntR基因的VP在低酸，高盐、去污剂的作用下存活能力明显下降，同时运动能力和生物被膜形成能力也显著降低；此外，进一步发现VP-GntR参与VP对宿主细胞的毒性作用和对小鼠致病过程。（2）VP-GntR蛋白负调控VP组氨酸代谢途径中的重要酶类基因，同时正调控与VP转运、生物被膜形成和毒力相关的基因。通过细菌转录组测序技术比较分析VP野生株和VP-GntR基因缺失株的转录差异基因，运用RT-PCR对结果进行验证，结果发现表达差异显著的基因有19个，其中上调基因4个，下调基因基因15个。KEGG pathway代谢通路分析和GO功能分析，结合EMSA试验结果表明VP-GntR直接负调控VP组氨酸代谢途径中的4个基因，包括VP1273，VP1274，VP1275和VP127。此外，下调基因主要有VP0356，VPA0015，VPA0343等及一些未知蛋白，功能分析提示它们主要参与VP生物膜形成、代谢作用、氧化分解、生物学结合等过程。（3）VP-GntR调控的靶基因参与VP运动性、生物被膜形成和毒力。结合分子生物信息学，选定VP1273,VP1276,VPA0015,VPAA0343进行深入研究。研究发现这4个基因的缺失均不影响VP的生长特性，且它们均不参与VP耐盐和耐酸能力，但均显著影响VP生物被膜的形成能力。VP1273或VP1276基因缺失后VP的泳动运动能力显著下降，但浮游运动能力不受影响。相反的是，VPA0015或VPA0343基因缺失后，VP的浮游运动能力显著下降，泳动运动能力不受影响。与野生株相比，感染VP1273或VPA0015基因缺失株的小鼠存活率均明显提高。本项目研究结果初步阐明了VP-GntR调控VP致病性的分子机制，可加深人们对VP毒力调控机制的认识，同时为VP防控及药物开发提供新思路。