副溶血性弧菌中密度感应系统依赖的T3SS1和T6SS2调控机制研究

发布时间：2018-08-15 12:49

【摘要】：副溶血性弧菌（Vibrio parahaemolyticus，VP）是一种海洋微生物，主要生存于海水，港口水环境及附着在海产品上，，人们主要通过食用未煮熟的污染的海产品而感染，目前副溶血性弧菌已经成为引起食物中毒的首要病原菌。密度感应系统（quorum-sensing，QS）是细菌中广泛存在的细菌之间的信号传递机制，主要通过合成，释放及感应自身诱导因子（autoinducers，AI）来感受生存环境的变化，继而调控与细菌菌密度相关的系列基因的转录表达。副溶血性弧菌QS系统影响细菌很多生物学功能，如生物膜的形成，菌落透明度的变化，细菌的运动能力等。AphA和OpaR是副溶血性弧菌QS系统的2个主要转录调控子，分别在低菌密度（low cell density，LCD）和高菌密度（high celldensity，HCD）时高表达，并介导细菌在LCD和HCD之间进行转换。三型分泌系统（Type3Secretion Systems，T3SS）是细菌蛋白分泌装置，通过把毒力效应蛋白注射入宿主细胞内引起细胞损伤。副溶血性弧菌T3SS1以诱导宿主细胞程序性死亡或自噬的方式介导细菌的细胞毒性。副溶血性弧菌六型分泌系统Ⅱ（Type6Secretion Systems2，T6SS2）主要介导其对宿主细胞的粘附作用,粘附是细菌与宿主细胞相互作用的首要步骤，是引起致病的必要条件。副溶血性弧菌T3SS1和T6SS2是其关键的的毒力因子，参与其致病的多种生物学功能。本课题着重研究QS中心调控子AphA和OpaR对T3SS1和T6SS2的具体转录调控机制。副溶血性弧菌T3SS1主要受exsACDE操纵子的调控。在副溶血性弧菌近缘菌哈氏弧菌中，QS系统中心调控子LuxR通过抑制exsA基因的表达来间接抑制T3SS1的表达。但是在副溶血性弧菌中QS系统调控T3SS1的详尽机制并未报道。根据生物信息学预测，副溶血性弧菌中QS系统可能也是通过作用于exsACDE操纵子来调控T3SS1的转录；且根据操纵子结构实验我们确定了4个相应的靶基因进行研究，exsC、exsB、 exsD和VP1687（T3SS1效应蛋白基因）。副溶血性弧菌T6SS2受OpaR蛋白的转录调控，OpaR蛋白促进其表达，但是详细的调控机制并未报道。根据生物信息学及相关文献报道推测：QS系统可能通过直接作用于T6SS2的基因簇来调控其转录与表达。T6SS2基因簇由3个操纵子组成，我们选择了每个操纵子的首位基因作为靶基因来进行研究，即T6SS2的靶基因为VPA1027、VPA1043和VPA1044。本研究利用自杀质粒pDS132对aphA和opaR基因进行了缺失，并以pBAD33质粒为载体进行了相应基因的回补，利用pET蛋白表达系统在大肠杆菌BL21（λDE3）中获得了His-AphA和His-OpaR的重组蛋白，随后用凝胶阻滞实验（electrophoretic mobility shift assay，EMSA）和DNA酶I足迹实验（DNaseI footpriting assay）研究调控子与靶基因启动子区之间的关系，并用引物延伸实验和LacZ报告基因融合实验研究调控子对靶基因的调控关系，继而详细阐述调控机制。实验结果表明：AphA通过激活exsC、exsB和exsD基因的转录来促进T3SS1（VP1687）的转录，继而促进细菌对宿主细胞的细胞毒性；OpaR通过抑制exsC、exsB和exsD基因的转录来抑制T3SS1（VP1687）的转录，继而抑制细菌对宿主细胞的细胞毒性。AphA和OpaR结合在exsB基因的启动子区序列上，直接调控其的转录表达。AphA和OpaR对exsC和exsD的作用均是间接的。这个结果表明在菌密度较低时aphA大量表达从而促进T3SS1表达，致使微量的细菌就可以引起感染；在菌密度较高时opaR抑制T3SS1的表达，使细菌对宿主的致病力下降，进而促进其排出宿主体外，利于细菌的传播。 AphA间接负向调控VPA1027、VPA1043和VPA1044的转录从而抑制T6SS2的转录；OpaR结合于这3个靶基因的启动子区直接正向调控T6SS2的转录。这表明T6SS2可能在细菌感染的后期起主要作用，维持细菌的毒力。本研究首次利用生物信息学及经典的分子生物学实验详细的阐述了QS系统依赖的T3SS1和T6SS2转录调控机制，为重塑副溶血性弧菌基因调控网络，揭示其致病机理提供了理论基础。
[Abstract]:Vibrio parahaemolyticus (VP) is a kind of marine microorganism. It mainly exists in seawater, harbor water environment and attaches to seafood. People are infected mainly by eating uncooked contaminated seafood. At present, Vibrio parahaemolyticus has become the primary pathogen causing food poisoning. Sing (QS) is a signal transduction mechanism between bacteria. It mainly senses the changes of living environment by synthesizing, releasing and sensing autoinducers (AI), and then regulates the transcription and expression of a series of genes related to bacterial density. AphA and OpaR are the two major transcription regulators in the QS system of Vibrio parahaemolyticus, which are highly expressed at low cell density (LCD) and high cell density (HCD), respectively, and mediate the transformation between LCD and HCD.
Type 3 Secretion Systems (T3SS) is a bacterial protein secreting device that causes cell damage by injecting virulent effector proteins into host cells. Vibrio parahaemolyticus T3SS1 mediates cytotoxicity by inducing programmed cell death or autophagy. Vibrio parahaemolyticus type 6 secretion system II (T6Secr) Etion systems 2 (T6SS2) mainly mediates its adhesion to host cells. Adhesion is the first step in the interaction between bacteria and host cells and is a necessary condition for pathogenesis. Vibrio parahaemolyticus T3SS1 and T6SS2 are the key virulence factors involved in the pathogenesis of various biological functions. Specific transcriptional regulation mechanisms of A and OpaR on T3SS1 and T6SS2.
In Vibrio parahaemolyticus, the central regulator LuxR of the QS system indirectly inhibits the expression of T3SS1 by inhibiting the expression of exsA gene. However, the detailed mechanism of the QS system regulating T3SS1 in Vibrio parahaemolyticus has not been reported. The QS system in Vibrio parahaemolyticus may also regulate the transcription of T3SS1 by acting on exsACDE operon, and we identified four corresponding target genes, exsC, exsB, exsD and VP1687 (T3SS1 effector protein gene).
Vibrio parahaemolyticus T6SS2 is regulated by the transcription of OpaR protein. OpaR protein promotes its expression, but the detailed regulatory mechanism has not been reported. The target genes of T6SS2 were VPA1027, VPA1043 and VPA1044.
In this study, the suicide plasmid pDS132 was used to delete the aphA and opaR genes, and pBAD33 plasmid was used as the vector to replenish the corresponding genes. The recombinant proteins of his-AphA and his-OpaR were obtained by pET protein expression system in E. coli BL21 (lambda DE3). Then the recombinant proteins of his-AphA and his-OpaR were tested by electrophoretic mobility shift assay (EMSA). And the DNA enzyme I footprint assay (DNase I footprint assay) was used to study the relationship between the regulator and the promoter region of the target gene, and the primer extension test and LacZ reporter gene fusion test were used to study the regulatory relationship between the regulator and the target gene, and then the regulatory mechanism was elaborated in detail.
The results showed that AphA promoted the transcription of T3SS1 (VP1687) by activating the transcription of exsC, exsB and exsD genes, and then promoted the cytotoxicity of bacteria to host cells; OpaR inhibited the transcription of T3SS1 (VP1687) by inhibiting the transcription of exsC, exsB and exsD genes, and then inhibited the cytotoxicity of bacteria to host cells. The effect of AphA and OpaR on exsC and exsD is indirect. This result indicates that aphA overexpression promotes T3SS1 expression at low bacterial density, resulting in infection of microorganisms; opaR inhibits the expression of T3SS1 at high bacterial density. The pathogenicity of the host decreases, and then promotes the excretion of the host in vitro, which is beneficial to the spread of bacteria.
AphA indirectly negatively regulates the transcription of VPA1027, VPA1043 and VPA1044, thereby inhibiting the transcription of T6SS2. OpaR binds to the promoter region of these three target genes and directly regulates the transcription of T6SS2. This suggests that T6SS2 may play a major role in the later stage of bacterial infection and maintain the virulence of bacteria.
In this study, the transcriptional regulation mechanism of QS system-dependent T3SS1 and T6SS2 was elaborated in detail by using bioinformatics and classical molecular biology experiments for the first time, which provided a theoretical basis for remodeling the gene regulatory network of Vibrio parahaemolyticus and revealing its pathogenic mechanism.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R155.5

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