【摘要】：副溶血性弧菌（Vibrio parahaemolyticus，VP）是一種海洋微生物，主要生存于海水，港口水環(huán)境及附著在海產(chǎn)品上，，人們主要通過食用未煮熟的污染的海產(chǎn)品而感染，目前副溶血性弧菌已經(jīng)成為引起食物中毒的首要病原菌。密度感應系統(tǒng)（quorum-sensing，QS）是細菌中廣泛存在的細菌之間的信號傳遞機制，主要通過合成，釋放及感應自身誘導因子（autoinducers，AI）來感受生存環(huán)境的變化，繼而調(diào)控與細菌菌密度相關(guān)的系列基因的轉(zhuǎn)錄表達。副溶血性弧菌QS系統(tǒng)影響細菌很多生物學功能，如生物膜的形成，菌落透明度的變化，細菌的運動能力等。AphA和OpaR是副溶血性弧菌QS系統(tǒng)的2個主要轉(zhuǎn)錄調(diào)控子，分別在低菌密度（low cell density，LCD）和高菌密度（high celldensity，HCD）時高表達，并介導細菌在LCD和HCD之間進行轉(zhuǎn)換。 三型分泌系統(tǒng)（Type3Secretion Systems，T3SS）是細菌蛋白分泌裝置，通過把毒力效應蛋白注射入宿主細胞內(nèi)引起細胞損傷。副溶血性弧菌T3SS1以誘導宿主細胞程序性死亡或自噬的方式介導細菌的細胞毒性。副溶血性弧菌六型分泌系統(tǒng)Ⅱ（Type6Secretion Systems2，T6SS2）主要介導其對宿主細胞的粘附作用,粘附是細菌與宿主細胞相互作用的首要步驟，是引起致病的必要條件。副溶血性弧菌T3SS1和T6SS2是其關(guān)鍵的的毒力因子，參與其致病的多種生物學功能。本課題著重研究QS中心調(diào)控子AphA和OpaR對T3SS1和T6SS2的具體轉(zhuǎn)錄調(diào)控機制。 副溶血性弧菌T3SS1主要受exsACDE操縱子的調(diào)控。在副溶血性弧菌近緣菌哈氏弧菌中，QS系統(tǒng)中心調(diào)控子LuxR通過抑制exsA基因的表達來間接抑制T3SS1的表達。但是在副溶血性弧菌中QS系統(tǒng)調(diào)控T3SS1的詳盡機制并未報道。根據(jù)生物信息學預測，副溶血性弧菌中QS系統(tǒng)可能也是通過作用于exsACDE操縱子來調(diào)控T3SS1的轉(zhuǎn)錄；且根據(jù)操縱子結(jié)構(gòu)實驗我們確定了4個相應的靶基因進行研究，exsC、exsB、 exsD和VP1687（T3SS1效應蛋白基因）。 副溶血性弧菌T6SS2受OpaR蛋白的轉(zhuǎn)錄調(diào)控，OpaR蛋白促進其表達，但是詳細的調(diào)控機制并未報道。根據(jù)生物信息學及相關(guān)文獻報道推測：QS系統(tǒng)可能通過直接作用于T6SS2的基因簇來調(diào)控其轉(zhuǎn)錄與表達。T6SS2基因簇由3個操縱子組成，我們選擇了每個操縱子的首位基因作為靶基因來進行研究，即T6SS2的靶基因為VPA1027、VPA1043和VPA1044。 本研究利用自殺質(zhì)粒pDS132對aphA和opaR基因進行了缺失，并以pBAD33質(zhì)粒為載體進行了相應基因的回補，利用pET蛋白表達系統(tǒng)在大腸桿菌BL21（λDE3）中獲得了His-AphA和His-OpaR的重組蛋白，隨后用凝膠阻滯實驗（electrophoretic mobility shift assay，EMSA）和DNA酶I足跡實驗（DNaseI footpriting assay）研究調(diào)控子與靶基因啟動子區(qū)之間的關(guān)系，并用引物延伸實驗和LacZ報告基因融合實驗研究調(diào)控子對靶基因的調(diào)控關(guān)系，繼而詳細闡述調(diào)控機制。 實驗結(jié)果表明：AphA通過激活exsC、exsB和exsD基因的轉(zhuǎn)錄來促進T3SS1（VP1687）的轉(zhuǎn)錄，繼而促進細菌對宿主細胞的細胞毒性；OpaR通過抑制exsC、exsB和exsD基因的轉(zhuǎn)錄來抑制T3SS1（VP1687）的轉(zhuǎn)錄，繼而抑制細菌對宿主細胞的細胞毒性。AphA和OpaR結(jié)合在exsB基因的啟動子區(qū)序列上，直接調(diào)控其的轉(zhuǎn)錄表達。AphA和OpaR對exsC和exsD的作用均是間接的。這個結(jié)果表明在菌密度較低時aphA大量表達從而促進T3SS1表達，致使微量的細菌就可以引起感染；在菌密度較高時opaR抑制T3SS1的表達，使細菌對宿主的致病力下降，進而促進其排出宿主體外，利于細菌的傳播。 AphA間接負向調(diào)控VPA1027、VPA1043和VPA1044的轉(zhuǎn)錄從而抑制T6SS2的轉(zhuǎn)錄；OpaR結(jié)合于這3個靶基因的啟動子區(qū)直接正向調(diào)控T6SS2的轉(zhuǎn)錄。這表明T6SS2可能在細菌感染的后期起主要作用，維持細菌的毒力。 本研究首次利用生物信息學及經(jīng)典的分子生物學實驗詳細的闡述了QS系統(tǒng)依賴的T3SS1和T6SS2轉(zhuǎn)錄調(diào)控機制，為重塑副溶血性弧菌基因調(diào)控網(wǎng)絡，揭示其致病機理提供了理論基礎(chǔ)。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R155.5

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