双组分信号转导系统PhoQ/PhoP调控志贺菌毒力的分子机制

PhoQ/PhoP, a two component signal transduction system, plays an important regulation roles in gram negative bacteria. It acts as a virulence regulator in pathogenic bacteria, mediating the adoption of bacterial cells to low Mg2+ environments, and regulating other bacterial biological activities. However, molecular regulatory mechanism of PhoQ/PhoP in Shigella remains to be clarified, although knockout of phoP or inhibition of PhoQ histidine kinase activity have resulted in attenuated of Shigella virulence in animal infection model or cell invasion. Based on what we have studied about inhibition of S.flexineri virulence by blocking PhoQ activity, we further investigate the roles of PhoQ/PhoP in Shigella with phoQ/phoP knockout strain (S.flexineri 2a 301) by homologous recombination. The phoQ/phoP knockout strain‘s abilities of cell invasion, intracellular survival, reaction to external stress and inducing conjunctivitis of mice or guinea pig are compared with that of wild-type strain. It will be confirmed by transfection of wild-type strain with anti-RNA expression plasmids. Transcriptional profile of PhoQ/PhoP knockout strain is compared with that of wild type by using gene chips, and the differentially expressed gene will be confirmed by RT-PCR. Then, based on transcriptome data, we predicts PhoQ/PhoP regulated gene with bioinformatics, and confirmed by promoters binding activity of re-PhoP with EMSA. The binding motif in the region of promoters are analyzed by DNase foot-printing. In the PhoQ/PhoP regulon, we will focus on the genes related with virulence as well as stress reactions to environments in shigella. The PhoQ/PhoP regulated genes will be further knocked out by homologous recombination, or the genes expression be knocked down by transfection of anti-RNA plasmid. The study will shed a light on PhoQ/PhoP regulation pathways in shigella virulence and stress reaction.

PhoQ/PhoP双组份信号传导系统在数种革兰阴性菌中具有控制细菌毒力、介导细菌适应低镁环境、调节细菌多种生物学活性等功能。虽然志贺菌的PhoP基因单敲除或抑制PhoQ组氨酸激酶可致细菌毒力下降，但其在志贺菌中调控的分子机制尚不清楚。本项目在PhoQ抑制剂的基础上，将深入研究PhoQ/PhoP调控志贺菌的毒力及细菌应激的机制：利用同源重组技术构建志贺菌PhoP/PhoQ基因突变株，比较突变株和野生株在外环境压力中的存活能力、细胞侵袭和胞内存活能力、引起感染动物角膜炎症的能力等；结合基因芯片比较野生株与突变株细菌表型、基因转录谱的差异；进而结合生物信息学分析和凝胶阻滞试验（EMSA）及DNA酶指纹法确定反应调节蛋白PhoP与相关毒力基因启动子区域的结合序列，发现并确证受其调控的下游毒力相关基因及其生物学功能，阐明PhoQ/PhoP双组分信号系统在志贺菌中调控毒力及应激相关基因表达的通路。

PhoQ/PhoP双组份信号传导系统在其他革兰氏阴性菌中具有调控细菌毒力、适应低镁环境等多种生物学活性等功能，但其在志贺菌中调控的分子机制尚不清楚。本课题通过同源重组技术构建志贺菌PhoP/PhoQ基因突变株，比较了突变株和野生株的毒力及在外环境压力下的存活能力、细胞侵袭和胞内存活能力。发现phoQ/phoP基因敲除株在细胞水平（Hela细胞感染模型）和动物水平（豚鼠角结合膜感染模型）上毒力明显下降，回复突变株可使毒力回复至野生株的水平。PhoP/Q可调控细菌与环境压力有关基因表达，有利于细菌在不良环境压力下（酸性、氧应激、多粘菌素等）中生存和繁殖。利用基因芯片进行突变株/野生株基因转录谱的比较，利用生物信息学方法预测 PhoQ/PhoP 下游调控基因，并通过凝胶迁移滞后试验，验证 PhoP与下游基因启动子结合，证明PhoP蛋白可与phoP, mgtA, slyB, icsA, shf, rstA, yoaE, hdeA, yrbL, yhiW及pagP。利用 DNase I Foot -printing 试验分析 PhoP 蛋白与靶基因启动子结合的共有序列5’ [T/G]GTTTA- 5nt - [T/G]GTTTA 3’，与大肠埃希菌报道的PhoP box结合模式一致。进而，我们侧重分析和研究与毒力和应激应答相关的关键差异表达基因。在环境压力下phoP、shf及icsA在低Mg2+、酸性及抗菌肽环境下转录水平均有较明显的上调。构建phoP、shf及icsA的LacZ报告菌株，研究结果显示PhoQ/PhoP正调控该类基因的转录。通过同源重组构建了福氏志贺菌icsA敲除株及其回复突变株，并将icsA互补质粒转入phoPQ敲除株，证实phoPQ-icsA对志贺菌毒力的影响。志贺菌双组分信号系统phoP/Q调控细菌与环境压力有关基因表达，有利于细菌在不良环境（酸性、氧应激、多粘菌素等）中生存和繁殖，同时还可以调控细菌的毒力，有利于细菌侵袭宿主细胞。