志贺氏菌毒力大质粒调控ATP合成酶表达

Shigella is the pathogen of bacillary dysentery. It shares a high degree of homology and similarity of genome with E. coli, the main difference between the two spieces lies in the presence or absence of the virulence plasmid. At the same time, Shigella is a typical example of co-evolution and mutual adaptation between virulence plasmid genes and chromosomal genes. However, the regulation of virulence plamid on bacterial genome is poorly understood. We compared soluble protein complexes of wild-type strain with virulence plasmid deletion mutant of Shigella, using BN/SDS-PAGE 2-D electrophoresis system. We found deletion of virulence plasmid increased the expression of ATP synthase. The project plans to detect the effect of virulence plasmid on the expression of ATP synthase subunits and interaction among them using western blot and immunoprecipitation. With the aim of mechanisms underlying regulation of virulence plasmid on the expression of ATP synthase, we plan to build virulence plasmid large fragment mutants library and screen ATP synthase regulatory proteins. We want to detect ATP synthase activity and acid resistance of shigella to explore the biological significance of the regulation of virulence plasmid on ATP synthase expression. This study will further reveal the regulation of virulence plasmid on chromosome, provide theoretical basis for the research of Shigella evolution process and development of new vaccines and antibiotics.

志贺氏菌是细菌性痢疾的病原菌，它与大肠杆菌的基因组有高度的同源性和相似性，主要区别在于毒力大质粒的有无。志贺氏菌是毒力大质粒和染色体协同进化、相互适应的典范，然而毒力大质粒对细菌基因组的调控还知之甚少。我们采用BN/SDS-PAGE 2-D电泳系统，对比了志贺氏菌野生株和毒力大质粒缺失突变株的可溶性蛋白复合物，发现毒力大质粒能够调控ATP合成酶的表达。本项目拟采用Western blot和免疫共沉淀等方法分别检测毒力大质粒对ATP合成酶各亚基的表达以及它们之间相互作用的影响，同时构建毒力大质粒大片段突变体库筛选ATP合成酶的调控相关蛋白，从而明确毒力大质粒调控ATP合成酶表达的具体机制。通过ATP合成酶活性检测及对菌株耐酸能力的分析，探索毒力大质粒调控ATP合成酶表达的生物学意义。本研究将进一步揭示毒力大质粒对染色体的调控作用，为志贺氏菌进化过程研究以及疫苗和新型抗生素的研制提供理论依据。

志贺氏菌是细菌性痢疾的病原菌，它与大肠杆菌的基因组有高度的同源性和相似性，主要区别在于毒力大质粒的有无。毒力质粒的获取是志贺氏菌从无毒的大肠杆菌向致病菌进化的关键步骤，此外，细菌染色体和毒力质粒的共同进化和相互协调也是进化过程中很重要的因素。志贺氏菌是毒力大质粒和染色体协同进化、相互适应的典范，然而毒力大质粒对细菌基因组的调控还知之甚少。我们采用BN/SDS-PAGE 2-D电泳系统，对比了志贺氏菌野生株和毒力大质粒缺失突变株的可溶性蛋白复合物，发现毒力大质粒能够调控ATP合成酶的表达。AtpA/D与细菌内质子流和pH调控有关，酸抗性的差异显示毒力质粒降低了上述菌株在胃酸等强酸情况下的存活率。这些结果拓展了我们对非致病菌向致病菌进化过程的理解，同时也显示出细菌进化过程中异源基因和宿主染色体相互协作的重要性。