Transcription Regulation in Pseudomonas aeruginosa

描述（由申请人提供）：铜绿假单胞菌是一种重要的人类机会性病原体，是导致囊性纤维化（CF）患者发病和死亡的主要原因；铜绿假单胞菌在CF肺部的慢性定植通常会导致进行性肺损伤，最终导致呼吸衰竭和死亡。提出的工作将阐明Tex的功能和作用机制，Tex是一种来自铜绿假单胞菌的转录因子，是慢性疾病模型中宿主肺部感染所必需的。Tex在细菌中高度保守，是真核转录延伸因子Spt6的同源物。这项研究的基本假设是，Tex通过影响毒力基因的表达在铜绿假单胞菌的发病机制中发挥重要作用，而毒力基因本身对宿主的感染或存活很重要。为了支持这一观点，初步实验表明，Tex与基因表达的中心酶RNA聚合酶（RNAP）相关，并且Tex可以影响P. aeruginosa中一部分基因的表达。此外，这些实验表明，Tex不仅与RNAP相关，还与一种假定的多亚基复合物（称为RNA降解体）的组分相关。RNA降解体是一种含有核糖核酸酶的复合物，首次在大肠杆菌中被描述，预计主要参与mRNA的降解。这些发现提出了这种转录机制可能与铜绿假单胞菌的RNA降解机制在操作上相关联的可能性。可以想象，这种联系可以促进特定转录物的任何必要处理或降解，并可能代表引发基因控制的新机制。拟议的研究将确定表达受Tex影响的基因，确定与Tex共同纯化的rna，并测试Tex如何影响基因表达的特定模型。以Tex的高分辨率晶体结构为指导，我们将探索Tex的哪些结构特征对其功能很重要。此外，我们将进一步研究含有Tex的蛋白质复合物的性质，并研究Tex与P. aeruginosa中RNAP和推定降解体组分的相互作用。本实验不仅可以揭示Tex在发病机制中的重要作用的分子基础，还可以促进开发新的治疗药物，用于对抗CF肺内铜绿假单胞菌感染。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an important opportunistic pathogen of humans that is notorious for being the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients; chronic colonization of the CF lung by P. aeruginosa typically leads to progressive lung damage, and eventually respiratory failure and death. The proposed work will elucidate the function and mechanism of action of Tex, a putative transcription factor from P. aeruginosa that is required for infection of the host lung in a chronic disease model. Tex is highly conserved amongst bacteria, and is a homolog of the eukaryotic transcription elongation factor Spt6. The underlying hypothesis for the proposed study is that Tex plays an important role in P. aeruginosa pathogenesis by influencing the expression of virulence genes that are themselves important for infection of, or survival within, the host. In support of this idea, preliminary experiments indicate that Tex associates with RNA polymerase (RNAP), the central enzyme of gene expression, and that Tex can influence the expression of a subset of genes in P. aeruginosa. Furthermore these experiments reveal that Tex associates not only with RNAP but also with components of a putative multi-subunit complex, termed the RNA degradosome: a ribonuclease-containing complex first described in E. coli that is predicted to be primarily involved in the degradation of mRNA. These findings raise the possibility that the transcription machinery may be operationally linked to the RNA degradation machinery in P. aeruginosa. Such a link could conceivably facilitate any requisite processing or degradation of a particular transcript and may represent a novel mechanism for eliciting gene control. The proposed studies will identify genes whose expression is influenced by Tex, identify RNAs that co-purify with Tex, and test specific models for how Tex might influence gene expression. With a high-resolution crystal structure of Tex as a guide, we will explore which structural features of Tex are important for its function. Moreover, we will investigate further the nature of those protein complexes that contain Tex and investigate the interactions of Tex with both RNAP and components of the putative degradosome in P. aeruginosa. The proposed experiments should not only shed light on the molecular basis for the importance of Tex in pathogenesis, but may also facilitate the development of novel therapeutic agents that can be used to combat P. aeruginosa infection in the CF lung.