Transcription Regulation in Pseudomonas aeruginosa

描述(申请人提供)：铜绿假单胞菌是一种重要的人类条件致病菌，是囊性纤维化患者发病和死亡的主要原因。铜绿假单胞菌对囊性纤维化患者肺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。这项拟议的工作将阐明TeX的功能和作用机制，TeX是铜绿假单胞菌的一种假定转录因子，在慢性疾病模型中是宿主肺感染所必需的。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，毒力基因本身对宿主的感染或生存至关重要。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)有关，并且TeX可以影响铜绿假单胞菌中一组基因的表达。此外，这些实验表明，TeX不仅与RNAP有关，而且与一个可能的多亚单位复合体的成分有关，该复合体被称为RNA降解体：一种首次在大肠杆菌中描述的含有核糖核酸酶的复合体，据预测主要参与mRNA的降解。这些发现提出了这样一种可能性，即转录机制可能在操作上与铜绿假单胞菌的RNA降解机制有关。可以想象，这种连接可以促进特定转录本的任何必要的处理或降解，并可能代表一种引发基因控制的新机制。拟议的研究将确定其表达受Tex影响的基因，识别与Tex共同净化的RNA，并测试Tex可能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具有重要意义。此外，我们将进一步研究那些含有TeX的蛋白质复合体的性质，并研究TeX与RNAP和铜绿假单胞菌中假定的降解体成分的相互作用。拟议的实验不仅应该阐明TeX在发病机制中的重要性的分子基础，而且还可能促进可用于对抗CF肺中铜绿假单胞菌感染的新型治疗剂的开发。

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.