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.
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