An essential RNA chaperone in Pseudomonas aeruginosa

Boston Children'S Hospital, Boston MA

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Abstract

Abstract Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients and a major cause of hospital-acquired pneumonia. We are proposing to study Hfq, a highly conserved RNA chaperone that is thought to function through its interactions with small regulatory RNAs (sRNAs) and mRNAs. We have obtained evidence that Hfq is essential in P. aeruginosa strain PAO1 and that cells containing a deletion of hfq carry suppressors that allow these cells to grow. Here we propose to determine what roles Hfq plays in cells of P. aeruginosa and determine the molecular basis for the essentiality of Hfq in this organism. In particular, in Aim 1 we propose to identify sRNAs and mRNA transcripts that Hfq associates with using RNA immunoprecipitation coupled with high-throughput DNA sequencing (RIP-Seq). In Aim 2 we propose to use a ClpXP protease-based depletion system coupled with RNA-Seq to determine what effect Hfq depletion has on transcript abundance on a genome-wide scale in P. aeruginosa. Our preliminary RIP-Seq studies indicate that Hfq associates with the transcripts of several key regulators of virulence gene expression in P. aeruginosa, and as part of the second aim we propose to determine whether this association might exert any regulatory effect at the level of either transcript or protein abundance. In Aim 3 we will identify those mutations that can suppress the effects of a hfq mutation. We will then attempt to determine what effect these mutations have on gene expression in both the presence and absence of Hfq. Our studies have the potential to reveal the basis for the essentiality of a key RNA chaperone in P. aeruginosa. Because Hfq appears to be critical for the growth of P. aeruginosa, the work we propose might aid in the development of novel therapeutics for the treatment of patients that suffer from P. aeruginosa infections.