Global post-transcriptional regulators in P. aeruginosa

Boston Children'S Hospital, Boston MA

Investigators

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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 persons with cystic fibrosis (pwCF), is a major cause of hospital-acquired pneumonia and is particulary problematic in burn wound infections. Hfq is a conserved global post-transcriptional regulator that is required for the virulence of P. aeruginosa. In other organisms Hfq is best known for its ability to promote the base-pairing between small regulatory RNAs (sRNAs) and their target transcripts, with Hfq-promoted interaction between an sRNA and its mRNA target typically functioning to repress target translation. Although approximately 150 different sRNAs are thought to exist in P. aeruginosa, for most of these we understood little about their possible regulatory roles because we did not know which transcripts they targeted. We have recently identified the targets for approximately 90 Hfq-bound sRNAs in P. aeruginosa using an approach called RNA interaction through ligation and sequencing (RIL-seq). These studies revealed that a single sRNA called PhrS largely dominates the RNA-RNA interaction landscape in P. aeruginosa by pairing with close to 800 targets, which is an unusually large number of targets for an sRNA. In Aim 1 we propose to use Ribo-seq to determine which of the many targets of PhrS are controlled at the level of translation by this sRNA. We will also determine whether the extensive target repertoire we observe for PhrS in one of the main laboratory strains of P. aeruginosa is conserved in clinical isolates, where little is known about the regulatory roles of sRNAs. In Aim 2 we propose to determine whether an sRNA called CrcZ, which acts as a molecular decoy for Hfq, functions as a global regulator of sRNA-dependent control in P. aeruginosa. We will also determine whether putative base-pairing interactions we have uncovered between CrcZ and specific sRNAs make them particularly sensitive to the regulatory effects of CrcZ. Our study therefore has the potential to illuminate an additional mechanism through which CrcZ acts. Finally, we have uncovered an sRNA that controls virulence gene expression in P. aeruginosa and influences the ability of the organism to compete with another bacterial species. In Aim 3 we propose to determine how this sRNA exerts its myriad regulatory effects. Our proposed studies might help explain why mutations that abolish production of this sRNA occur in pwCF during the course of an infection. The experiments described in this proposal are expected to reveal how three conserved Hfq-bound sRNAs exert key regulatory effects in P. aeruginosa, and are relevant for understanding post-transciptional control exerted by sRNAs in clinical isolates of this bacterium.