Evaluation of the infection‐relevant role of small RNA‐based regulatory systems in the opportunistic pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic pathogen in immune-compromised and cystic fibrosis patients. Emerging candidates for the role of molecular regulators of virulence in P. aeruginosa are small RNAs (sRNAs), which in other bacterial pathogens have been shown to play key roles in modulating cellular processes linked to pathogenesis. We have performed a comparative sRNA profiling via deep-sequencing of P. aeruginosa PAO1 and PA14 strains which share the same host range but differ in pathogenicity, PA14 being considerably more virulent in several model organisms. Altogether, we have identified more than 150 novel candidate sRNAs and validated a third of them by Northern blotting (1). We now aim at identifying infection-relevant small RNA-based regulatory systems. To this end, we are focusing on a short list of 8 validated sRNAs which showed, at preliminary screenings, potential virulence hallmarks such as: specific expression in PA14 or PAO1; responsiveness to infection-relevant host stimuli (e.g. oxygen availability, temperature shift) or invasion signals (e.g. quorum sensing). Our approach is to generate sRNA deletion mutants and test them for i) canonical virulence phenotype(s) such as motility, protease and siderophore secretion, mucoidy, biofilm formation, haemolytic/autolytic activity, rhamnolipids production; ii) the ability to invade human respiratory epithelial cells and stimulate an immune response. Virulence/attenuation of those sRNA mutants showing altered virulence traits in vitro will be tested in both acute and chronic murine models of airways infection by P. aeruginosa. The above tests are accompanied by in silico, genetic and transcriptomics-based screenings for the genome-wide search of sRNAs target genes. 1. Ferrara S, Brugnoli M, De Bonis A, Righetti F, Delvillani F, Deho G, Horner D, Briani F, Bertoni G (2012) Comparative profiling of Pseudomonas aeruginosa strains reveals differential expression of novel unique and conserved small RNAs. PLoS One 7: e36553.