Targeting the alternative sigma factor RpoN to combat virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative sigma factor 54 (σ⁵⁴, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro, we explored its effects in a Caenorhabditis elegans-P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.