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

Abstract

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.

Blocking the alternative sigma factor RpoN reduces virulence ofPseudomonas aeruginosaisolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain

“…SCH0057-7, SCH0256-1, SCH0338-58, and SCH0354-1 were transformed with a plasmid expressing RpoN* or the empty vector and selected with gentamicin. If RpoN* affected transcription of virulence-related genes in different genetic backgrounds as previously reported (38), we expected attenuation of virulence-related phenotypes in P. aeruginosa CF patient isolates. RpoN* significantly reduced colony diameter in all four patient isolates in the swimming motility assay (Student’s t-test, ** p≤0.01, ***p≤0.0001) (Fig 4A).…”

“…Antibiotic resistance is a problem in CF patients with P. aeruginosa infections (47–49). We previously reported that RpoN* alters transcription of several genes involved in multidrug efflux pumps that confer natural resistance (38). Additionally, RpoN is implicated in tolerance to various classes of antibiotics (34–36).…”

“…The specific and conserved nature through which RpoN controls its regulon led us to develop the RpoN molecular roadblock, RpoN*. RpoN* is a cis-acting peptide that specifically binds the −24 site of RpoN consensus promoters, blocking transcription by RpoN and other factors (38). When RpoN* is expressed in P. aeruginosa laboratory strains, transcription is affected globally and virulence is attenuated (38).…”

“…RpoN* is a cis-acting peptide that specifically binds the −24 site of RpoN consensus promoters, blocking transcription by RpoN and other factors (38). When RpoN* is expressed in P. aeruginosa laboratory strains, transcription is affected globally and virulence is attenuated (38). RpoN* also affects virulence in an RpoN deletion strain of P. aeruginosa PAO1, demonstrating its ability to attenuate gene expression by repressing expression of genes located downstream of RpoN promoters (38).…”

“…When RpoN* is expressed in P. aeruginosa laboratory strains, transcription is affected globally and virulence is attenuated (38). RpoN* also affects virulence in an RpoN deletion strain of P. aeruginosa PAO1, demonstrating its ability to attenuate gene expression by repressing expression of genes located downstream of RpoN promoters (38). This strategy of blocking multiple promoters throughout the P. aeruginosa genome may be an effective method to combat virulence and evade development of resistance.…”