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.

Figure 1

Schematic of σ⁵⁴ (RpoN) and the molecular roadblock, RpoN*. (a) σ⁵⁴ (RpoN) is composed of three regions. Region III is highly conserved, and is necessary for recognizing and binding −24/−12 promoter elements. (b) Amino acid sequence of RpoN*. Peptide sequence includes AA376-400 of RpoN Region III from A. aeolicus and includes amino acids (▴) that specifically bind to the −24 promoter DNA. Point mutation (Y48A, asterisk) attenuates binding and transcriptional activity of RpoN*. (c) Schematic of the interaction between RpoN* and the −24 element of the σ⁵⁴ consensus promoter.

Figure 2

Summary of genes affected by expression of RpoN* in P. aeruginosa PAO1. Expression of RpoN* interferes with the ability of RNA polymerase and native RpoN to bind to sig54 promoters altering the transcriptional profile of P. aeruginosa PAO1. Summaries of areas of gene expression that are specifically altered by RpoN* are shown and include the genes encoding the transcriptional regulators RhlR, Anr, GbdR, and their respective regulons. These results are consistent with RpoN binding sites identified by ChIP-seq.. Expression of RpoN* was shown to alter expression of a number of other genes including hypothetical (~250) and ribosomal (~70) genes. A full list of these genes is available in Supplementary Table S2.

Figure 3

Model of predicted RpoN* mechanism of action. (a) RpoN*, a cis-acting peptide, binds -24 promoter sites, blocking gene transcription by RpoN. (b) Additional sigma factors can compensate for absence of native RpoN, resulting in transcription of associated genes. In the absence of native RpoN, the RpoN* blocks transcription by other sigma factors with a promoter binding site at the same gene. (c) RpoN* blocks transcription by obstructing RpoN:RNAP binding, as well as halting transcription of other sigma factors binding at the same gene.

Figure 4

RpoN* expression reduced virulence phenotypes. (a–d) Photograph (top) plus bioluminescent overlay (bottom) for phenotype assays: (a,a’) twitching, or pili, motility conducted on semi-hard agar; (b,b’) swimming, or flagellar, motility assays conducted on soft agar; (c,c’) protease assays conducted on milk agar; (d,d’) elastase assays conducted on LB agar with elastin. Strains used: P. aeruginosa PA19660 (a-f) and PAO1 (g-i) wild-type (empty vector), RpoN*, Y48A* point mutant, and P. aeruginosa PAO1 ΔlasR. All assays were conducted at 37 °C for 24–48 h, with 30 mg/L gentamicin and 2 mM IPTG. Colony diameter of P. aeruginosa strains in twitching (e) and swimming (f) motility assays, with (+) and without (−) 2 mM IPTG. Pyocyanin and pyoverdine (g) production assays conducted in LB or King’s B broth, respectively, with 30 mg/L gentamicin and 1 mM IPTG. Elastase (h) production assay in peptone-tryptic soy broth with 1 mM IPTG. Growth kinetics (i) with RpoN* expression induced with 1 mM IPTG at 0.5 OD₆₀₀. Statistics used were Student’s t-test (***p ≤ 0.0001; **p ≤ 0.01; *p ≤ 0.05). Bars indicate mean colony diameter of replicates; error bars represent one standard deviation of the mean. n = 4 to 7 replicates per assay.

Figure 5

RpoN* decreases biofilm formation in vitro. (a–d) Air-Liquid Interface (ALI) biofilm formation assay conducted in well bottoms of 6-well microtiter plates for 24 h and visualized by phase contrast microscopy at 200x. Strains used: P. aeruginosa PA14 wild type (a), empty vector (b), RpoN* (c), and Y48A* point mutant (d). Images shown are representative fields of view from two independent experiments with three technical replicates for each strain. (e) Biofilms formed in 96-well microtiter plates were stained with crystal violet, solubilized, and quantified at OD₅₅₀ (n = 28). Data presented as mean ± SEM. Student’s t test performed (****p ≤ 0.0001).

Figure 6

RpoN* increases C. elegans survival in C. elegans – P. aeruginosa infection assays. (a) Brain-heart infusion media with bacto-agar was supplemented with 30 mg/L gentamicin and at least 1 mM IPTG. All paralytic killing assays were conducted at room temperature (22–24 °C), and scored every 2 h. Strains used: E. coli (▪, n = 195), P. aeruginosa ΔlasR (⚫, n = 197), wild-type P. aeruginosa PAO1 (Manoil strain) (empty vector, Δ, n = 205), RpoN* (▴, n = 301), and Y48A* point mutant (▾, n = 376). (b) Slow killing agar (0.35% bactopeptone, 2% bactoagar) was supplemented with 30 mg/L gentamicin and at least 1 mM IPTG, when necessary. Slow killing assays were conducted at 20 °C and scored every 24 h. Strains used: E. coli (▪, n = 90), P. aeruginosa ΔlasR (⚫, n = 90), wild-type P. aeruginosa PA19660 Xen5 (empty vector, Δ, n = 171), RpoN* (▴, n = 160). (c) Appearance of worms fed on P. aeruginosa wild type (upper panels) or RpoN* (lower panels). All images taken on an Olympus stereoscope (mag. 4.5x). Scale bars are 100 μm. Points on the Kaplan-Meier survival curves represent the combined survival of three or more separate assays. Mantel-Cox log-rank tests performed to analyze curves (***p ≤ 0.0001).