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Influence of sRNA PA0805.1 on motility and virulence

ABSTRACT: Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues. The small non-coding RNA sRNA PA0805.1 was shown to be 5-fold upregulated under swarming conditions. This sRNA was cloned, transformed into PAO1 WT and overexpressed, leading to broad phenotypic changes including reduced swarming, swimming and twitching motility, as well as increased adherence, cytotoxicity, and tobramycin resistance. The strain overexpressing PA0805.1 was compared against an empty vector (EV) strain under swarming conditions using TMT10 labeled differential protein expression analysis. The TMT10 reporter channels were sequentially assigned in increasing reporter mass as TMT0..TMT9. We assigned four TMT10 channels (TMT0..TMT3) to biological replicate samples from the EV strain, and three TMT10 channels (TMT7..TMT9) to biological replicate samples from the PA0805.1 strain. Data was acquired over a single 180-minute HPLC-MS run, with peptides identified using X!tandem (cyclone 2012.10.01.1). Protein level expression values were computed using the sum of the reporter channel values from their member peptides, with protein assignment handled by X!tandem. Expression values were transformed into a log2 scale for differential analysis. The seven channel-specific expression vectors were each normalized to put them into a common scale. Differential analysis between the two strains yielded 925 proteins at p<0.05 (Welch t-test, uncorrected).

INSTRUMENT(S): Orbitrap Q Exactive HF-X

ORGANISM(S): Pseudomonas Aeruginosa Pao1 (ncbitaxon:208964)

SUBMITTER: Robert E.W. Hancock

PROVIDER: MSV000084373 | MassIVE |

REPOSITORIES: MassIVE

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Project description:Purpose: Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues, i.e., a semisolid surface with amino acids as a nitrogen source (relevant to the human lung). Swarming is an intricately regulated process, but to date post-transcriptional regulation has not been extensively investigated. Small non-coding RNAs (sRNA) are hypothesized to play post-transcriptional regulatory roles, largely through suppression of translation, and we previously demonstrated 20 sRNA species that were dysregulated under swarming conditions. Overexpressing these revealed one sRNA, PA0805.1, that when cloned, transformed into PAO1 WT and overexpressed led to broad phenotypic changes. Methods: The sRNA PA0805.1 was cloned and overexpressed in PAO1 WT. Phenotypic screens including motility, cytotoxicity and adherence assays were performed. RNA-Seq and proteomics were performed under swarming conditions compared to the empty vector strain to discover which genes were influenced by the overexpression of PA0805.1 Results: Phenotypic screens revealed that PA0805.1 resulted in reduced swarming, swimming and twitching motility, as well as increased adherence, cytotoxicity, and tobramycin resistance. A deletion mutant ∆PA0805.1 was supersusceptible to tobramycin under swarming conditions. The strain overexpressing PA0805.1 was compared to the empty vector strain by RNA-Seq and proteomics under swarming conditions to determine sRNA targets. Broad transcriptional and proteomic profiles were revealed, encompassing 1121 differentially expressed genes and 258 proteins with significantly different abundance. Importantly, these included 106 transcriptional regulators, two-component regulatory systems, sigma and anti-sigma factors. Downstream of these regulators were found downregulated type IV pilus genes, many upregulated adherence and virulence factors, and two multidrug efflux systems, mexXY and mexGHI-opmD. Conclusions: The sRNA PA0805.1 appears to regulate diverse bacterial lifestyles, most likely through a regulatory cascade.

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Influence of sRNA PA0805.1 on motility and virulence

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