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The small RNAs PA2952.1 and PrrH as regulators of virulence, motility and iron metabolism in Pseudomonas aeruginosa

ABSTRACT: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome encoding hundreds of potential intergenic small RNAs (sRNAs), P. aeruginosa can easily adapt to different conditions and stresses. Overexpression of the previously uncharacterized PA2952.1 resulted in decreased swarming and swimming motility, increased gentamicin and tobramycin resistance under swarming conditions, and increased trimethoprim susceptibility. RNA-Seq and proteomics were performed on the wildtype overexpressing PA2952.1 cf. the empty vector control 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 (TMT4..TMT6) to biological replicate samples from the PA2952.1 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.

INSTRUMENT(S): Orbitrap Q Exactive HF-X

ORGANISM(S): Pseudomonas Aeruginosa

SUBMITTER: Robert E.W. Hancock

PROVIDER: MSV000085955 | MassIVE | Sat Aug 15 18:25:00 BST 2020

REPOSITORIES: MassIVE

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Influence of sRNA PA0805.1 on motility and virulence

Project description: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).

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