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

ABSTRACT: Purpose: 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. We previously identified 20 sRNAs dysregulated under swarming conditions and here provide phenotypic characterization for these sRNAs. Methods: Here, these sRNA were overexpressed in strain PAO1 and subjected to an array of phenotypic screens. Results: Overexpression of prrH resulted in decreased swimming motility, while a ∆prrH mutant had decreased cytotoxicity and increased pyoverdine production. 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, and revealed the differential expression of 784 genes and differential abundance of 445 proteins. Amongst these were found 82 transcriptional regulators, two-component systems, sigma and anti-sigma factors. Downstream effectors included downregulated pili, dysregulated flagellar genes, the upregulated efflux pump MexGHI-OpmD, the upregulated arn operon, and downregulated DNA synthesis genes. Genes involved in iron and zinc uptake were also dysregulated, and certain pyoverdine and phenazine genes were upregulated. Conclusions: Overall, the sRNAs PA2952.1 and prrH appeared to be involved in regulating virulence-related programs in P. aeruginosa, including iron acquisition and motility.

ORGANISM(S): Pseudomonas aeruginosa PAO1

PROVIDER: GSE146765 | GEO | 2020/11/04

REPOSITORIES: GEO

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

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