Project description:Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyper swarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. Swarming inhibition did not mediate rhamnolipid production, which regulates swarming motility in P. aeruginosa. Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and pyochelin, flagellar, and pili synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa.
Project description:Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyper swarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. Swarming inhibition did not mediate rhamnolipid production, which regulates swarming motility in P. aeruginosa. Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and pyochelin, flagellar, and pili synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa. In total 4 samples: gene expressions of P. aeruginosa with (2 samples) or without (2 samples) 1-naphthol
Project description:Abstract: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen found ubiquitously in the environment. Responsible for considerable human morbidity and mortality, particularly in nosocomial infections and individuals with cystic fibrosis, P. aeruginosa can adapt to surface growth by undergoing swarming motility. In P. aeruginosa, swarming motility is a rapid multicellular movement that occurs on soft surfaces of appropriate viscosity with amino acids as a nitrogen source. Here we tested the small synthetic host defense peptide, innate defense regulator 1018, and found that it inhibited swarming motility at concentrations as low as 0.75 μg/ml, well below the MIC for planktonic cells. A screen of the PA14 transposon insertion mutant library revealed twenty-nine mutants that demonstrated partial tolerance to 1018 under swarming conditions. Two of these mutants, in the genes anr and rhlB (a regulator of anaerobic metabolism and protein involved in rhamnolipid production, respectively), were complemented to restore susceptibility to 1018. RNA-Seq of peptide-treated cells under swarming conditions revealed the dysregulation of 1,190 genes compared to the untreated swarm front, and 67% of these genes were similarly dysregulated at the untreated swarm centre. In contrast, expression of 70 Anr-regulated genes was upregulated by peptide treatment, and 45 genes showed differential or opposite regulation of expression in peptide-treated and swarm centre cells. Many transcriptional regulators required for swarming were dysregulated in peptide-treated cells, indicative of a mechanism by which 1018 may inhibit swarming motility. Overall, this study illustrates a use for peptide 1018 in inhibiting swarming surface motility, an important bacterial adaptation.
Project description: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.
Project description:Surfing motility is a complex adaptation that is different from swarming motility and requires the stringent stress response in Pseudomonas aeruginosa LESB58 Cystic fibrosis (CF) is a genetic disease that affects mucin-producing body organs such as the lungs. Characteristic of CF is the production of thick and sticky mucus that can lead to progressive airway obstruction. The glycoprotein mucin is the major macromolecular component of mucus. Recently, we identified that the presence of mucin induced a rapid surface adaptation termed surfing motility in motile bacteria. P. aeruginosa, the main colonizing pathogen in CF employs several stress coping mechanisms to survive the highly viscous environment of the CF lung. Here, RNA-Seq was used to study the stringent stress response in the hypervirulent CF isolate LESB58 (Liverpool Epidemic Strain) via transcriptional profiling. As the stringent response is regulated by relA and spoT, we created a double knockout of these genes in LESB58 to study the impact of these stress regulators on surfing motility using RNA-Seq.
Project description:In this experiment the transcriptional profile of the Pseudomonas aeruginosa PA14 two-component sensor kinase PA4398 was investigated under swarming conditions using DNA microarrays. To this aim three independent cultures of the PA14 wild-type and the PA4398 mutant were grown until mid-log phase in Luria-Bertani broth following an incubation on BM2-swarm plates containing 0.1 % (wt/vol) casaminoacids and 0.5 % (wt/vol) agar for 20 h at 37 °C. Subsequent total RNA was extracted from the leading edge of dendritic swarm colonies and analyzed by microarrays.
Project description:The ParS/ParR two component regulatory system plays important roles for multidrug resistance in Pseudomonas aeruginosa. In this study we report RNA-seq analyses of the transcriptomes of P. aeruginosa PAO1 wild type and par mutants growing in a minimal medium containing 2% casamino acids. This has allowed the quantification of PAO1 transcriptome, and further defines the regulon that is dependent on the ParS/ParR system for expression. Our RNA-seq analysis produced the first estimates of absolute transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished the expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to affecting drug resistance genes, transcripts of quorum sensing genes (rhlIR and pqsABCDE-phnAB), were significantly up-regulated in both parS and parR mutants. Consistent with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of par genes also lead to overproduction of phenazines and increased swarming motility, consistent with the up-regulation of quorum sensing genes. Our results established a link among ParS/ParR, MexEF-OprN and quorum sensing in Pseudomonas aeruginosa. Based on these results, we propose a model to illustrate the relationship among these regulatory systems in P. aeruginosa. A total of 9 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas aeruginosa PAO1 wild type strain (3 replicates); Pseudomonas aeruginosa parS mutant (3 replicates); Pseudomonas aeruginosa parR mutant (3 replicates).