Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome wide transcriptional analysis of P. aeruginosa PAO1 response to pH at 25 mM phosphate background

ABSTRACT: During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal P. aeruginosa to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation. We used a microarray to define the virulence-related genes in P. aeruginosa grown as lawns on NGM at pH7.5 vs pH6.0 All samples for gene expression analysis were prepared in triplicate. P. aeruginosa MPAO1 cells collected from lawns grown on NGM/[Pi]25 mM, pH 6.0 or NGM/[Pi]25, pH 7.5 were used for RNA isolation as previously described. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility

ORGANISM(S): Pseudomonas aeruginosa PAO1

SUBMITTER: Olga ZABORINA

PROVIDER: E-GEOD-29789 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosa can be achieved without iron provision by maintaining local phosphate abundance: role of pH.

Romanowski Kathleen K Zaborin Alexander A Fernandez Hoylan H Poroyko Valeriy V Valuckaite Vesta V Gerdes Svetlana S Liu Donald C DC Zaborina Olga Y OY Alverdy John C JC

BMC microbiology 20110926

<h4>Background</h4>During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse ...[more]

PMID: 21943078

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Project description:Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response resulting in a colony morphology and phenotype referred to as mucoid. However how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar (PIA) supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, transcriptomics, and in a murine acute virulence model. The PA14 non-redundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan, uptake of phosphate and iron, phenazines biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa growing in the presence of vanadate caused differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study. Strain PAO1 was cultured on both PIA and PIAAMV for 18 hr. Three separate plates were used for each media. The cells from each plate were scraped into 4 ml of RNA Protect (Qiagen) and stored immediately at -80°C. Cells were treated with RNAprotect (Qiagen) and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. RNA isolation, cDNA preparation, labeling and microarray analysis were performed as previously described in reference: Damron, F. H., J. P. Owings, Y. Okkotsu, J. J. Varga, J. R. Schurr, J. B. Goldberg, M. J. Schurr, and H. D. Yu. 2012. Analysis of the Pseudomonas aeruginosa Regulon Controlled by the Sensor Kinase KinB and Sigma Factor RpoN. J Bacteriol 194:1317-30.

Dataset Information

Genome wide transcriptional analysis of P. aeruginosa PAO1 response to pH at 25 mM phosphate background

Publications

Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosa can be achieved without iron provision by maintaining local phosphate abundance: role of pH.

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