Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Dictyostelium discoideum in response to infection with Pseudomonas aeruginosa PA01 and PA14 strains which differ in virulence

ABSTRACT: Pseudomonas aeruginosa is an opportunistic human pathogen, infecting immuno-compromised patients and causing persistent respiratory infections in people affected from cystic fibrosis. Pseudomonas strain Pseudomonas aeruginosa PA14 shows higher virulence than Pseudomonas aeruginosa PAO1 in a wide range of hosts including insects, nematodes and plants but the precise cause of this difference is not fully understood. Little is known about the host response upon infection with Pseudomonas and whether or not transcription is being affected as a host defense mechanism or altered in the benefit of the pathogen. In this context the social amoeba Dictyostelium discoideum has been described as a suitable host to study virulence of Pseudomonas and other opportunistic pathogens.

ORGANISM(S): Dictyostelium discoideum

SUBMITTER: Gareth Bloomfield

PROVIDER: E-TABM-464 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Among multiple interconnected pathways for L-Lysine (L-Lys) catabolism in pseudomonads, Pseudomonas aeruginosa PAO1 employed the decarboxylase and the transaminase pathways. However, up till now several genes involved in the operation and regulation of these pathways were still missing. Transcriptome analyses coupled with promoter activity measurements and mutant growth phenotype analysis lead us to identify several new members of the L-Lys and D-Lys catabolic pathways and their regulatory elements, including argR to trigger lysine decarboxylation into cadaverine, PauR for the Î³-glutamylation pathway of polyamine catabolism into 5-aminovalerate, gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for D-Lys catabolism, lysR-lysXE for L-Lys efflux, and lysP for L-Lys uptake. Gene expression microarray, including probe preparation, hybridization, fluidics run and chip scan, was performed by Georgia State University DNA/Protein Core Facility. P. aeruginosa PAO1 was grown aerobically in minimal medium P with 350 rpm shaking at 37C, in the presence of 10mM L-glutamate supplemented with 10 mM L-lysine, cadaverine, 5-amino valerate, glutaric acid, D-lysine or 5mM L-pipecolate. Cells were harvested when the optical density at 600 nm reached 0.5~0.6 by centrifugation for 5 minutes at 4C. Total RNA samples were isolated by RNeasy purification kit following instructions of the manufacturer (Qiagen). Reverse transcription for cDNA synthesis, fragmentation by DNase I treatment, cDNA probe labeling and hybridization were performed according to the instructions of GeneChip manufacturer (Affymetrix). Data were processed by Microarray Suite 5.0 software normalizing the absolute expression signal values of all chips to a target intensity of 500. GeneSpring software (Silicon Genetics) was used for expression pattern analysis and comparison. Data collection was carried out using GCOS 1.4 software (Affymetrix). Probe intensity values were normalized to a target value of 500 with normalization factor equal to 1. Data analysis was performed using GeneSpring GX 11 Software (Aglient, Palo Alto,CA). Related Research Papers: 1. Indurthi SM, Chou HT, Lu CD. Molecular Characterization of lysR-lysXE, gcdR-gcdHG, and amaR-amaAB Operons for Lysine Export and Catabolism: A Comprehensive Lysine Catabolic Network in Pseudomonas aeruginosa PAO1. Microbiology. 2015 Submitted [EMID:04803de65c782] 2. Chou HT, Li J, and Lu CD. Functional Characterization of the agtABCD and agtSR Operons for Î³-Aminobutyrate and Î´-Aminovalerate Uptake and Regulation in Pseudomonas aeruginosa PAO1. Curr. Microbiology. 2014 Jan;68(1):59-63. 3. Chou HT, Li JY, Peng YC, Lu CD. Molecular characterization of PauR and its role in control of putrescine and cadaverine catabolism through the Î³-glutamylation pathway in Pseudomonas aeruginosa PAO1. J Bacteriol. 2013 Sep; 195(17):3906-13. 4. Chou HT, Hegazy M, Lu CD. L-lysine Catabolism is Controlled by Arginine/ArgR in Pseudomonas aeruginosa PAO1. J Bacteriol. 2010 Nov;192(22):5874-80

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Transcription profiling of Dictyostelium discoideum in response to infection with Pseudomonas aeruginosa PA01 and PA14 strains which differ in virulence

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