The Indispensible and Global Role of NAD Metabolism for the Lifestyle and Virulence of Pseudomonas aeruginosa
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ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa TBCF10839
SUBMITTER: Sarah Frank
PROVIDER: E-GEOD-39867 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Phagocytosis and killing of the opportunistic pathogen Pseudomonas aeruginosa by polymorphonuclear neutrophils (PMNs) is the major antipseudomonal host defense of vertebrates. By screening a transposon library of the clinical P. aeruginosa isolate TBCF10839 that can grow and replicate in PMNs, a mutant was identified that was most strongly sensitized to killing by PMNs. The inactivated gene PA1572 termed nadK1 was found to encode an ATP:NAD kinase. The transcriptomes of the TBCF10839 wild type and nadK1 mutant were investigated in the presence of PMNs or H2O2. Exposure to H2O2 led to diametrical mRNA expression profiles. H2O2-degrading enzymes were upregulated by wild type, but not by nadK1 mutant. This data demonstrates that NadK1 is crucial for the response of P. aeruginosa to reactive oxygen species. The transcriptomes of Pseudomonas aeruginosa TBCF10839 wild type and nadK1 mutant were comparatively investigated in the presence of polymorphonuclear neutrophils or H2O2 in order to investigate the role of NadK1 in oxidative stress response. These samples represent test samples. The expression profile of Pseudomonas aeruginosa strain TBCF10839 wild type cells, isolated from a Cystic Fibrosis patient, was investigated under standard growth conditions in batch culture in Luria broth (LB) with no treatment. These samples represent reference samples. All samples were analyzed in duplicate.
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Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators. We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments. Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 µM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed. First comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions. Second comparison: Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions. Third comparison: Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.
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