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Global regulation of gene expression by OxyR in an important human opportunistic pathogen.

ABSTRACT: Most bacteria control oxidative stress through the H(2)O(2)-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that ?70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well.

SUBMITTER: Wei Q

PROVIDER: S-EPMC3378865 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other

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Global regulation of gene expression by OxyR in an important human opportunistic pathogen.

Wei Qing Q Minh Phu Nguyen Le PN Dötsch Andreas A Hildebrand Falk F Panmanee Warunya W Elfarash Ameer A Schulz Sebastian S Plaisance Stéphane S Charlier Daniel D Hassett Daniel D Häussler Susanne S Cornelis Pierre P

Nucleic acids research 20120124 10

Most bacteria control oxidative stress through the H(2)O(2)-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of ...[more]

PMID: 22275523

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Project description:BACKGROUND: Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. The bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation. RESULTS: We examined gene expression by intracellular B. cenocepacia using selective capture of transcribed sequences (SCOTS) combined with microarray analysis. We identified 767 genes with significantly different levels of expression by intracellular bacteria, of which 330 showed increased expression and 437 showed decreased expression. Affected genes represented all aspects of cellular life including information storage and processing, cellular processes and signaling, and metabolism. In general, intracellular gene expression demonstrated a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. Deletion of various SCOTS-identified genes affected bacterial entry into macrophages and intracellular replication. We also show that intracellular B. cenocepacia is cytotoxic towards the macrophage host, and capable of spread to neighboring cells, a role dependent on SCOTS-identified genes. In particular, genes involved in bacterial motility, cobalamin biosynthesis, the type VI secretion system, and membrane modification contributed greatly to macrophage entry and subsequent intracellular behavior of B. cenocepacia. CONCLUSIONS: B. cenocepacia enters macrophages, adapts to the phagosomal environment, replicates within a modified phagosome, and exhibits cytotoxicity towards the host cells. The analysis of the transcriptomic response of intracellular B. cenocepacia reveals that metabolic adaptation to a new niche plays a major role in the survival of B. cenocepacia in macrophages. This adaptive response does not require the expression of any specific virulence-associated factor, which is consistent with the opportunistic nature of this microorganism. Further investigation into the remaining SCOTS-identified genes will provide a more complete picture of the adaptive response of B. cenocepacia to the host cell environment.

Project description:UnlabelledPseudomonas aeruginosa uses N-acyl-homoserine lactone (AHL)-dependent quorum sensing (QS) systems to control the expression of secreted effectors. These effectors can be crucial to the ecological fitness of the bacterium, playing roles in nutrient acquisition, microbial competition, and virulence. In this study, we investigated the metabolic consequences of AHL-dependent QS by monitoring the metabolic profile(s) of a lasI rhlI double mutant (unable to make QS signaling molecules) and its wild-type progenitor as they progressed through the growth curve. Analysis of culture supernatants by (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy revealed that at the point where AHL concentrations peaked in the wild type, the metabolic footprints (i.e., extracellular metabolites) of the wild-type and lasI rhlI mutant diverged. Subsequent gas chromatography-mass spectrometry (GC-MS)-based analysis of the intracellular metabolome revealed QS-dependent perturbations in around one-third of all identified metabolites, including altered concentrations of tricarboxylic acid (TCA) cycle intermediates, amino acids, and fatty acids. Further targeted fatty acid methyl ester (FAME) GC-MS-based profiling of the cellular total fatty acid pools revealed that QS leads to changes associated with decreased membrane fluidity and higher chemical stability. However, not all of the changes we observed were necessarily a direct consequence of QS; liquid chromatography (LC)-MS analyses revealed that polyamine levels were elevated in the lasI rhlI mutant, perhaps a response to the absence of QS-dependent adaptations. Our data suggest that QS leads to a global readjustment in central metabolism and provide new insight into the metabolic changes associated with QS during stationary-phase adaptation.ImportanceQuorum sensing (QS) is a transcriptional regulatory mechanism that allows bacteria to coordinate their gene expression profile with the population cell density. The opportunistic human pathogen Pseudomonas aeruginosa uses QS to control the production of secreted virulence factors. In this study, we show that QS elicits a global "metabolic rewiring" in P. aeruginosa. This metabolic rerouting of fluxes is consistent with a variety of drivers, ranging from altered QS-dependent transcription of "metabolic genes" through to the effect(s) of global "metabolic readjustment" as a consequence of QS-dependent exoproduct synthesis, as well as a general stress response, among others. To our knowledge, this is the first study of its kind to assess the global impact of QS on the metabolome.

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Global regulation of gene expression by OxyR in an important human opportunistic pathogen.

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Global regulation of gene expression by OxyR in an important human opportunistic pathogen.

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