Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of Pseudomonas Aeruginosa grown under sublethal doses of UVA or in the dark up to early logarithmic phase

ABSTRACT: Ultraviolet A (UVA) radiation is the major fraction of UV radiation reaching the Earth’s surface; its harmful effects on microorganisms obey mainly to oxidative damage, with the consequent loss of bacterial viability. In this work, the global transcriptional response of Pseudomonas aeruginosa exposed to UVA was analyzed. P. aeruginosa is an opportunistic human pathogen, also present in terrestrial and aquatic environments; its high ubiquity and versatily obeys to a complex regulatory network wich allows it to adapt to stressful conditions. To conduct this study, the PAO1 strain was grown in under sublethal doses of UVA or in the dark up to early logarithmic phase, and total RNA was obtained and sequenced by the RNA-seq technique. The analysis of the results, taking as significant a factor change ≥ 2 between irradiated and control samples, indicated that a total of 298 genes were regulated by UVA, representing 5.36 % of the total P. aeruginosa genome; half of these were induced and the other half were repressed. Data obtained by the transcriptomic study were validated by using RT qPCR of selected genes. The results presented in this study suggest that one of the main UVA targets are proteins carrying [Fe-S] clusters since several genes involved in the processes of synthesis, trafficking and assembly of these structures were upregulated. The management of intracellular iron levels also seems to be a robust response to this stress factor. The strong induction of genes involved in denitrification led us to suggest that this pathway and/or reactive nitrogen species such as nitric oxide could have a role in the response to this radiation. DNA also demonstrated to be an important UVA target as observed by the induction of SOS, prohage and pyocins genes. On the other hand, the down-regulation of genes involved in the biosynthesis of PQS, a quorum sensing signal of P. aeruginosa with several functions, could be beneficial given its role as endogenous photosensitizer. The study of the effects of UVA radiation is interesting due to its ecological consequences in natural environments. In addition, taking into account the high sensitivity of P.aeruginosa to UVA radiation and the issues with using traditional antibacterial products by the intrinsec or acquired resistance of P. aeruginosa to these agents, specially in the case of biofilms, UVA studies could have important implications for human health, industrial facilities and environmental management.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Magdalena Pezzoni

PROVIDER: E-MTAB-13681 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed âantioxidant responseâ or âPhase II detoxificationâ. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental âimageâ of UV generated lipid mediators. Experiment Overall Design: we profiled global mRNA expression levels in human dermal fibroblasts that had been treated with either UVA-1 or oxidized lipids. To investigate the effect of oxidized phospholipids on gene regulation, we used two preparations, which differed in their degree of oxidation; the minimally oxidized UV-PAPC resulting from UVA-1 irradiation of PAPC, and air-oxidized PAPC (OxPAPC), which represents the full spectrum of oxidation products (Gruber 07) (Reis et al., 2005). We irradiated dermal fibroblasts with UVA-1 (40J/cmÂ²) or treated them with UV-PAPC, OxPAPC or native PAPC (100Âµg/ml each). We analyzed global gene expression four hours after stimulation with gene arrays (Affymetrix U133A Plus 2.0 Gene Chips).

Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed “antioxidant response” or “Phase II detoxification”. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental “image” of UV generated lipid mediators.

Project description:Ultraviolet (UV) wavebands in sunlight are immunomodulatory. About half the amount of UVA within a minimum erythemal dose of sunlight is systemically immunosuppressive, while higher doses protect from UVB immunosuppression in mice. We have previously shown that these responses to UVA are genetically restricted as they occur in C57BL/6 but not Balb/c mice. We used gene set enrichment analysis of microarray data and real-time RT-PCR confirmation to determine the molecular mechanisms associated with UVA immunomodulation. We found up-regulation of mRNA for the alternative complement pathway. The core-enriched genes complement component 3, properdin and complement factor B were all activated by the immunosuppressive dose of UVA only in UVA-responsive C57BL/6 but not unresponsive BALB/c mice. This therefore matched the genetic restriction and dose responsiveness of UVA immunosuppression. The immune-protective higher UVA dose prevented UVB from down regulating chemokine receptor 7 and IL-12B, and decreased IL-10, supporting previous identification of IL-12 and IL-10 in high dose UVA protection from UVB immunosuppression. Our study has identified activation of the alternative complement pathway as a trigger of UVA-induced systemic immunosuppression and suggests that this pathway is likely to be an important sensor of UVA-induced damage to the skin. 24 hours after UVA, UVB and ssUV irradiation, a 1 cm2 uniform section of skin was excised from the dorsal surface of irradiated and control mice. Total RNA was then extracted from the whole skin using TRIzol reagent (Gibco Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturerâ??s instructions, purified, DNase treated and reverse transcribed into cDNA. For the microarray study a direct incorporation of Cyanine 3-dCTP and Cyanine 5-dCTP fluorescent dyes (Perkin Elmer Life Sciences, Inc. Boston, MA, USA) was used for cDNA synthesis. For each UV dose, a reference design was used to compare an unirradiated control against an irradiated sample. Microarray experiments used compugen 22k mouse oligonucleotide microarray slides (The Clive and Vera Ramaciotti Centre for Gene Function Analysis, Sydney Australia (http://www.ramaciotti.unsw.edu.au). Lower and higher UVA doses were used. C57BL/6 mice were irradiated with lower UVA, higher UVA, UVB, or ssUV; Balb/C mice were irradiated with lower or higher UVA. Experiments were replicated 6 times for each UV dose. A fluorescent dye swap was done for each alternate hybridisation to reduce systematic dye bias of incorporated fluorescent dyes.

Dataset Information

RNA-seq of Pseudomonas Aeruginosa grown under sublethal doses of UVA or in the dark up to early logarithmic phase

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