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Metabolic analysis of the response of Pseudomonas putida DOT-T1E strains to toluene using Fourier transform infrared spectroscopy and gas chromatography mass spectrometry

ABSTRACT: To elucidate any observable metabolic alterations during interactions of several strains of Pseudomonas putida (DOT-T1E, and its mutants DOT-T1E-PS28 and DOT-T1E-18) with the aromatic hydrocarbon toluene, metabolomic approaches were employed. Initially, Fourier-transform infrared (FT-IR) spectroscopy, which provided a rapid, high-throughput metabolic fingerprint of P. putida strains, was used to investigate any phenotypic changes resulting from exposure to toluene. Principal component discriminant function analysis (PC-DFA) allowed the differentiation between different conditions of toluene on bacterial cells, which indicated phenotypic changes associated with the presence of the solvent within the cell. Examination of PC-DFA loading plots suggested that the protein and fatty acids groups were responsible for discrimination of responses by P. putida strains to toluene. To identify metabolites of interest, the polar extracts of P. putida cells were analysed using gas chromatography-mass spectrometry (GC-MS) and 15 metabolites of P. putida central metabolic pathways were detected. Multi-block principal component analysis (MB-PCA) indicated that P. putida cultures challenged with toluene were differentially clustered away from the non-challenged cells. Investigation of MB-PCA loading plots and N-way ANOVA for condition | strain×time blocking (dosage of toluene) suggested ornithine as the most significant compound that increased upon solvent exposure. Ornithine presents itself as a major feature which may have important functions in toluene stress tolerance mechanisms.

INSTRUMENT(S): Exactive (Thermo Scientific)

SUBMITTER: Drupad Trivedi

PROVIDER: MTBLS319 | MetaboLights | 2016-08-23

REPOSITORIES: MetaboLights

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Project description:Background: Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we provide initial evidence for potential roles of AKR1C3 in PCa progression. Methods: Spatial distribution of AKR1C3 was analyzed using immunohistochemical staining in prostate adenocarcinoma tissue array. Human PCa PC-3 cells were stably transfected with AKR1C3 cDNA to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analyses were performed to identify pathways that are activated by elevated AKR1C3 expression in PCa cells. Functional confirmation of microarray and bioinformatics results was performed by immunoblot analysis and an in vitro Matrigel angiogenesis assay. Results: Elevated AKR1C3 expression was specifically limited to human prostate adenocarcinoma. Microarray and bioinformatics analysis suggested that elevated AKR1C3 expression in PC-3 cells modulates estradiol and androgen metabolism and activates insulin growth factor (IGF)-1 and Akt signaling pathways. Immunoblots confirmed that phosphorylated levels of IGF-1 receptor (IGF-1R) and Akt are significantly up-regulated in PC3-AKR1C3 as compared to mock transfectants. PC3-AKR1C3 transfectants promoted endothelial cell tube formation in Matrigel as compared to parental PC-3 cells and mock transfectants. Conclusion: Microarray and bioinformatics data followed by biological analyses suggest that elevated AKR1C3 expression in PC-3 cells promotes PCa angiogenesis and aggressiveness. These results suggest AKR1C3 can promote the aggressiveness of PCa through modulating estrogen and androgen metabolism with subsequent activation of growth factor IGF-1 and cytoplasmic Akt signaling pathways. Total RNA from mock- and ACR1C3 transfected PC-3 cells was isolated, with 2 or 3 biological replicates each. Gene expression data from AKR1C3 transfected PC-3 cells were compared with mock-transfected data.

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Metabolic analysis of the response of Pseudomonas putida DOT-T1E strains to toluene using Fourier transform infrared spectroscopy and gas chromatography mass spectrometry

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