Decreased coenzyme A levels in ridA mutant strains of Salmonella enterica result from inactivated serine hydroxymethyltransferase.
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ABSTRACT:
SUBMITTER: Flynn JM
PROVIDER: S-EPMC4041533 | biostudies-literature | 2013 Aug
REPOSITORIES: biostudies-literature
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Decreased coenzyme A levels in ridA mutant strains of Salmonella enterica result from inactivated serine hydroxymethyltransferase.
Flynn Jeffrey M JM Christopherson Melissa R MR Downs Diana M DM
Molecular microbiology 20130719 4
The RidA/Yer057/UK114 family of proteins is well represented across the domains of life and recent work has defined both an in vitro activity and an in vivo role for RidA. RidA proteins have enamine deaminase activity, and in their absence the reactive 2-aminoacrylate (2-AA) accumulates and inactivates at least some pyridoxal 5'-phosphate (PLP)-containing enzymes in Salmonella enterica. The conservation of RidA suggested that 2-AA was a ubiquitous cellular stressor that was generated in central ...[more]
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Project description:The reactive intermediate deaminase RidA (EC 3.5.99.10) is conserved across all domains of life and deaminates reactive enamine species. When Salmonella enterica ridA mutants are grown in minimal medium, 2-aminoacrylate (2AA) accumulates, damages several pyridoxal 5'-phosphate (PLP)-dependent enzymes, and elicits an observable growth defect. Genetic studies suggested that damage to serine hydroxymethyltransferase (GlyA), and the resultant depletion of 5,10-methelenetetrahydrofolate (5,10-mTHF), was responsible for the observed growth defect. However, the downstream metabolic consequence from GlyA damage by 2AA remains relatively unexplored. This study sought to use untargeted proton nuclear magnetic resonance (1H NMR) metabolomics to determine whether the metabolic state of an S. enterica ridA mutant was accurately reflected by characterizing growth phenotypes. The data supported the conclusion that metabolic changes in a ridA mutant were due to the IlvA-dependent generation of 2AA, and that the majority of these changes were a consequence of damage to GlyA. While many of the metabolic differences for a ridA mutant could be explained, changes in some metabolites were not easily modeled, suggesting that additional levels of metabolic complexity remain to be unraveled.IMPORTANCE The accumulation of the reactive enamine intermediate 2-aminoacrylate (2AA) elicits global metabolic stress in many prokaryotes and eukaryotes by simultaneously damaging multiple pyridoxal 5'-phosphate (PLP)-dependent enzymes. This work employed 1H NMR to expand our understanding of the consequence(s) of 2AA stress on metabolite pools and effectively identify the metabolic changes stemming from one damaged target: GlyA. This study shows that nutrient supplementation during 1H NMR metabolomics experiments can disentangle complex metabolic outcomes stemming from a general metabolic stress. Metabolomics shows great potential to complement classical reductionist approaches to cost-effectively accelerate the rate of progress in expanding our global understanding of metabolic network structure and physiology. To that end, this work demonstrates the utility in implementing nutrient supplementation and genetic perturbation into metabolomics workflows as a means to connect metabolic outputs to physiological phenomena and establish causal relationships.
| S-EPMC7065518 | biostudies-literature
Project description:The reactive intermediate deaminase RidA (EC: 3.5.99.10) is conserved across all domains of life and deaminates reactive enamine species. When S. enterica ridA mutants are grown in minimal medium, 2-aminoacrylate (2AA) accumulates, damages several pyridoxal 5’-phosphate (PLP)- dependent enzymes, and elicits an observable growth defect. Genetic studies suggested that damage to serine hydroxymethyltransferase (GlyA), and the resultant depletion of 5,10-methelenetetrahydrofolate (5,10-mTHF), was responsible for the observed growth defect. However, the downstream metabolic consequence from GlyA damage by 2AA remains relatively unexplored. This study sought to use untargeted 1H NMR metabolomics to determine whether the metabolic state of a S. enterica ridA mutant was accurately reflected by characterizing growth phenotypes. The data supported the conclusion that metabolic changes in a ridA mutant were due to the IlvA-dependent generation of 2AA, and that the majority of these changes were a consequence of damage to GlyA. While many of the shifts in the metabolome of a ridA mutant could be explained, changes in some metabolites were not easily modeled, suggesting that additional levels of metabolic complexity remain to be unraveled.
2020-01-22 | ST001308 | MetabolomicsWorkbench
Project description:A major Salmonella component involved in cellular activation is the lipopolysaccharide (LPS) molecule which can act as a dendritic cell (DC) stimulator. The structure of the lipid A domain of the LPS molecule dictates its immunostimulatory capacity with various cell types. In this study, the role of lipid A as an integral component of Salmonella in stimulating murine DCs was studied by using a Salmonella enterica serovar Typhimurium lpxM mutant with defective lipid A. This study revealed that a mutation in lpxM did not significantly affect the ability of bacteria to activate DCs. Although the lpxM mutant less tumor necrosis factor alpha, interleukin-1beta, and inducible nitric oxide synthase than the parental strain, this was only seen at lower multiplicities of infection (MOIs). Both strains upregulated surface molecule expression on DCs and augmented the T-cell-stimulating capacity of these cells in an MOI-independent manner. Thus, the lpxM mutation did not appear to affect the stimulatory capacity of the Salmonella mutant.
| S-EPMC219569 | biostudies-other
Project description:Purpose: Members of the broadly distributed Rid/YER057c/UK114 protein family have imine/enamine deaminase activity, notably on a 2-aminoacrylate (2AA). Strains of Salmonella enterica, and other organisms lacking RidA, have diverse growth phenotypes, attributed to the accumulation of 2AA. The goal of this study was to use transcriptional differences between S. enterica wild-type and ridA strains to explore the breadth of the cellular consequences that resulted from accumulation of 2AA. Methods: S. enterica LT2 mRNA profiles of wild-type (WT) and ridA mutant cells were generated by Next Generation Sequencing, in biological quadruplicate, using Illumina Nextseq (150 cycles) Mid Output Flowcell in the paired end mode with a read length of 75bp. The sequence reads that passed quality filters were analyzed by DEseq to call for statistically significant (FDR < 0.05) differential gene expression. qRT–PCR experiemnts were performed using SYBR Green assays to corroborate the differential expression for 9 of the genes identified as differentially expressed. Results: Pair-wise comparison of gene expression identified 413 genes with statistically significant changes (186 higher expression, and 227 lower expression) in transcript abundance between ridA mutant and wild-type strains (FDR < 0.05). Of these loci,when genes with no known function (STMxxxx and yxxX locus tags--120 genes) and genes showing less than a two-fold difference in expression were excluded, 84 genes remained which showed differential expression between strains. RNA-seq data had a linear relationship with qRT–PCR for four genes (napF, metH, dadX, and fliI) belonging to the 84 gene dataset, one gene (thiF) with an FDR < 0.05, but failing the two-fold cutoff threshold and one gene (sdaC) that did not meet the statistical cutoff (FDR > 005). Pearson's correlation coefficient (R2) for the relationship between RNAseq and qRT-PCR data was measured at 0.96. Analysis of the differentially expressed genes uncovered several areas of metabolism and physiology which may be affected by RidA elimination, and subsequent 2-aminoacrylate accumulation. Conclusions: Our study represents the first detailed analysis on the transcriptional effect RidA elimination has in S. enterica LT2, using biologic replicates, and generated by RNA-seq technology. Our results uncovered a motility effect associated with the accumulation of 2-aminoacrylate in a ridA mutant as well as identified additional areas of metabolism impacted by the metabolic stress associated with 2AA accumulation. These data also emphasized the value of integrating global approaches with biochemical genetic approaches to understand the complex system of microbial metabolism.
2017-08-28 | GSE103146 | GEO
Project description:BACKGROUND: Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT), but their roles have not been fully investigated. METHODS: The presence of Plasmodium SHMT isoforms in the intra-erythrocytic stage was assessed based on their gene expression using reverse transcription PCR (RT-PCR). Localization studies of Plasmodium SHMT isoforms were performed by transfection of fluorescent-tagged gene constructs into P. falciparum and expressions of fluorescent fusion proteins in parasites were observed using a laser scanning confocal microscope. Genetic targeting through homologous recombination was used to study the essentiality of SHMT in Plasmodium spp. RESULTS: Semi-quantitative RT-PCR revealed the expression of these two genes throughout intra-erythrocytic development. Localization studies using P. falciparum expressing fluorescent-tagged SHMT showed that PfcSHMT-red fluorescent fusion protein (PfcSHMT-DsRed) is localized in the cytoplasm, while PfmSHMT-green fluorescent fusion protein (PfmSHMT-GFP) co-localized with Mitotracker™-labelled mitochondria as predicted. The essentiality of plasmodial cSHMT was inferred from transfection experiments where recovery of viable knock-out parasites was not achieved, unless complemented with a functional equivalent copy of shmt. CONCLUSIONS: Distinct compartment localizations of PfSHMT were observed between cytoplasmic and mitochondrial isoforms, and evidence was provided for the indispensable role of plasmodial cSHMT indicating it as a valid target for development of novel anti-malarials.
| S-EPMC3521198 | biostudies-literature
Project description:Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ?glyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8? resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT.
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Project description:The plant-like, bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from malaria parasites has been a good target for drug development. Dihydrofolate reductase (DHFR) is inhibited by clinically established antimalarials, pyrimethamine and cycloguanil. Thymidylate synthase (TS) is the target of potent experimental antimalarials such as 5-fluoroorotate and 1843U89. Another enzyme in folate recycling, serine hydroxymethyltransferase (SHMT), produces 5,10-methylenetetrahydrofolate which, in many cells, is required for the de novo, biosynthesis of thymidine and methionine. Thus, the biochemical characterization of malarial SHMT was of interest. The principle, active Plasmodium falciparum SHMT (PfSHMT) was expressed in E. coli and purified using an N-terminal histidine tag. Unlike the plant enzyme, but like the host enzyme, PfSHMT requires the cofactor pyridoxal 5'-phosphate for enzymatic activity. The substrate specificities for serine, tetrahydrofolate, and pyridoxal 5'-phosphate were comparable to those for SHMT from other organisms. Antifolates developed for DHFR and TS inhibited SHMT in the mid-micromolar range, offering insights into the binding preferences of SHMT but clearly leaving room for improved new inhibitors. As previously seen with P. falciparum DHFR-TS, PfSHMT bound its cognate mRNA but not control RNA for actin. RNA binding was not reversed with enzyme substrates. Unlike DHFR-TS, the SHMT RNA-protein interaction was not tight enough to inhibit translation. Another gene PF14_0534, previously proposed to code for an alternate mitochondrial SHMT, was also expressed in E. coli but found to be inactive. This protein, nor DHFR-TS, enhanced the catalytic activity of PfSHMT. The present results set the stage for developing specific, potent inhibitors of SHMT from P. falciparum.
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Project description:Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. Recombinant attenuated bacterial vector systems based on genetically engineered Salmonella have been developed as highly potent vaccines. Due to the pathogenic properties of Salmonella, efficient attenuation is required for clinical applications. Since the hallmark study by Hoiseth and Stocker in 1981 (S. K. Hoiseth and B. A. D. Stocker, Nature 291:238-239, 1981, http://dx.doi.org/10.1038/291238a0), the auxotrophic ΔaroA mutation has been generally considered safe and universally used to attenuate bacterial strains. Here, we are presenting the remarkable finding that a deletion of aroA leads to pronounced alterations of gene expression, metabolism, and cellular physiology, which resulted in increased immunogenicity, virulence, and adjuvant potential of Salmonella. These results suggest that the enhanced immunogenicity of aroA-deficient Salmonella strains might be advantageous for optimizing bacterial vaccine carriers and immunotherapy. Accordingly, we demonstrate a superior performance of ΔaroA Salmonella in bacterium-mediated tumor therapy. In addition, the present study highlights the importance of a functional shikimate pathway to sustain bacterial physiology and metabolism.
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Project description:Transcriptional profiling of Salmonella enterica serovar Typhimurium SL1344 cells comparing wildtype with ΔfabR mutant. Salmonella strains were cultured under free-living TSB conditions (TSB 1/20, 200 rpm, 25 °C) for 6h (ca. 2 x 108 cells/ml).
2014-12-31 | GSE52880 | GEO
Project description:BACKGROUND:Serine hydroxymethyltransferase 1 (SHMT1) is an enzyme involved in folic acid metabolism and is known to contribute to the development of hypertension. We evaluated the relationship between SHMT1 promoter methylation and essential hypertension (EH). METHODS:Quantitative methylation-specific polymerase chain reaction was used to measure the SHMT1 promoter methylation level in 241 EH patients and 288 age- and gender-matched healthy individuals. The diagnostic value of SHMT1 promoter hypermethylation was analyzed using a receiver operating characteristic (ROC) curve. The Gene Expression Omnibus (GEO) database and dual-luciferase reporter assay were used to validate our findings. RESULTS:Compared with the control group, significant differences in SHMT1 promoter methylation were found in both EH and hyperhomocysteinemia groups (P < 0.001 and P = 0.029, respectively). The area under the curve of the diagnosis of SHMT1 promoter hypermethylation for EH was 0.808, with a sensitivity and specificity of 73.9% and 77.8%, respectively. The risk of SHMT1 promoter hypermethylation was significantly higher in the >65-year group than in the ?65-year group (odds ratio = 3.925; 95% confidence interval = 2.141-7.196). In addition, GEO database analysis showed that 5-aza-deoxycytidine increased gene expression in several carotid endothelial cell lines. A dual-luciferase reporter assay revealed that the target sequence in the SHMT1 promoter upregulated gene expression. CONCLUSION:Our findings indicate that SHMT1 promoter hypermethylation increases the risk of EH and may be a promising biomarker for EH.
| S-EPMC6818578 | biostudies-literature