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Possible links between stress defense and the tricarboxylic acid (TCA) cycle in Francisella pathogenesis.

ABSTRACT: Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. In vivo, this facultative intracellular bacterium survives and replicates mainly in the cytoplasm of infected cells. We have recently identified a genetic locus, designated moxR that is important for stress resistance and intramacrophage survival of F. tularensis. In the present work, we used tandem affinity purification coupled to mass spectrometry to identify in vivo interacting partners of three proteins encoded by this locus: the MoxR-like ATPase (FTL_0200), and two proteins containing motifs predicted to be involved in protein-protein interactions, bearing von Willebrand A (FTL_0201) and tetratricopeptide (FTL_0205) motifs. The three proteins were designated here for simplification, MoxR, VWA1, and TPR1, respectively. MoxR interacted with 31 proteins, including various enzymes. VWA1 interacted with fewer proteins, but these included the E2 component of 2-oxoglutarate dehydrogenase and TPR1. The protein TPR1 interacted with one hundred proteins, including the E1 and E2 subunits of both oxoglutarate and pyruvate dehydrogenase enzyme complexes, and their common E3 subunit. Remarkably, chromosomal deletion of either moxR or tpr1 impaired pyruvate dehydrogenase and oxoglutarate dehydrogenase activities, supporting the hypothesis of a functional role for the interaction of MoxR and TPR1 with these complexes. Altogether, this work highlights possible links between stress resistance and metabolism in F. tularensis virulence.

SUBMITTER: Dieppedale J

PROVIDER: S-EPMC3734585 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Possible links between stress defense and the tricarboxylic acid (TCA) cycle in Francisella pathogenesis.

Dieppedale Jennifer J Gesbert Gael G Ramond Elodie E Chhuon Cerina C Dubail Iharilalao I Dupuis Marion M Guerrera Ida Chiara IC Charbit Alain A

Molecular & cellular proteomics : MCP 20130513 8

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. In vivo, this facultative intracellular bacterium survives and replicates mainly in the cytoplasm of infected cells. We have recently identified a genetic locus, designated moxR that is important for stress resistance and intramacrophage survival of F. tularensis. In the present work, we used tandem affinity purification coupled to mass spectrometry to identify in vivo interacting partners of three pr ...[more]

PMID: 23669032

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Project description:Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo. Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δψ) and cell ATP levels were assayed using fluorometric and luminescent test. Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δψ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.

Dataset Information

Possible links between stress defense and the tricarboxylic acid (TCA) cycle in Francisella pathogenesis.

Publications

Possible links between stress defense and the tricarboxylic acid (TCA) cycle in Francisella pathogenesis.

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