Impaired succinate oxidation prevents growth and influences drug susceptibility in Mycobacterium tuberculosis

Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the TCA cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bi-directional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M. tuberculosis have relied on the use of single-gene deletion mutants, raising the possibility that the remaining enzymes could catalyze succinate oxidation in the absence of the other. To address this, we report on the use of mycobacterial CRISPR interference (CRISPRi) to construct single, double, and triple transcriptional knockdowns of sdhA1, sdhA2, and frdA in M. tuberculosis. We show that the simultaneous knockdown of sdhA1 + sdhA2 is required to prevent succinate oxidation and overcome the functional redundancy within these enzymes. Succinate oxidation was demonstrated to be essential for the optimal growth of M. tuberculosis, with the combined knockdown of sdhA1 + sdhA2 significantly impairing the activity of the respiratory chain and preventing growth on a range of carbon sources. Moreover, impaired succinate oxidation was shown to influence the activity of several antitubercular drugs against M. tuberculosis, including potentiating the activity of bioenergetic inhibitors and attenuating the activity of cell wall inhibitors. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility.

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Metabolic and Developmental Effects Resulting from Deletion of the citA Gene Encoding Citrate Synthase in Aspergillus nidulans

Eukaryotic Cell ◽

10.1128/ec.00373-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 656-666 ◽

Cited By ~ 9

Author(s):

Sandra L. Murray ◽

Michael J. Hynes

Keyword(s):

Aspergillus Nidulans ◽

Fluorescent Protein ◽

Citrate Synthase ◽

Glyoxylate Cycle ◽

Single Gene ◽

Carbon Sources ◽

Tca Cycle ◽

Poor Growth ◽

Content Type ◽

The Glyoxylate Cycle

ABSTRACT Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsAΔ citAΔ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.

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Rapid drug-susceptibility testing of Mycobacterium tuberculosis clinical isolates to first-line antitubercular drugs by nitrate reductase assay: A comparison with proportion method

International Journal of Mycobacteriology ◽

10.1016/j.ijmyco.2016.06.006 ◽

2016 ◽

Vol 5 (4) ◽

pp. 469-474 ◽

Cited By ~ 6

Author(s):

Amrish Kohli ◽

Gulnaz Bashir ◽

Akeela Fatima ◽

Abiroo Jan ◽

Nayeem-u-din Wani ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nitrate Reductase ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

First Line ◽

Antitubercular Drugs ◽

Nitrate Reductase Assay ◽

Proportion Method

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Role of O2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate

Journal of Bacteriology ◽

10.1128/jb.00572-16 ◽

2016 ◽

Vol 199 (1) ◽

Cited By ~ 10

Author(s):

Rachel M. Wheatley ◽

Vinoy K. Ramachandran ◽

Barney A. Geddes ◽

Benjamin J. Perry ◽

Chris K. Yost ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

Regulatory Protein ◽

Carbon Sources ◽

Cytosine Deaminase ◽

Optimal Growth ◽

Tca Cycle ◽

Cell Physiology ◽

Type I ◽

Content Type ◽

Glutamate Synthesis

ABSTRACT Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547 [phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O2 (pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O2 and cell death. IMPORTANCE Rhizobium leguminosarum, a soil bacterium that forms N2-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O2 tensions during growth in soil and N2 fixation in symbiosis with legumes.

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Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain

Biochemical Journal ◽

10.1042/bj3480607 ◽

2000 ◽

Vol 348 (3) ◽

pp. 607-614 ◽

Cited By ~ 910

Author(s):

Mark R. OWEN ◽

Elena DORAN ◽

Andrew P. HALESTRAP

Keyword(s):

Respiratory Chain ◽

Rat Hepatocytes ◽

Mitochondrial Respiratory Chain ◽

Time Dependent ◽

Succinate Oxidation ◽

Isolated Rat Hepatocytes ◽

Peripheral Tissues ◽

Dependent Inhibition ◽

Insulin Dependent ◽

Mitochondrial Oxidation

Although metformin is widely used for the treatment of non-insulin-dependent diabetes, its mode of action remains unclear. Here we provide evidence that its primary site of action is through a direct inhibition of complex 1 of the respiratory chain. Metformin (50 μM) inhibited mitochondrial oxidation of glutamate+malate in hepatoma cells by 13 and 30% after 24 and 60 h exposure respectively, but succinate oxidation was unaffected. Metformin also caused time-dependent inhibition of complex 1 in isolated mitochondria, whereas in sub-mitochondrial particles inhibition was immediate but required very high metformin concentrations (K0.5, 79 mM). These data are compatible with the slow membrane-potential-driven accumulation of the positively charged drug within the mitochondrial matrix leading to inhibition of complex 1. Metformin inhibition of gluconeogenesis from L-lactate in isolated rat hepatocytes was also time- and concentration-dependent, and accompanied by changes in metabolite levels similar to those induced by other inhibitors of gluconeogenesis acting on complex 1. Freeze-clamped livers from metformin-treated rats exhibited similar changes in metabolite concentrations. We conclude that the drug's pharmacological effects are mediated, at least in part, through a time-dependent, self-limiting inhibition of the respiratory chain that restrains hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues. Lactic acidosis, an occasional side effect, can also be explained in this way.

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The probable site of action of thenolytrifluoracetone on the respiratory chain

Biochemical Journal ◽

10.1042/bj1640617 ◽

1977 ◽

Vol 164 (3) ◽

pp. 617-620 ◽

Cited By ~ 40

Author(s):

W J Ingledew ◽

T Ohnishi

Keyword(s):

Succinate Dehydrogenase ◽

Respiratory Chain ◽

Close Association ◽

Succinate Oxidation ◽

Paramagnetic Resonance ◽

Spin Interactions ◽

Site Of Action ◽

Transfer Inhibitor ◽

Probable Site ◽

Electron Paramagnetic

1. It is shown that the electron-transfer inhibitor thenoyltrifluoroacetone abolishes a respiratory-chain electron-paramagnetic-resonance absorbance due to spin-spin interactions of ubisemiquinones at concentrations similar to those required for inhibition of succinate oxidation. 2. A specific site of interaction of thenoyltrifluoroacetone with the respiratory chain is proposed to be on the ubisemiquinone with which succinate dehydrogenase reacts. 3. Our results further demonstrate the close association of the HiPIP (high-potential iron-sulphur) centre of succinate dehydrogenase with ubisemiquinone.

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Comparison of Proportion and Resistance Ratio Methods for Drug Susceptibility Testing of Mycobacterium tuberculosis Isolated from Patients Attending National Tuberculosis Centre, Nepal

SAARC Journal of Tuberculosis Lung Diseases and HIV/AIDS ◽

10.3126/saarctb.v5i1.3078 ◽

2010 ◽

Vol 5 (1) ◽

pp. 13-20

Author(s):

S Acharya ◽

P Ghimire ◽

DK Khadka ◽

S Nepali

Keyword(s):

Mycobacterium Tuberculosis ◽

Fluorescence Microscopy ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Resistance Ratio ◽

National Tuberculosis ◽

Smear Negative ◽

Culture Positive ◽

Good Agreement

Background: Tuberculosis (TB) is among the most serious infectious cause of global morbidity and mortality. Emergence of Multi-drug resistant tuberculosis (MDR-TB) is posing an increased threat to TB control programs. Drug susceptibility testing (DST) of Mycobacterium tuberculosis (M. tuberculosis) isolates is important for tackling such problems. Setting: National Tuberculosis Centre (NTC), Thimi, Bhaktapur, Nepal. Objectives: Comparative evaluation of two in vitro DST methods in determining susceptibility of M. tuberculosis isolates from patients attending NTC, to front-line anti-TB drugs: (Isoniazid-INH, Rifampicin-RFP, Streptomycin-SM, and Ethambutol-EMB). Methodology: This study was conducted from Sep 2006-Jun 2007. A total of 862 sputum samples (diagnosis or follow up cases) collected from patients (type of patients or their categories was not differentiated in this study) attending NTC bacteriology lab for sputum direct smear microscopy were analyzed using fluorescence microscopy. All smear positive samples, smear negative samples requested for culture were cultured. All culture positive samples confirmed as M. tuberculosis by biochemical tests were processed for DST by both proportion (PR) and resistance ratio (RR) methods. Results: Out of 862 sputum samples analyzed, 226 (26.2%) samples were positive for Acid Fast Bacilli (AFB) by fluorescence microscopy. Among 323 samples 226 smear positive samples and 97 smear negative samples requested for culture), 221 (68.4%) were culture positive, 92 (28.5%) were culture negative and 10 (3.1%) were contaminated. Out of 221 isolates of M. tuberculosis, 57.5% were resistant to one or more drugs by the PR method and 56.6% by the RR method. Similarly, MDR isolates were 29.9% and 29% by PR and RR methods respectively. On correlation analysis using Mc Nemar Chi-square test, no significant difference between the two tests were observed (p>0.05). The results showed high agreement between both methods and agreement rates to INH, RFP, SM and EMB were 93.2%, 93.7%, 93.2% and 94.1% respectively. Similarly, the agreement rates between both methods using kappa analysis showed kappa (k) value of 0.86, 0.85, 0.86 and 0.84 for INH, RFP, SM and EMB respectively, which is believed to be good agreement between both methods (k=0.80 to 1.00: Very good agreement). Conclusion: In conclusion, this study showed that both the Proportion and Resistance ratio methods are equally good for determining drug susceptibility of M. tuberculosis. Keywords: Mycobacterium tuberculosis; Drug Susceptibility Testing; Proportion Method; Resistance Ratio Method. DOI: 10.3126/saarctb.v5i1.3078 SAARC J. Tuber. Lung Dis. HIV/AIDS 2008 Vol.5(1) 13-20

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Comparison of the Performances of Two In-House Rapid Methods for Antitubercular Drug Susceptibility Testing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00960-08 ◽

2008 ◽

Vol 53 (2) ◽

pp. 808-810 ◽

Cited By ~ 6

Author(s):

Agustina I. de la Iglesia ◽

Emma J. Stella ◽

Héctor R. Morbidoni

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Sensitivity And Specificity ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Antitubercular Drug ◽

Rapid Methods ◽

Resistance Assessment

ABSTRACT Resistance to rifampin (rifampicin), isoniazid, and streptomycin of 69 Mycobacterium tuberculosis isolates was analyzed by an in-house method based on mycobacteriophage D29 and a colorimetric micromethod. Both methods showed sensitivity and specificity values ranging from 93% to 100%. These simple methods offer an option for drug resistance assessment of M. tuberculosis.

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The Cloning and Mapping of ADR6, a Gene Required for Sporulation and for Expression of the Alcohol Dehydrogenase II Isozyme From Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/116.4.531 ◽

1987 ◽

Vol 116 (4) ◽

pp. 531-540

Author(s):

Aileen K W Taguchi ◽

Elton T Young

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcohol Dehydrogenase ◽

Single Gene ◽

Carbon Sources ◽

Transcription Unit ◽

Yeast Cells ◽

Recessive Mutation ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Activating Sequence ◽

A Site

ABSTRACT The alcohol dehydrogenase II (ADH2) gene of the yeast, Saccharomyces cerevisiae, is not transcribed during growth on fermentable carbon sources such as glucose. Growth of yeast cells in a medium containing only nonfermentable carbon sources leads to a marked increase or derepression of ADH2 expression. The recessive mutation, adr6-1, leads to an inability to fully derepress ADH2 expression and to an inability to sporulate. The ADR6 gene product appears to act directly or indirectly on ADH2 sequences 3' to or including the presumptive TATAA box. The upstream activating sequence (UAS) located 5' to the TATAA box is not required for the Adr6- phenotype. Here, we describe the isolation of a recombinant plasmid containing the wild-type ADR6 gene. ADR6 codes for a 4.4-kb RNA which is present during growth both on glucose and on nonfermentable carbon sources. Disruption of the ADR6 transcription unit led to viable cells with decreased ADHII activity and an inability to sporulate. This indicates that both phenotypes result from mutations within a single gene and that the adr6-1 allele was representative of mutations at this locus. The ADR6 gene mapped to the left arm of chromosome XVI at a site 18 centimorgans from the centromere.

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