ABSTRACT
In fungi, ergosterol is an essential component of the plasma membrane. Its biosynthesis from acetyl-CoA is the primary target of the most commonly used antifungal drugs. Here, we show that the pantothenate kinase Cab1p, which catalyzes the first step in the metabolism of pantothenic acid for CoA biosynthesis in budding yeast (Saccharomyces cerevisiae), significantly regulates the levels of sterol intermediates and the activities of ergosterol biosynthesis-targeting antifungals. Using genetic and pharmacological analyses, we show that altered pantothenate utilization dramatically alters the susceptibility of yeast cells to ergosterol biosynthesis inhibitors. Genome-wide transcription and MS-based analyses revealed that this regulation is mediated by changes both in the expression of ergosterol biosynthesis genes and in the levels of sterol intermediates. Consistent with these findings, drug interaction experiments indicated that inhibition of pantothenic acid utilization synergizes with the activity of the ergosterol molecule-targeting antifungal amphotericin B and antagonizes that of the ergosterol pathway-targeting antifungal drug terbinafine. Our finding that CoA metabolism controls ergosterol biosynthesis and susceptibility to antifungals could set the stage for the development of new strategies to manage fungal infections and to modulate the potency of current drugs against drug-sensitive and -resistant fungal pathogens.
Subject(s)
Drug Resistance, Fungal/genetics , Ergosterol/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/genetics , Sterols/metabolism , Amphotericin B/pharmacology , Antifungal Agents/pharmacology , Coenzyme A/biosynthesis , Coenzyme A/drug effects , Ergosterol/biosynthesis , Ergosterol/genetics , Gene Expression Regulation, Fungal/drug effects , Genome, Fungal/drug effects , Pantothenic Acid/biosynthesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sterols/biosynthesis , Terbinafine/pharmacologyABSTRACT
The aim of this study was to determine seizure-induced oxidative stress by measuring hippocampal glutathione (GSH) and glutathione disulfide (GSSG) levels in tissue and mitochondria. Kainate-induced status epilepticus (SE) in rats resulted in a time-dependent decrease of GSH/GSSG ratios in both hippocampal tissue and mitochondria. However, changes in GSH/GSSG ratios were more dramatic in the mitochondrial fractions compared to hippocampal tissue. This was accompanied by a mild increase in glutathione peroxidase activity and a decrease in glutathione reductase activity in hippocampal tissue and mitochondria, respectively. Since coenzyme A (CoASH) and its disulfide with GSH (CoASSG) are primarily compartmentalized within mitochondria, their measurement in tissue was undertaken to overcome problems associated with GSH/GSSG measurement following subcellular fractionation. Hippocampal tissue CoASH/CoASSG ratios were decreased following kainate-induced SE, the time course and magnitude of change paralleling mitochondrial GSH/GSSG levels. Cysteine, a rate-limiting precursor of glutathione was decreased following kainate administration in both hippocampal tissue and mitochondrial fractions. Together these changes in altered redox status provide further evidence for seizure-induced mitochondrial oxidative stress.
Subject(s)
Glutathione Disulfide/metabolism , Glutathione/metabolism , Mitochondria/metabolism , Oxidative Stress , Seizures/metabolism , Status Epilepticus/metabolism , Animals , Coenzyme A/drug effects , Coenzyme A/metabolism , Cysteine/drug effects , Cysteine/metabolism , Disease Models, Animal , Glutathione/drug effects , Glutathione Disulfide/drug effects , Glutathione Peroxidase/drug effects , Glutathione Peroxidase/metabolism , Glutathione Reductase/drug effects , Glutathione Reductase/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Kainic Acid , Male , Mitochondria/drug effects , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Rats , Rats, Sprague-Dawley , Seizures/chemically induced , Status Epilepticus/chemically induced , Time FactorsABSTRACT
The intracellular concentration of CoA metabolites and nucleotides was determined in batch cultures of Methylobacterium rhodesianum grown on methanol and shifted to growth on fructose. The intracellular concentration of CoA decreased from a high value of 0.6 nmol/mg poly(beta-hydroxybutyrate)-free bacterial dry mass during growth on methanol to a low value of 0.03 nmol/mg poly(beta-hydroxybutyrate)-free bacterial dry mass after a shift to fructose as a carbon source. The levels of NADH, NADPH, and acetyl-CoA were also lower. Under these conditions, acetyl-CoA was metabolized by both citrate synthase and beta-ketothiolase, and poly(beta-hydroxybutyrate) synthesis and growth occurred simultaneously during growth on fructose. Moreover, the level of ATP was approximately 50% lower during growth on fructose, supporting the hypothesis of a bottleneck in the energy supply during the growth of M. rhodesianum with fructose.
Subject(s)
Fructose/pharmacology , Gram-Negative Aerobic Rods and Cocci/drug effects , Gram-Negative Aerobic Rods and Cocci/metabolism , Hydroxybutyrates/metabolism , Methanol/pharmacology , Solvents/pharmacology , 3-Hydroxybutyric Acid , Acetyl Coenzyme A/drug effects , Acetyl Coenzyme A/metabolism , Acetyl-CoA C-Acyltransferase/drug effects , Acetyl-CoA C-Acyltransferase/metabolism , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Alcohol Oxidoreductases/drug effects , Alcohol Oxidoreductases/metabolism , Cell Division/drug effects , Citrate (si)-Synthase/drug effects , Citrate (si)-Synthase/metabolism , Coenzyme A/drug effects , Coenzyme A/metabolism , Culture Media/pharmacology , Gram-Negative Aerobic Rods and Cocci/enzymology , NAD/drug effects , NAD/metabolism , NADP/drug effects , NADP/metabolismABSTRACT
Ammonia toxicity and relative sodium benzoate toxicity alters the energy metabolism, leading to a decrease of adenosine triphosphate and free coenzyme A levels. The object of the present study was to analyze the hepatic and muscular acyl-coenzyme A profiles in chronically hyperammonemic mice treated with varying doses of the sodium benzoate. An enzymatic method was used for the measurement of free coenzyme A, acetyl-coenzyme A, and medium and long chain acyl-coenzyme A. Untreated chronic hyperammonemia resulted in a decrease in acetyl-coenzyme A and an increase in the long chain acyl-coenzyme A in the liver, accompanied by an increase in total coenzyme A in the muscular tissues. Treatment with sodium benzoate at moderate doses, caused a decrease in the hepatic free and esterified coenzyme A while these were increased at higher doses. We conclude that chronic hyperammonemia is responsible for qualitative changes in the free and esterified coenzyme A profile in the liver, while causing qualitative and quantitative changes in the muscular tissue, probably due to an inhibition of mitochondrial oxidation. The sodium benzoate had a biphasic effect on the hepatic content of free and esterified coenzyme A, suggesting a degradation of coenzyme A at moderate doses. However, at a higher dose of benzoate, the possibility of glycine mobilization and/or a significant formation of acylcarnitines is proposed as an important factor in an increase of the hepatic total coenzyme A.