A Fox2-dependent fatty acid ß-oxidation pathway coexists both in peroxisomes and mitochondria of the ascomycete yeast Candida lusitaniae.

Frédéric Gabriel,Stephen Manon,Bénédicte Salin,Jean-Jacques Bessoule,Karine Dementhon,Thierry Noël,Marine Lucas-Guérin,Isabelle Accoceberry

doi:10.1371/journal.pone.0114531

Abstract

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying 14Cα-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.

Highlights

Lipid metabolism in fungal pathogenesis has recently attracted growing interest because of its possible multiple implications during host-pathogen interactions [1, 2]
The unique genes ICL1 encoding the isocitrate lyase, FOX2 encoding the multifunctional protein of b-oxidation, and PXA1 encoding part of an ABC transporter responsible for peroxisomal long chain fatty acids (FA) uptake, were identified in the genome of C. lusitaniae with a Basic Local Alignment Search Tool (BLAST) analysis [36] using as query the orthologous proteins of C. albicans
We tested the ability of the C. lusitaniae icl1D and fox2D mutants to grow on minimal medium (YNB agar) containing either glucose, glycerol, acetate, saturated short-chain FA (C10:0), saturated medium-chain FA (C12:0, C14:0), saturated long-chain FA (C16:0, C18:0), or unsaturated long-chain FA (C18:1, C22:1), as the exclusive carbon source (Table 1)

Summary

Introduction

Lipid metabolism in fungal pathogenesis has recently attracted growing interest because of its possible multiple implications during host-pathogen interactions [1, 2]. One particular issue addresses the role of the catabolism of fatty acids (FA) as a source of nutrient when a fungal pathogen invades the host [2, 3]. Fatty acids are activated to fatty acyl-coenzyme A esters (FA-CoA) by acylCoA synthetases, and shortened between carbon 2 and 3 through the boxidation spiral, yielding acetyl-CoA in four steps (Fig. 1): i/an acyl–CoA–oxidase (Pox1p alias Fox1p) converts FA-CoA into trans-2-enoyl-CoA and transfers electrons directly to oxygen generating H2O2 [4], which is detoxified by the Cta1p catalase [13]; ii/and iii/a multifunctional enzyme (Fox2p), which has both enoylCoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, converts trans-2enoyl-CoA into 3-ketoacyl-CoA [14, 15]; iv/a 3-ketoacyl-CoA thiolase (Pot1p alias Fox3p) converts 3-ketoacyl-CoA into acetyl-CoA and FA-CoA, shortened by two carbon units, which can undergo an additional b-oxidation cycle. Acetyl units may integrate the glyoxylate cycle [16], or be exported outside the peroxisome by the carnitine acetyl transferase shuttle system, or be exported as citrate or malate [3]

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