Peroxysomal Carnitine Acetyl Transferase Influences Host Colonization Capacity in Sclerotinia sclerotiorum

In lower eukaryotes, the glyoxylate cycle allows cells to utilize two-carbon compounds when simple sugars are not available. In filamentous fungi, glyoxylate metabolism is coupled with β-oxidation of fatty acids, and both are localized to ubiquitous eukaryotic organelles called peroxisomes. Acetyl coenzyme A (acetyl-CoA) produced during β-oxidation is transported via the cytosol into mitochondria for further metabolism. A peroxisomal-specific pathway for acetyl-CoA transport requiring peroxisomal carnitine acetyl transferase (CAT) activity has been identified in Magnaporthe grisea peroxisomes. Here, we report that a Sclerotinia sclerotiorum ortholog of the M. grisea peroxisomal CAT-encoding gene Pth2 (herein designated Ss-pth2) is required for virulence-associated host colonization. Null (ss-pth2) mutants, obtained by in vitro transposon mutagenesis, failed to utilize fatty acids, acetate, or glycerol as sole carbon sources for growth. Gene expression analysis of these mutants showed altered levels of transcript accumulation for glyoxylate cycle enzymes. Ss-pth2 disruption also affected sclerotial, apothecial, and appressorial development and morphology, as well as oxalic acid accumulation when cultured with acetate or oleic acid as sole carbon nutrient sources. Although mutants were able to penetrate and initially colonize host tissue, subsequent colonization was impaired. Genetic complementation with the wild-type Ss-pth2 restored wild-type virulence phenotypes. These findings suggest an essential role in S. sclerotiorum for the peroxisomal metabolic pathways for oxalic acid synthesis and host colonization.

Effects of conjugated linoleic acid (CLA) isomers on lipid levels and peroxisome proliferation in the hamster

Effects of the conjugated linoleic acid (CLA) isomers cis-9, trans-11 (c9, t11 CLA) and trans-10, cis-12 (t10, c12 CLA) on lipid metabolism and markers of peroxisome proliferation were investigated in hamsters fed on purified diets containing 30 % energy as fat and 0·1 g cholesterol/kg for 8 weeks. Four groups (n 32 each) received diets without CLA (control), with a mixture of equal amounts of c9, t11 and t10, c12 CLA (CLA mix), with c9, t11 CLA, and with t10, c12 CLA. The total amount of CLA isomers was 1·5 % energy or 6·6 g/kg diet. CLA was incorporated into glycerides and exchanged for linoleic acid in the diet. Compared with the control, the CLA mix and t10, c12 CLA decreased fasting values of LDL- (21 and 18 % respectively) and HDL-cholesterol (8 and 11 %), increased VLDL-triacylglycerol (80 and 61 %), and decreased epididymal fat pad weights (9 and 16 %), whereas c9, t11 CLA had no significant effects. All CLA preparations increased liver weight, but not liver lipids. However, the increase in liver weight was much less in the c9, t11 CLA group (8 %) than in the other two groups (25 %) and might have been caused by the small amount of t10, c12 CLA present in the c9, t11 CLA preparation. Liver histology revealed that increased weight was due to hypertrophy. Markers of peroxisome proliferation, such as cyanide-insensitive palmitoyl CoA oxidase (EC 1.3.3.6) and carnitine acetyl transferase (EC 2.3.1.7) activities, were not increased by CLA. Both c9, t11 CLA and t10, c12 CLA were incorporated into phospholipids and triacylglycerols, but t10, c12 CLA only about half as much as c9, t11 CLA. In addition, linoleic acid and linolenic acid concentrations were lower in lipids of the t10, c12 CLA group compared with the c9, t11 CLA group. These data suggest that t10, c12 CLA stimulated the oxidation of all C18 polyunsaturated fatty acids. The results indicate that the t10, c12 CLA isomer, and not the so-called natural CLA isomer (c9, t11), is the active isomer affecting lipid levels in hamsters.