C9-methylated glucosylceramide is a fungus-specific sphingolipid. This lipid is a major membrane component in the cell and is thought to play important roles in the growth and virulence of several fungal species. To investigate the importance of the methyl branch of the long-chain base in glucosylceramides in pathogenic fungi, we identified and characterized a sphingolipid C9-methyltransferase gene (MTS1, C9-MethylTransferase for Sphingolipid 1) in the pathogenic yeast Candida albicans. The mts1 disruptant lacked (E,E)-9-methylsphinga-4,8-dienine in its glucosylceramides and contained (E)-sphing-4-enine and (E,E)-sphinga-4,8-dienine. Reintroducing the MTS1 gene into the mts1 disruptant restored the synthesis of (E,E)-9-methylsphinga-4,8-dienine in the glucosylceramides. We also created a disruptant of the HSX11 gene, encoding glucosylceramide synthase, which catalyses the final step of glucosylceramide synthesis, in C. albicans and compared this mutant with the mts1 disruptant. The C. albicans mts1 and hsx11 disruptants both had a decreased hyphal growth rate compared to the wild-type strain. The hsx11 disruptant showed increased susceptibility to SDS and fluconazole, similar to a previously reported sld1 disruptant that contained only (E)-sphing-4-enine in its glucosylceramides, suggesting that these strains have defects in their cell membrane structures. In contrast, the mts1 disruptant grew similarly to wild-type in medium containing SDS or fluconazole. These results suggest that the C9-methyl group of a long-chain base in glucosylceramides plays an important role in the hyphal elongation of C. albicans independent of lipid membrane disruption.

Malassezia yeasts are part of the cutaneous microflora commonly found on animals and human and may sometimes cause various opportunistic skin diseases. As most of Malassezia species show lipid-dependency, lipolytic enzymes such as lipase and phospholipase are necessary for them to obtain useful lipids from the environment. Consequently, these enzymes are thought to play an important role in the growth and pathogenicity of Malassezia. Here we analyze and compare extracellular lipase and phospholipase activities of several Malassezia species cultivated under common growth conditions. M. globosa showed the highest lipase activity of all of the Malassezia species included in our studies. The lipid-independent M. pachydermatis also showed high lipase and phospholipase activity. These results indicate that this Malassezia species are capable of utilizing lipids well in contrast to the other lipid-dependent species of the genus. Our data suggest that lipase may be a pathogenic factor in the skin disease associated with Malassezia and provide an explanation as to why M. globosa is an important pathogenic species in several human skin diseases despite its slow rate of growth.

Ceramides and glycosylceramides, including desaturated long-chain bases, are present in most fungi as well as animals and plants. However, as the budding yeast Saccharomyces cerevisiae is not capable of desaturating long-chain bases, little is known about the physiological roles of these compounds in fungi. To investigate the necessity of desaturation of long-chain backbones in ceramides and glucosylceramides in fungal cells, we have identified and characterized a sphingolipid D 8-desaturase (SLD) gene from the pathogenic yeast Candida albicans. Gene disruption of the C. albicans SLD homologue led to the accumulation of (E)-sphing-4-enine, a main substrate for the SLD enzyme. Introducing the Candida SLD gene homologue into these mutant cells resulted in the recovery of synthesis of (4E, 8E)-sphinga-4,8-dienine and this gene homologue was therefore identified as a Ca-SLD gene. Additionally, the sld disruptant of C. albicans had a decreased hyphal growth rate compared with the wild-type strain. These results suggest that D 8 -desaturation of long-chain bases in ceramides plays a role in the morphogenesis of C. albicans.

SummaryDuring disseminated infection by the opportunistic pathogen Candida glabrata, uptake of sterols such as serum cholesterol may play a significant role during pathogenesis. The ATP-binding cassette transporter Aus1p is thought to function as a sterol importer and in this study, we show that uptake of exogenous sterols occurred under anaerobic conditions in wildtype cells of C. glabrata but not in AUS1-deleted mutant (aus1D) cells. In aerobic cultures, growth inhibition by fluconazole was prevented in the presence of serum, and AUS1 expression was upregulated. Uptake of sterol by azole treated cells required the presence of serum, and sterol alone did not reverse FLC inhibition of growth. However, if iron availability in the growth medium was limited by addition of the iron chelators ferrozine or apo-transferrin, growth of wild-type cells, but not aus1D cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1D strain caused a significantly decreased kidney fungal burden than the wild-type strain or a strain in which AUS1 was restored. We conclude that sterol uptake in C. glabrata can occur in iron poor environment of host tissues and thus may contribute to C. glabrata pathogenesis.

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