Unraveling lipid metabolism in lipid-dependent pathogenic Malassezia yeasts

Malassezia yeasts are lipid-dependent fungal species that are common members of the human and animal skin microbiota. The lipid-dependency is a crucial trait in the adaptation process to grow on the skin but also plays a role in their pathogenic life style. Malassezia species can cause several skin infections like dandruff or seborrheic dermatitis but also bloodstream infections. Understanding the lipid metabolism in Malassezia is essential to understand its life style as skin commensal and pathogen, however, many aspects about the lipid-synthesis pathways remain inconclusive. M. pachydermatis was considered to be the only lipid independent Malassezia species due to the capacity to grow on Sabouraud agar medium. Genome analysis showed the absence of the FA synthase genes as was shown before in other Malassezia species. This suggested that M. pachydermatis could not grow in the absence of a lipid source. Physiological evaluations showed palmitic acid have a fungicidal effect on M. pachydermatis, while oleic acid had a fungistatic effect. However, mixtures of saturated and unsaturated FA did sustain growth. These results showed that this species needs a combination of different FAs to support full growth. The results underscore that M. pachydermatis is not able to grow in the absence of FAs while palmitic acid, the major FA in Sabouraud, does by itself not support growth of this species. We combined genome sequencing and in- silico lipid-synthesis pathways reconstruction revealing differences in the metabolism of fungal steroids and degradation of CoA-activated long-chain FAs, arachidonic acid, and butanoate metabolism between Malassezia yeasts. We predicted defects in the assimilation of palmitic acid in M. globosa, M. sympodialis, M. pachydermatis, and the atypical variant of M. furfur, but not in M. furfur. These predictions were validated via physiological characterization, confirming these in-silico predictions. Differences in the assimilation of saturated and unsaturated fatty acids were found among the different species. These differences were also connected with the storage of neutral lipids in lipid droplets (LD), which could be related as a mechanism to obtain and exploit lipid sources during starvation conditions. Lipidomic analysis showed that these lipid droplets contain triglycerides but no sterol esters (SEs). However genome analysis indicating the presence of genes encoding proteins for esterification of ergosterol, indicating that regulation prevents SE storage. In addition, genomic analysis confirmed the presence of all genes required for the formation of the main precursor for synthesis of triglycerides. New insights in the pathophysiology of Malassezia spp obtained by genome sequencing and metabolic reconstruction require experimental validation. To this end, genetic tools are needed. We implemented a highly efficient Agrobacterium-mediated genetic transformation system for M. furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in these yeasts by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. The T-DNA was mitotically stable in approximately 80 % of the transformants after 10 times sub-culturing in the absence of hygromycin selection