Characterization of growth of lipid-dependent Malassezia yeast species, members of the skin mycobiome

Objective: Malassezia species are lipid-dependent due to the lack of cytosolic fatty acid synthase required for De novo synthesis of fatty acids (FAs) and these yeasts are part of the skin mycobiome. Pathogenicity of Malassezia has been related to several factors including the ability to produce enzymes such as esterases, lipases, lipoxygenases and proteases which enable growth on the host skin and lead to changes in sebum (skin fat) composition. The skin functions in the innate defense against pathogens due to its low water content, acidic pH, its microbiota, and antimicrobial compounds like free fatty acids. Understanding lipid dependency of Malassezia will help to understand how these yeasts establish themselves as part of the skin microbiota, which adaptation mechanisms are involved, and how, and whether, lipid metabolism impacts the shift to pathogenicity. The complex nutritional requirements of Malassezia have delayed the full comprehension of its lipid metabolism. Reconstruction of the lipid-synthesis pathways of Malassezia species in silico predicted amongst others a defect in the assimilation of palmitic acid in M. globosa, M. sympodialis, M. pachydermatis and an atypical isolate of M. furfur, but not in M. furfur. This prediction was validated by physiological characterization in chemically defined media (MM) using different lipid sources. Methods: Growth on FAs in liquid MM: Strains were first grown for 7 days at 33°C in lipid-rich mDixon medium. To prevent subsequent growth in MM due to the presence of residual lipids we performed a two-phase growth in MM. First, cells were diluted into MM containing specific lipids. After 3 days, these cells were diluted again in fresh MM with the same lipids. Growth was monitored for 7 days by determining OD600 nm and CFU by plating on mDixon plates. Results: M. furfur could assimilate palmitic acid or oleic acid as well as all Tween variants tested. The atypical M. fufur strain could assimilate only Tween 80, Tween 20, and oleic acid. M. pachydermatis, M. globosa, and M. sympodialis were able to grow in the first step in MM but not in the second step in MM with any of the lipid sources tested. Only M. furfur was able to maintain growth in MM with palmitic acid in the second growth step. Both M. pachydermatis and atypical M. furfur could sustain growth in MM with a mixture of palmitic acid and oleic acid. Conclusion: 1. A new culturing method for Malassezia spp. in chemically defined media was developed. 2. In silico predicted assimilation defects of palmitic acid for Malassezia spp. was confirmed. 3. Palmitic acid is fungicidal for a subset of Malassezia spp. but not for M. furfur. 4. FAs that induce lipid toxicity and do not affect the skin cells and microbiome harmony might have a therapeutic use.