Identification and characterization of lipid droplets in Malassezia furfur

Abstract

Objective: Malassezia furfur is a lipid-dependent yeast that is part of the human skin microbiota. Mechanisms underlying growth on skin including its lipid metabolism are largely unknown. Lipid droplets (LDs), also known as lipid particles, lipid bodies, oil bodies, or oleosomes, are highly dynamic organelles that can be produced in nearly all cells. The presence of LDs in Malassezia (Pityrosporum orbiculare) have been reported, but their role has not been studied. LDs contribute to a variety of processes including lipid storage, cell signaling, temporary protein storage, cell lipid homeostasis, prevention of lipotoxicity, biosynthesis and secretion of inflammatory mediators such as prostaglandins and leukotriene's, interferon responses, and antigen cross presentation. Lipid droplets are expected to play an important role in Malassezia spp during their growth and/or adaptation on the skin yet this has not been investigated. The purpose of this study is to characterize LD of M. furfur in complex medium (mDixon) and minimal medium supplemented with sources of palmitic acid (PA; C16:0) and/or oleic acid (OA; C18:1). We addressed the presence of LD during starvation for fatty acids, the composition of the lipid droplets using lipidomics and we used genomic analysis to identify the pathways involved in neutral lipid synthesis in this yeast. Methods: Lipid droplets (LDs) were identified in M. furfur growing in mDixon broth or MM with or without PA and/or OA through confocal microscopy using nile red staining. LD dynamics and cell viability was monitored upon transferring M. furfur cells that had been pre-grown in MM with PA and / or OA to MM without fatty acids. Lipidomic analyses were performed on chloroform/methanol extracted lipids from LD isolated via density gradient centrifugation. Results: CFUs strongly decreased upon lipid starvation irrespective of the type(s) of fatty acid present in the pre-culture and no CFUs were found after 160 h. Interestingly, cells observed in the 3 cultures after 160 h still contained LDs with distribution and morphology indistinguishable form those at the 72 h time point. Yet, they were less abundant as compared to those observed at t = 0 h. Lipidomic analyses revealed the presence of phospholipids as well as triglycerides (TGs) in LDs of M. furfur but sterol esters (SEs) were not detected. TG species 52:4, 54:3, 54:4, and 54:5 were enriched in LDs after growth in the presence of Tween 80 or oleic acid (both donors of C18:1), while TG species 50:1 and 52:2 were enriched after growth in the presence of Tween 40 (donor of C16:0). All enzymes involved in TG and SE synthesis were detected in the genome of M. furfur. Conclusion: Remarkably, LD remain present in furfur cells even when starved for 160 min in MM lacking fatty acids. Lipidomic analyses of LD of Malassezia furfur indicated the presence of triglycerides but absence of SEs. Genomic analyses showed the presence of genes encoding proteins for esterification of ergosterol, indicating that regulation prevents SE storage and confirmed the presence of all genes required for the formation of the main precursor for synthesis of triglycerides.