Identification and characterization of lipid droplets in Malassezia  furfur

Celis AM1, 3, Triana S1, Ibarra H1, Cardona J2, Restrepo S1,1, Gonzalez, A2, Wösten HAB3, de Cock H3

1. Laboratorio de Micología y Fitopatología, Departamento de Ciencias Biológicas Universidad de Los Andes, Bogotá, Colombia. 
2. Grupo de Diseño de Productos y Procesos (GDPP), Departamento de Ingeniería Química, Universidad de los Andes.
3. Microbiology, Department of Biology, Utrecht University, Utrecht, The Netherlands 

Objectives
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 address the presence of LD during starvation for fatty acids, the composition of the lipid droplets using lipidomics and we use 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 throught 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 was 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 analysis 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).

Conclusion
1.	Remarkably, LD remain present in M. furfur cells even when starved for 160 min in MM lacking fatty acids.
2.	of  Lipidomic analysis of LD of Malassezia furfur indicated the presence of triglycerides but absence of SEs. 
3.	Genome analysis showed the presence of genes encoding proteins for esterification of ergosterol, indicating that regulation prevents SE storage.
4.	Genomic analysis confirmed the presence of all genes required for the formation of the main precursor for synthesis of triglycerides.