Stage-Specific De Novo Synthesis of Very-Long-Chain Dihydroceramides Confers Dormancy to Entamoeba Parasites

Amoebiasis is a parasitic disease caused by Entamoeba histolytica infection and is a serious public health problem worldwide due to ill-prepared preventive measures as well as its high morbidity and mortality rates. Amoebiasis transmission is solely mediated by cysts. Cysts are produced by the differentiation of proliferative trophozoites in a process termed “encystation.” Entamoeba encystation is a fundamental cell differentiation process and proceeds with substantial changes in cell metabolites, components, and morphology, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites that function as key factors for encystation. However, a comprehensive lipid analysis has not been reported, and the involved lipid metabolic pathways remain largely unknown. Here, we exploited the state-of-the-art untargeted lipidomics and characterized 339 molecules of 17 lipid subclasses. Of these, dihydroceramide (Cer-NDS) was found to be among the most induced lipid species during encysta- tion. Notably, in encysting cells, amounts of Cer-NDS containing very long N-acyl chains ($26 carbon) were more than 30-fold induced as the terminal product of a de novo metabolic pathway. We also identified three ceramide synthase genes responsible for producing the very-long-chain Cer-NDS molecules. These genes were upregulated during encystation. Furthermore, these ceramide species were shown to be indispensable for generating membrane impermeability, a prerequisite for becoming dormant cyst that shows resistance to environmental assault inside and outside the host for transmission. Hence, the lipid subclass of Cer-NDS plays a crucial role for Entamoeba cell differentiation and morphogenesis by alternating the membrane properties. IMPORTANCE Entamoeba is a protozoan parasite that thrives in its niche by alternating its two forms between a proliferative trophozoite and dormant cyst. Cysts are the only form able to transmit to a new host and are differentiated from trophozoites in a process termed “encystation.” During Entamoeba encystation, cell metabolites, components, and morphology drastically change, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites. However, the involved lipid species and their metabolic pathways remain largely unknown. Here, we identified dihydroceramides (Cer-NDSs) containing very long N-acyl chains (C₂₆ to C₃₀) as a key metabolite for Entamoeba encystation by our state-of-the-art untargeted lipidomics. We also showed that these Cer-NDSs are critical to generate the membrane impermeability, a prerequisite for this parasite to show dormancy as a cyst that repels substances and prevents water loss. Hence, ceramide metabolism is essential for Entamoeba to maintain the parasitic lifestyle.