Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major

ABSTRACTSterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm-). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm- mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm- mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm-. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.AUTHOR SUMMARYSterols are well recognized for their stabilizing effects on the plasma membrane, but their functions in intracellular organelles are under explored, which hampers the development of sterol synthesis inhibitors as drugs. Our previous studies have demonstrated significant plasma membrane instability in the sterol biosynthetic mutant c14dm- in Leishmania major, a pathogenic protozoan responsible for cutaneous leishmaniasis causing 1-1.5 million infections a year. While the plasma membrane defects have undoubtedly contributed to the reduced virulence exhibited by c14dm- mutants, it was not clear whether other cellular processes were also affected. In this study, we revealed profound mitochondrial dysfunctions and elevated level of reactive oxygen species in c14dm- mutants. These sterol mutants rely heavily on glycolysis to generate energy and are extremely sensitive to glucose restriction. In addition, the accumulation of oxidants appears to be responsible (at least in part) for the previously observed heat sensitivity in c14dm- mutants. Thus, genetic or chemical inactivation of C14DM can influence the functions of cellular organelles beyond the plasma membrane. These findings shed light on the mechanism of action for azole compounds and provide new insight into the roles of sterol biosynthesis in Leishmania parasites.