Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress in Leishmania major

Abstract

Lathosterol oxidase (LSO) catalyzes the formation of C5-C6 double bond in the synthesis of various types of sterols in mammals, fungi, plants and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C5-C6 desaturation, we generated a LSO-null mutant line (lso-) in Leishmania major, the causative agent for cutaneous leishmaniasis. Lso- parasites lacked the ergostane-based sterols commonly found in wild type L. major and instead accumulated equivalent sterol species without the C5-C6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso- mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alteration in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C5-C6 double bond is vital for the structure of sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCESterols are essential membrane components in eukaryotes and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. Lathosterol oxidase (aka sterol C5-desaturase) is required for the formation of C5-C6 double bond in the sterol core structure in mammals, fungi, protozoans, plants and algae. Functions of this C5-C6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that the C5-C6 double bond is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.