Investigating the roles of lathosterol oxidase and sterol C4-demethylase in Leishmania survival, stress response and virulence

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2020-12

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Abstract

Sterols are essential membrane components in eukaryotes and important precursors for the synthesis of bioactive molecules. Sterol synthesis inhibitors have shown efficacy against pathogenic fungi and trypanosomatids. Understanding the roles of sterol biosynthetic enzymes can guide the development of new drugs and counter drug resistance. Previous studies from our laboratory have shown that sterol C14-demethylase and sterol methyl transferase are involved in heat tolerance and mitochondria functions in Leishmania major (L. major). In this study, I first characterized the lathosterol oxidase (LSO) which catalyzes the formation of C5-C6 double bond on sterol intermediates in L. major. LSO-null mutants were viable and replicative in culture, indicating that this gene is not essential. However, LSO is required for acid resistance, intracellular pH maintenance and the expression of a vacuolar-type H+-pyrophosphatase. Besides, deletion of LSO interfered with the modification of short side chain sugar residues in lipophosphoglycan, a major virulence factor preferentially located in the sterol-rich membrane domains. Finally, LSO-null mutants exhibited moderate mitochondrial defects, increased resistance to amphotericin B and attenuated virulence. Together, this study reveals the biological roles of LSO in L. major parasites. Second, I investigated the functions of sterol C4-demethylase (C4DM), which removes the two C4 methyl groups in sterol synthesis. There are several candidate genes for the L. major C4DM. Deletion studies of putative ERG25 and LSO suggest that neither encode the true C4DM despite showing significant degree of similarity to the C4DMs in other organisms. Another potential candidate for C4DM is CYP5122A1 (22A1), an underexplored cytochrome P450-dependent enzyme in Leishmania. To characterize the function of 22A1, its essentiality was investigated in the promastigote and amastigote stages of Leishmania. My data indicated that the L. major chromosomal 22A1 could only be deleted in the presence of episomally expressed 22A1. After strong negative selection, these chromosomal 22A1-null mutants still retained the episomal copy of 22A1 in both promastigote and amastigote stages, demonstrating the essentiality of 22A1 in L. major survival in both stages. Besides, 22A1 overexpressors were resistant to a putative C4DM inhibition drug DB766. Together, 22A1 is a potential therapeutic target in Leishmania. Overall, my study shed new light on the importance of sterol metabolism in Leishmania by elucidating the roles of LSO and 22A1, two underexplored sterol biosynthetic enzymes. In addition, this work critically evaluated LSO and 22A1 as potential new drug targets and provided clues to enhance drug efficacy and prevent resistance.


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Leishmania, Sterols, Lathosterol oxidase, Sterol C4-demethylase, Stress response

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