Sterol methyltransferase is required for optimal mitochondrial function and virulence in Leishmania major

Abstract

Summary Limited knowledge on the exact functions of ergostane-based sterols has hampered the application of sterol synthesis inhibitors against trypanosomatid parasites. Sterol methyltransferase (SMT) is directly involved in the synthesis of parasite specific C24-methylated sterols including ergosterol and 5-dehydroepisterol. While pharmacological studies hint at its potential as a drug target against trypanosomatids, direct evidence for the cellular function and essentiality of SMT is lacking. Here we characterized the SMT knockout mutants and their complemented strains in Leishmania major, the causative agent for cutaneous leishmaniasis. Deletion of SMT alleles led to a complete loss of C24-methylated sterols, which were replaced by cholestane-based sterols. SMT-null mutants were fully viable and replicative in culture but showed increased sensitivity to sphingolipid synthesis inhibition. They were not particularly vulnerable to heat, acidic pH, nitrosative or oxidative stress, yet exhibited high mitochondrial membrane potential and increased superoxide generation indicating altered physiology of the mitochondria. Despite possessing high levels of GPI-anchored glycoconjugates, SMT-null mutants showed significantly attenuated virulence in mice. In total, our study reveals that the biosynthesis of ergostane-based sterols is crucial for the proper function of mitochondria and the proliferation of Leishmania parasites in mammals.

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

“…1B-F), suggesting that LSO is primarily located at the ER. This result is similar to the localizations of C14DM and SMT in Leishmania (18, 19), as well as LSO in T. thermophila (28).…”

“…ergosterol and 5-dehydroepisterol, represented by peaks 1 and 2 in Fig. 2A, respectively (18, 19). Interestingly, all the sterols from lso – promastigotes were shifted to the right in the GC chromatogram including two dominant peaks 1’ and 2’ (Fig.…”

“…Interestingly, both c14dm – and smt – mutants show altered expression of lipophosphoglycan (LPG), a GPI-anchored virulence factor (20). Compared to L. major wild type (WT) parasites, the cellular level of LPG appears to be much lower in c14dm – but higher in smt – mutants (18, 19). These findings suggest that loss-of-function mutations in C14DM and SMT can lead to Amp B resistance but result in significant defects in stress response and virulence.…”

“…To interrogate the roles of these enzymes in L. major , we generated null mutants of C14DM ( c14dm – ) and SMT ( smt – ) using the targeted gene deletion approach (17). Both c14dm – and smt – mutants lack ergostane-based sterols but are viable in culture and highly resistant to Amp B (18, 19). C14dm – mutants are extremely sensitive to heat and highly attenuated in virulence (19).…”

“…They also display altered morphology, cytokinesis defects and increased plasma membrane fluidity (19). By comparison, defects exhibited by smt – mutants, including elevated mitochondrial membrane potential and superoxide level, are less drastic (18). Interestingly, both c14dm – and smt – mutants show altered expression of lipophosphoglycan (LPG), a GPI-anchored virulence factor (20).…”