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

Abstract

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.

Figure 1.. SMT is not required for the viability, replication or differentiation of L. major promastigotes in culture.

(A) Genomic DNA from WT, smt+/− (heterozygotes, three independent clones) and smt⁻ (homozygotes, four independent clones) parasites was analyzed by Southern blot using a radioactive probe recognizing an upstream region of the SMT ORFs (SMT80 + SMT90). Replacements of SMT ORFs with PAC or BSD resistance genes are indicated. (B) Promastigotes (●: WT, ○: smt⁻, ▼: smt⁻/+SMT80) were inoculated in the complete M199 medium at 1.0 × 10⁵ cells/ml and cell densities were determined daily. The arrow indicates the start of stationary phase. During the stationary phase, percentages of dead cells (C) and metacyclics (D) were determined daily. (E) Promastigotes (●: WT, ○: smt⁻, ▼: smt⁻/+SMT80) were inoculated in a FBS-free M199 medium at 2.0 × 10⁵ cells/ml and cell densities were determined daily.

Figure 2.. Depletion of ergostane-based sterols and accumulation of cholestane-based sterols in smt⁻ promastigotes.

(A–E) Partial GC-MS spectra of total sterols extracted from WT (A), smt⁻ (B), smt⁻/+SMT80 (C), smt⁻/+SMT90 (D), and smt⁻/+SMT80&90 (E) promastigotes. The X axis represents retention time (RT) in minutes and the Y axis indicates relative abundance of lipid species. Cholesta-3,5-diene (RT = 11.85–11.87) was added prior to lipid extraction as a standard and marked by arrowheads (⧩). Major sterol species (marked by arrows) include: VI: cholesta-7,24-dienol (RT = 15.00–15.02), VIII: cholesta-5,7,24-trienol (RT = 15.00–15.02), IX: 5-dehydroepisterol (RT = 15.37–15.39), X: ergosterol (RT = 14.75–14.77) and XI: cholesterol (RT = 13.84–13.85). Sterol compositions in WT and SMT-mutants are summarized in (F).

Figure 3.. Smt⁻ mutants show increased resistance to Amp B.

Log phase promastigotes (●: WT, ○: smt⁻, ▼: smt⁻/+SMT80) were inoculated in M199 media containing various concentrations of Amp B (A), ITZ (B) or myriocin (C). Culture densities were measured after 48 hours and percentages of growth were calculated using cells grown in the absence of drugs as controls.

Figure 4.. Smt⁻ mutants contain increased levels of GPI-anchored virulence factors.

In (A)-(B), whole cell lysates or culture supernatants (supe.) from log phase or day 3 stationary phase promastigotes were analyzed by western blot. Lane 1: WT, Lane 2: smt⁻, Lane 3: smt⁻/+SMT80. Expression levels of LPG and GP63 in whole cell lysates were normalized using α-tubulin as the loading control. Relative abundance of LPG (C) and GP63 (D) was determined by averaging three independent experiments.

Figure 5.. Smt⁻ mutants are not hypersensitive to heat stress.

(A–B) Promastigotes (day 1 stationary phase) were incubated at 27 °C (A) or 37 °C/5% CO₂ (B) and cell viability were measured at 0, 18 and 38 hours post inoculation (Black bars: WT, white bars: smt⁻, grey bars: smt⁻/+SMT80). (C) WT or smt⁻ parasites grown in the presence of absence of 0.5 μM of ITZ were incubated at 37 °C/5% CO₂. Cell viability was measured after 8 hours. (D) Log phase promastigotes (black bars: WT; white bars: smt⁻; grey bars : smt⁻/ +SMT80) were incubated with 0.5 μM of TMA-DPH for 20 minutes at 25 °C or 37 °C and the fluorescent depolarization was determined using a spectrofluorometer. Plasma membrane anisotropy was calculated as previously described (Xu et al., 2014).

Figure 6.. Smt⁻ mutants exhibit higher mitochondrial membrane potential and ROS level.

(A) To measure mitochondrial membrane potential, log and stationary phase parasites (day 1–4) were labeled with 100 nM of TMRE for 15 minutes and mean fluorescence intensities (MFI) were determined by flow cytometry. As controls, log phase WT or smt⁻ parasites were pretreated with 75 μM of CCCp prior to TMRE staining. (B) To measure ROS accumulation in mitochondria, log and stationary phase parasites (day 1–4) were treated with 5 μM of MitoSox Red for 25 minutes and MFIs were determined by flow cytometry. Relative mitochondrial ROS levels were plotted in comparison to WT levels. As a control, log phase WT parasites were pretreated with 10 μM of antimycin A prior to MitoSox Red labeling. (C) smt⁻ mutants show lower oxygen consumption. Log phase promastigotes were resuspended in a respiration buffer and oxygen consumption was measured fluorometrically at 90s intervals for 60 minutes (described in Experimental Procedures).

Figure 7.. Smt⁻ mutants display altered mitochondria morphology

(A) Log phase WT, smt⁻ or smt⁻/+SMT80 parasites were stained with 100 nM of Mitotracker CMXRos followed by DNA staining with DAPI. About 200 cells were examined for each parasite line and representative images (70%−90% of all analyzed cells) are shown here. DIC: differential interference contrast. WT parasites pretreated with CCCp were included as controls. (B) Representative transmission electron micrographs of log phase WT, smt⁻ and smt⁻/+SMT80 parasites showing normal kinetoplasts (top panel) and swollen mitochondria in the smt⁻ mutant (lower panel, arrows). Scale bars in B: 500 nm.

Figure 8.. Smt⁻ mutants show attenuated virulence in mice.

BALB/c (A–D) or C57BL6 (E–F) mice were infected in the footpads with metacyclics (A–B, E–F) or lesion-derived amastigotes (C–D). Footpad lesions were recorded weekly in A, C, and E (●: WT, ○: smt⁻, ▼: smt⁻/+SMT80). Parasite numbers in the infected footpads were determined by limiting dilution assay at the indicated days post infection and summarized in B, D, and F (black bars: WT, white bars: smt⁻, grey bars: smt⁻/+SMT80).