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

Sumit Mukherjee; Samrat Moitra; Wei Xu; Veronica Hernandez; Kai Zhang

doi:10.1371/journal.ppat.1008810

. 2020 Aug 20;16(8):e1008810. doi: 10.1371/journal.ppat.1008810

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

Sumit Mukherjee ^1,^¤a, Samrat Moitra ¹, Wei Xu ^1,^¤b, Veronica Hernandez ¹, Kai Zhang ^1,^*

Editor: Michael P Barrett²

PMCID: PMC7462297 PMID: 32817704

Abstract

Sterol 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 summary

Sterols 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.

Introduction

Parasitic protozoa of the genus Leishmania are the causative agents for a group of diseases in humans and animals known as leishmaniasis [1, 2]. During their life cycle, Leishmania parasites alternate between flagellated, extracellular promastigotes in sandfly vectors and non-flagellated, intracellular amastigotes in vertebrates. Current drugs for leishmaniasis are plagued with high toxicity, low efficacy and resistance is on the rise [3]. Therefore, research involving the identification and characterization of metabolic pathways that could provide new drug targets is of considerable value.

While mammalian cells synthesize cholesterol as the main sterol, Leishmania promastigotes primarily make ergostane-based sterols such as ergosterol and 5-dehydroepisterol [4–6]. Ergosterol is distinguished from cholesterol by the presence of an unsaturated side chain with a methyl group at the C-24 position and an additional double bond at C-7 to C-8 in the B ring of the sterol core. Differing from promastigotes, Leishmania amastigotes (which reside in macrophages) mainly salvage cholesterol from the host, while downregulating the de novo ergosterol synthesis (supplemental Figure S14 and S15 in [7]). Despite the low abundance of ergostane-based sterols in amastigotes, sterol synthesis inhibitors and amphotericin B have shown promise as anti-leishmaniasis drugs [5, 8–10], suggesting that the residual amount of endogenous sterols are crucial for amastigotes and/or for the binding of amphotericin B. Among these sterol synthesis inhibitors are azole compounds which primarily target the cytochrome P450 enzyme sterol 14-α-demethylase (C14DM) [5, 11, 12]. Contribution of sterol biosynthesis to Leishmania biology is not fully understood, which hinders the development and improvement of azoles as anti-leishmaniasis drugs.

C14DM was found to be refractory to genetic deletion in Leishmania donovani, the causative agent for visceral leishmaniasis [13]. In contrast, our earlier study showed that this enzyme was dispensable in Leishmania major (responsible for cutaneous leishmaniasis in the old world) [7]. Thus, different Leishmania species may possess distinct requirements for sterol synthesis. Importantly, although L. major C14DM-null mutants (c14dm‾) are viable as promastigotes under normal tissue culture conditions, they show increased plasma membrane fluidity, disruption in lipid raft formation, and deficiency in lipophosphoglycan (LPG, a major surface glycoconjugate and virulence factor [14]) synthesis [7]. These plasma membrane defects probably contribute to the mutants’ hypersensitivity to heat and their severely attenuated virulence in mice, although other factors may be involved as well [7]. A later study on the sterol C-24-methyltransferase indicate that the accumulation of aberrant, C-14-methylated sterol intermediates, not the lack of ergostane-based sterols, is mainly responsible for the fitness loss displayed by c14dm‾ mutants [15].

While sterols are well recognized for their stabilizing effects on the plasma membrane [16, 17], their functions in intracellular organelles are poorly understood, as the end products of sterol synthesis are mainly found on the plasma membrane and secretory vesicles in mammalian cells and yeast [18–20]. In L. major, C14DM is primarily found in the ER but also present in the mitochondria suggesting that this enzyme is needed there [7]. An earlier study reported the presence of substantial amounts of endogenous and exogenous sterols in the mitochondria of Trypanosoma cruzi, a related trypanosomatid protozoan [21]. Application of sterol biosynthesis inhibitors such as ketoconazole and lovastatin altered the ultrastructure of mitochondria in T. cruzi, leading to intense proliferation of the inner-mitochondrial membranes and necrotic cell death [22]. Similarly, RNAi knockdown of ergosterol pathway genes in Trypanosoma brucei resulted in disorganization of mitochondria structure and depletion of ATP [23]. In Leishmania, the lipid composition of mitochondrial membrane has yet to be determined. Nonetheless, structural and functional abnormalities were also observed among other defects following treatment with azole compounds. For example, in Leishmania amazonensis, combinatorial treatment of ketoconazole and terbinafine led to alterations in the distribution and appearance of cristae and the formation of paracrystalline arrays within the mitochondrial matrix [24]. Another study reported the collapse of mitochondrial membrane potential (ΔΨm) following posaconazole treatment in L. amazonensis [25]. Besides azoles, treatment of L. amazonensis and T. cruzi with various azasterols, which inhibit the sterol C-24-methyltransferase, induced a loss of mitochondrial matrix contents [22, 26, 27]. Although the off-target effects from inhibitors cannot be excluded, these observations suggest that sterol synthesis plays important roles in preserving the structure and function of mitochondria in trypanosomatids.

Compared to mammalian cells which usually possess multiple mitochondria per cell, trypanosomatids have only one, relatively large mitochondrion per cell [28]. In addition to energy production, mitochondria in trypanosomatids have been implicated in fatty acid synthesis and redox homeostasis during different stages of their life cycle [29, 30]. Mitochondrial malfunctions, e.g. changes in ΔΨm, disruption of mitochondrial membrane integrity, and increase in reactive oxygen species (ROS) production, are major mediators of cell death under various stress conditions [31–33].

In this study, we sought to address the following important questions. First, how does C14DM inactivation affect the integrity and functions of mitochondria in L. major? Second, besides heat sensitivity, do c14dm‾ mutants exhibit any other stress response defects? Third, how do mitochondrial deficiencies influence the fitness of c14dm‾ parasites? Our results showed that genetic or chemical disruption of C14DM severely compromised the mitochondria in L. major, leading to significant ROS accumulation and extreme vulnerability to biologically relevant stress conditions such as heat and glucose starvation. These findings may guide the development of new treatment strategies that exploit the fitness cost associated with drug resistance.

Results

C14dm‾ promastigotes show increased mitochondrial membrane potential (ΔΨm) and altered mitochondrial morphology

To examine if C14DM deletion affects the mitochondria in L. major, we labeled promastigotes with MitoTracker CMXRos, a fluorogenic dye that binds to mitochondria in a ΔΨm-dependent manner. As indicated in Fig 1A, c14dm‾ mutants showed an intense mitochondrial staining 2–4 times stronger than wild type (WT) and c14dm‾/+C14DM (add-back) parasites. In addition, the mitochondria of c14dm‾ appeared to be more swollen with larger volume and surface area based on transmission electron microscopy and quantitative analysis by fluorescence microscopy (Fig 1B and S1A Fig). To examine whether the mitochondrial membrane in c14dm‾ was compromised, we analyzed the presence of cytochrome c in mitochondrial fraction and cytosolic fraction following extraction with digitonin, with ISCL and HSP83 being used as mitochondrial and cytosolic markers, respectively [34] [35] (S1B Fig). Similar to WT and c14dm‾/+C14DM parasites, cytochrome c in c14dm‾ remained in the mitochondrial enriched fractions when the digitonin extraction was performed at room temperature (S1B Fig). As a control, digitonin treatment at 37 °C disrupted the outer mitochondrial membrane, causing cytochrome c release into the cytosol [36] (S1B Fig). Thus, despite of its effects on ΔΨm and mitochondrial size, genetic ablation of C14DM does not affect mitochondrial membrane integrity at least with regard to cytochrome c localization under ambient conditions.

Fig 1 — (A) Log phase promastigotes were labeled with 100 nM of Mitotracker CMXRos followed by DAPI staining. WT parasites pretreated with CCCp were included as controls. DIC: differential interference contrast. (B) Representative transmission electron micrographs showing mitochondria in log phase promastigotes. Black arrows indicate areas containing kinetoplast (top panel) and tubular mitochondria (bottom panel). (C) To measure ΔΨm, log phase and stationary phase (day 1-day 4) promastigotes were labelled with 100 nM of TMRE for 15 min in PBS and mean fluorescence intensity (MFI) were determined by flow cytometry. Controls include WT and *c14dm*‾ parasites pretreated with CCCp and WT cells pretreated with 0.2 μM of ITZ. Bar values represent averages from three experiments and error bars represent standard deviations. (D) Live parasites imaging after TMRE labeling.

Consistent with the MitoTracker staining results, when live parasites were labeled with another ΔΨm-dependent dye tetramethylrhodamine ethyl ester (TMRE), c14dm‾ mutants retained 2–4 times more fluorescence than WT and c14dm‾/+C14DM cells as indicated by flow cytometry and microscopy (Fig 1C and 1D). The difference between WT and c14dm‾ parasites was more pronounced during the log phase when cells were metabolically active than stationary phase (Fig 1C). Similar results were observed after staining with rhodamine 123 (S2A Fig). As expected, pretreatment with the mitochondrial uncoupler CCCp (75 μM for 15 min) dramatically reduced the incorporation of TMRE and MitoTracker into WT and c14dm‾ parasites (Fig 1A and 1C), without significant effect on cell viability (<5% of cells were propidium iodide positive by flow cytometry). Furthermore, WT promastigotes grown in the presence of itraconazole (ITZ, a C14DM inhibitor) [7] for 48 hours at sublethal concentrations showed similar ΔΨm as c14dm‾ mutants (Fig 1C). Finally, we also examined if extracellular nutrient level affects ΔΨm by labelling promastigotes with TMRE in either complete M199 medium, M199 medium without fetal bovine serum, phosphate-buffered saline (PBS) supplemented with 5.5 mM of glucose (the glucose concentration in M199 medium), or PBS alone (Fig 2A). In these experiments, c14dm‾ mutants exhibited 2.5–4 times higher ΔΨm than WT/add-back parasites and no significant differences were detected between nutrient-replete and nutrient limiting conditions (Fig 2A). Collectively, these observations suggest that C14DM inhibition leads to significantly increased ΔΨm in L. major promastigotes regardless of extracellular nutrient levels.

Fig 2 — (A, B and F) Promastigotes were incubated in complete M199, serum-free M199, PBS contain 5.5 mM of glucose, or PBS only and labeled with 100 nM of TMRE (A, for 15 min), 5 μM of MitoSox Red (B, for 25 min) or 5 μM of DHE (F, for 30 min). MFIs were determined by flow cytometry. Effects of CCCp (75 μM, 15 min) were also monitored in A and B. (C) Log or stationary phase (day 1-day 4) promastigotes were labeled with 5 μM of MitoSox Red for 25 min in PBS and MFIs were determined by flow cytometry and normalized to values from log phase WT. WT cells grown in the presence of ITZ (for 48 hours) were also analyzed. (D and E) Live cell imaging of L. *major* promastigotes (D) or L. *mexicana* axenic amastigotes (grown in the absence or presence of 0.1 μM of ITZ for 48 hours, E) after labelling with MitoSox Red in PBS. White arrows indicate kDNA binding of MitoSox. (E) Live after labeling with MitoSox Red. In **A-C** and F, averaged values from five experiments are shown and error bars represent standard deviations.

Inactivation of C14DM leads to ΔΨm-dependent accumulation of ROS in the mitochondria

In mammalian cells, a high ΔΨm may cause accumulation of ROS in the mitochondria (mainly in the form of superoxide or O₂^•−) in part due to reverse electron flow from complex II to complex I of the electron transport chain (ETC) [37–39]. To assess the ROS level in c14dm‾ mutants, promastigotes were labelled with a mitochondria-specific ROS indicator MitoSox Red which binds to mitochondrial nucleic acid and fluoresces upon oxidation by superoxide [40]. In complete M199, c14dm‾ mutants showed 3.5 times more MitoSox labeling than WT and add-back cells (Fig 2B). Unlike ΔΨm, the mitochondrial ROS level in c14dm‾ was sensitive to nutrient availability (Fig 2B): PBS only (the highest) > PBS with glucose > M199 medium without fetal bovine serum > complete M199 (the lowest). Importantly, pretreatment of c14dm‾ parasites with CCCp significantly reduced MitoSox Red labeling, indicating that the high ΔΨm is mainly responsible for ROS accumulation (Figs 2A, 2B and 1C). These findings suggest that the mitochondria in c14dm‾ mutants are under significant oxidative stress during starvation.

Next we examined the mitochondrial ROS levels in log phase and stationary phase promastigotes after labeling them with MitoSox in PBS alone (Fig 2C). Compared to WT and add-back cells, c14dm‾ mutants contained 10–15 times more mitochondrial ROS by flow cytometry during log phase and early stationary phase, and the difference became less dramatic during late stationary phase when cells ceased proliferation (Fig 2C). Microscopic examination of MitoSox-labelled parasites revealed singular fluorescent spots in every cell representing the kinetoplast DNA, indicating the mitochondrial origin of ROS (Fig 2D). In these images, MitoSox signals from c14dm‾ were much more prominent than those from WT and c14dm‾/+C14DM cells (Fig 2D), which was in agreement with flow cytometry analysis (Fig 2B and 2C). Furthermore, when cultivated in the presence of ITZ (at 0.1–0.2 μM or near EC25 levels), L. major WT promastigotes and L. mexicana axenic amastigotes displayed high levels of MitoSox Red staining similar to c14dm‾ (Fig 2C and 2E), indicating that C14DM inhibition causes ROS built-up in the mitochondria of Leishmania parasites. In comparison to c14dm‾, mitochondrial defects exhibited by the sterol C-24-methyltransferase mutants (smt‾) were less profound (S2 Fig) [15], suggesting that the accumulation of C-14-methylated sterols is mainly responsible for these changes.

We then determined the cytoplasmic ROS level in log phase promastigotes using dihydroethidium (DHE), a cytosolic ROS indicator. As illustrated in Fig 2F, parasites showed high DHE staining under nutrient-limiting conditions. In addition, the amount of cytosolic ROS in c14dm‾ was about two-fold higher than WT and add-back parasites, which was not as dramatic as the increase of mitochondrial ROS (Fig 2B and 2F). As a control, treatment of WT parasites with hydrogen peroxide (100 μM for 10 min) led to significantly increased staining for DHE but not MitoSox Red, confirming the specificity of MitoSox Red for mitochondrial superoxide (S3 Fig). Together, these data suggest that genetic or chemical ablation of C14DM leads to significant accumulation of ROS inside the mitochondria.

Examination of superoxide dismutase (SOD) expression in c14dm‾ mutants

As byproducts of mitochondrial respiration, ROS (mainly the form of O₂^•−) can be generated from the partial reduction of oxygen by complexes I, II, and III of the ETC [41]. To limit the damaging effects of ROS, Leishmania cells utilize several antioxidant systems involving superoxide dismutases (SODs) and tryparedoxin peroxidases [42, 43]. The fact that c14dm‾ parasites accumulate high levels of O₂^•− suggests that either the ROS removal system is defective, or the production of O₂^•− is elevated due to excessive electron leakage from the ETC in these mutants. Here we examined whether a dysregulation of SOD, the primary enzyme to detoxify O₂^•−, is responsible for the accumulation of mitochondrial ROS in c14dm‾. Leishmania encode three iron-dependent SODs: the mitochondrial SODA and two glycosomal SODB (SODB1 and SODB2) [43, 44]. In whole cell lysate, we detected a 14–98% increase in SOD activity from c14dm‾ mutants and the increase was more pronounced during the stationary phase (Fig 3A). Western blot revealed that c14dm‾ mutants had similar levels of SODA (mitochondrial) and SODB (cytosolic) protein as WT parasites (Fig 3B). The increase in SOD activity in late stationary phase c14dm‾ coincided with the decrease of mitochondrial ROS (although still higher than WT and add-back parasites, Fig 2C, S2B Fig and Fig 3A), suggesting an adaptive response from the mutants to upregulate their antioxidant defense machinery. Collectively, these findings indicate that the ROS is accumulated in c14dm‾ mutants despite of near-normal SOD production.

Fig 3 — (A) Whole cell lysates from log phase and stationary phase (day 1-day 3) promastigotes were collected as described in **Materials and Methods**. SOD activity was measured using a SOD assay kit and results were normalized based on the amount of protein in each sample. Error bars represent standard deviations from two independent experiments. (B) Mitochondria enriched membrane fractions (Mem.) and cytosolic fractions (Sup.) were isolated from log phase promastigotes as described. Western blots were performed using antibodies against L. *amazonensis* SODA (mitochondrial) and SODB (both SODB1 and SODB2, glycosomal).

C14dm‾ mutants show reduced cellular respiration and increased glycolytic ATP production

The mitochondrial abnormalities in c14dm‾ prompted us to examine if these mutants could effectively carry on respiration and generate ATP. First, we measured the cellular oxygen consumption rate using the MitoXpress oxygen probe as described previously [45]. When incubated a mitochondrial respiration buffer made of Hanks’ balanced salt solution (HBSS) supplemented with sodium pyruvate and 2-deoxy-D-glucose (to block glycolysis) [46], c14dm‾ parasites showed a significant reduction (42–60%) in oxygen consumption compared to WT cells, and such defect was corrected in the c14dm‾/+C14DM parasites (Fig 4A and 4B). As expected, the addition of antimycin A, a respiratory inhibitor that blocks cytochrome c reductase or complex III [47], decreased oxygen consumption in WT parasites (Fig 4A). A similar experiment was performed when cells were incubated in the Dulbecco’s Modified Eagle’s Medium (DMEM) which contained 25 mM of glucose. While all cells exhibited higher oxygen consumption rates in DMEM than in HBSS, c14dm‾ mutants again used less oxygen than WT and c14dm‾/+C14DM parasites (Fig 4C). Given the considerable ROS accumulation in c14dm‾, the low oxygen consumption rate may protect these mutants from further oxidative damage.

Fig 4 — (**A-C**) Log phase promastigotes were incubated in a mitochondrial respiration buffer (A-B) or DMEM (C) and oxygen consumption rates (OCR) were determined using the MitoXpress probe as described in **Materials and Methods**. Buffer only and buffer with probe (no cells) were included as blanks (A). In A and C, fluorescence signals (in arbitrary units or A.U.) were measured every 90 s. In B, the relative OCR from the experiments in A was calculated by subtracting the 0 min reading from the 15 min reading. WT parasites treated with antimycin A (AA) were included as controls. (D) Cellular ATP contents in log phase promastigotes were measured after 1 hour incubation in an assay buffer (21 mM of HEPES, 0.7 mM of Na₂HPO4, and 137 mM of NaCl, pH 7.2) in the absence or presence of glucose (Glu, 5.5 mM), sodium pyruvate (Pyr, 5.5 mM), 2-deoxy-D-glucose (2DG, 5.5 mM), or sodium azide (NaN₃, 20 mM). All experiments were repeated three or four times and error bars represent standard deviations.

Next, we determined the ATP production from WT, c14dm‾ and c14dm‾/+C14DM parasites. When log phase promastigotes were incubated in an isotonic buffer (without glucose or any inhibitor) for 1 hour, a 30–40% increase in ATP content was observed in c14dm‾ mutants in comparison to WT or add-back parasites (Fig 4D, lane 1). Supplementation with glucose led to increased ATP production in all cells, presumably from both glycolysis and mitochondrial respiration (Fig 4D, lane 4). Again, despite the low oxygen consumption, c14dm‾ mutants produced 30–40% more ATP than WT and add-back parasites from glucose (Fig 4D, lane 4), suggesting that these cells have increased capacity to generate energy through glycolysis. Curiously, addition of sodium pyruvate had only modest effect on ATP production (Fig 4D, lane 7). Although Leishmania parasites possess putative plasma membrane-bound pyruvate transporters [48], the efficiency of pyruvate uptake is not known. Another possibility is that parasites can sense the lack of carbon sources besides pyruvate and regulate ATP biogenesis accordingly. As expected, addition of 2-deoxy-D-glucose (5.5 mM) significantly reduced ATP production in the absence of exogenous glucose (Fig 4D, lane 2 and 8). No significant reduction was detected when cells were incubated in the presence of glucose (5.5 mM), which might be due to inadequate competition on glucose uptake and utilization (Fig 4D, lane 5). Furthermore, addition of sodium azide which inhibits cytochrome c oxidase [46, 49] dramatically lowered ATP production in all cells (Fig 4D, lane 3, 6 and 9), suggesting that the glycolysis flux may be coupled to the efficiency of oxidative phosphorylation respiration in Leishmania so that cells can turn down glycolysis to prevent the buildup of toxic metabolites.

Glucose starvation has deleterious effect on c14dm‾ parasites

Our findings so far suggest that in comparison to WT cells, c14dm‾ mutants depend more on non-respiration pathways such as glycolysis to generate energy. If so, these mutants would be highly sensitive to glucose restriction. To test this possibility, we exposed parasites to glucose-limiting conditions and measured cell survival over time. As illustrated in Fig 5A, when incubated in HBSS, c14dm‾ mutants showed substantial cell death after 12 hours. At 20–24 hours, more than 90% of c14dm‾ cells were dead (Fig 5A and 5B). Similar results were observed when parasites were incubated in other neutral, isotonic buffers such as PBS (S4 Fig). In addition, L. major WT parasites cultivated in the presence of ITZ were highly vulnerable to glucose starvation like c14dm‾ mutants (S5 Fig). In these experiments, WT control, smt‾, smt‾/+SMT and c14dm‾/+C14DM parasites showed very good survival (<9% death after 24 hours), indicating this phenotype is caused by the ablation of C14DM (Fig 5B). Importantly, supplementation with glucose at 5.5 mM (the glucose concentration in M199 medium) completely rescued c14dm‾ mutants, whereas at the same molar concentration, sodium pyruvate (a respiration substrate), 2-deoxy-D-glucose (a non-metabolizable glucose analog), or aspartate (a gluconeogenic amino acid) failed to protect the mutants (Fig 5). Further tests revealed that glucose concentrations above 100 μM were sufficient to rescue c14dm‾ mutants (S4 Fig).

Fig 5 — Log phase promastigotes were inoculated in HBSS in the absence or presence of 2DG (5.5 mM), Pyr (5.5 mM), aspartate (Asp, 5.5 mM), Glu (5.5 mM), or glycerol (0.1 mM). In A, percentages of cell death for *c14dm‾* mutants were determined at the indicated time points. In B, percentages of cell death were determined after 24 hours for WT, *c14dm‾*, *c14dm‾/+C14DM*, *smt‾*, and *smt‾*/*+SMT* parasites. Error bars represent standard deviations from three independent experiments.

Recent studies have revealed that bloodstream forms of Trypanosoma brucei can use glycerol to support growth in the absence of glucose [50, 51]. To test if Leishmania parasites can utilize glycerol effectively, we attempted to rescue c14dm‾ mutants with glycerol. Remarkably, when provided at more than 100 μM, glycerol was able to protect c14dm‾ mutants under glucose-free conditions (Fig 5 and S4 Fig). Both glucose and glycerol were also able to alleviate the mitochondrial defects in c14dm‾ mutants (S6 Fig). Together, our results indicate that inactivation of C14DM leads to hypersensitivity to glucose starvation, which is likely caused by energy depletion and excessive ROS accumulation.

C14dm‾ parasites show increased uptake of glucose and glycerol

Next, we examined whether the uptake of glucose and glycerol is altered in c14dm‾ parasites using radiolabeled substrates as previously described [52, 53]. If respiration defects force c14dm‾ mutants to rely more on glycolysis to generate energy, these parasites may have higher demands for glucose or equivalent carbon sources. Trypanosomatid protozoans including Leishmania species possess a family of glucose transporters [54] and aquaglyceroporins [53] that can mediate nutrient uptake. As shown in Fig 6, c14dm‾ mutants were more efficient than WT and add-back parasites in the uptake of glucose and glycerol. The incorporation of glucose was much more robust at room temperature than at 4 °C (Fig 6A), whereas the incorporation of glycerol was less dependent on temperature (Fig 6B and 6C). The increased uptake of glucose and glycerol in c14dm‾ may serve as a compensatory mechanism to offset deficiencies in mitochondrial respiration.

Fig 6 — Incorporation of radiolabeled glucose (A) or glycerol (B and C) were determined as described under **Materials and Methods**. Transport assays were performed at room temperature (A and B unless specified) or 4 °C (C). Error bars represent standard deviations from three independent experiments.

ROS accumulation is partially responsible for the heat sensitivity in c14dm‾ mutants

We have previously shown that c14dm‾ parasites are extremely sensitive to heat stress (e.g. 37 °C treatment), and their increased plasma membrane fluidity (induced by the accumulation of C-14-methylated sterol intermediates) is a likely cause [7]. To investigate if the accumulation of ROS is also a contributing factor, WT and c14dm‾ parasites were incubated in PBS at either 27 °C (the regular temperature for promastigotes) or 37 °C. After 3 hours, a marked reduction in ΔΨm was detected in 30–35% of c14dm‾ parasites without substantial cell death (Fig 7A and 7B). No significant change in ΔΨm was observed in WT parasites (Fig 7B). To determine if the loss of ΔΨm was associated with compromised mitochondrial membrane integrity, we assessed the release of cytochrome c from mitochondria into the cytosol (Fig 7C and 7D). For WT cells, the majority of cytochrome c was found in the mitochondrial enriched membrane fractions under control (27 °C) and heat stress (37 °C, 3 hours) conditions (Fig 7C and 7D). In contrast, ~40% of cytochrome c was detected in the cytosolic fraction when c14dm‾ parasites were subjected to heat treatment (Fig 7C and 7D). Since 3 hours incubation at 37 °C did not induce substantial cell death (Fig 7A), this finding indicates that the mitochondrial disruption occurred when the majority of c14dm‾ mutants were viable. In addition, we measured the levels of cytosolic ROS in control and heat stressed cells. Unlike WT and add-back parasites which showed no significant change, c14dm‾ mutants displayed ~3-fold increase in cytosolic ROS following 37 °C treatment for 3 hours (Fig 7E). Together, these data suggest that heat stress causes a partial rupture of mitochondrial outer membrane in c14dm‾, leading to the dissemination of cytochrome c and ROS into the cytosol.

Fig 7 — (A-E) Log phase promastigotes were incubated in PBS at 27 °C or 37 °C for 3 hours. In A, percentages of cell death were determined by flow cytometry after PI staining. In B, ΔΨm was determined after TMRE labeling. Unlabeled WT parasites were included as a negative control (Neg). In C-D, parasites were lysed and cytosolic fractions (Sup.) were separated from mitochondrial fractions (Mem.) as described in **Materials and Methods**. Distribution of cytochrome c and HSP83 (a cytosolic protein marker) were determined by Western blots (C) and quantified (D). In E, parasites were labelled with the cytosolic ROS indicator DHE. Fluorescence intensities were measured by flow cytometry and relative cytosolic ROS levels were determined. (F) Log phase promastigotes were incubated at 37 °C in the absence or presence of reduced GSH. After 8 hours, mitochondrial ROS levels were measured as described. (G) *C14dm‾* parasites were incubated at 37 °C in the absence or presence of reduced GSH and percentages of cell death were determined at 0 and 8 hours. (H) WT parasites were incubated at 27 °C or 37 °C in the absence or presence of AA (10 μM) and percentages of dead cells were determined by flow cytometry at the indicated time points. †: Very few WT+AA cells were detectable after 36 hours at 37 °C. Error bars represent standard deviations from three independent experiments.

To further evaluate the contribution of ROS accumulation to the observed heat response phenotype, we treated c14dm‾ mutants with glutathione (GSH, an antioxidant) before exposing them to 37 °C. When administered at 5 mM, GSH treatment of c14dm‾ reduced the mitochondrial ROS by ~60% and the heat-induced cell death in 8 hours by ~50% (Fig 7F and 7G). The effect was much less prominent when GSH was provided at 1 mM (Fig 7F and 7G), indicating a dosage-dependent alleviation of ROS stress. Additionally, we tested the effect of antimycin A, an oxidative stress inducer (S3A Fig), on the heat sensitivity of WT parasites. At 27 °C, 10 μM of antimycin A had very mild effect on cell survival (<10% death after 36 hours) but at 37 °C, more than 60% of WT cells died after 8 hours (Fig 7H). From these studies, we conclude that in addition to plasma membrane defects, oxidative stress also makes c14dm‾ mutants vulnerable to heat.

C14dm‾ mutants exhibit increased sensitivity to pentamidine

Finally, to probe whether the mitochondrial defects in c14dm‾ can be exploited for developing better chemotherapy, we tested the susceptibility of these parasites to pentamidine, an antimicrobial agent with efficacy against trypanosomatids [55]. While the mode of action for pentamidine is not completely understood, previous reports suggest that this drug needs to be sequestered into the mitochondria of Leishmania in a ΔΨm-dependent manner to exert its activity [55, 56]. Thus, the elevated ΔΨm may make c14dm‾ more vulnerable to pentamidine. Indeed, in a 48 hours drug response assay [15], c14dm‾ mutants showed heightened growth inhibition by pentamidine, as their EC90, EC50 and EC25 (effective concentrations required to inhibit growth by 90%, 50% and 25% respectively) were 5–15 times lower than those of WT and add-back parasites (Fig 8A). WT cells grown in the presence of ITZ were also hypersensitive to pentamidine, suggesting that these two drugs can work synergistically (Fig 8A). In addition, we examined whether short time exposure to pentamidine can cause death or irreversible damage to c14dm‾ mutants by treating parasites with 2.5 μM or 25 μM of pentamidine for one hour, followed by two washes with PBS and then let cells grow in drug-free M199 media [57]. As shown in Fig 8B, exposure to 2.5 μM of pentamidine for one hour led to significant grow delay (2–3 days) in c14dm‾ and these mutants could only grow to 5–9 x 10⁶ cells/ml in six days (the maximal density for L. major promastigotes in complete M199 is ~3 x 10⁷ cells/ml). In contrast, for WT and add-back parasites, 2.5 μM of pentamidine had little impact and it took 25 μM of pentamidine to cause a similar degree of inhibition (Fig 8B and 8C). Meanwhile, 25 μM of pentamidine was sufficient to halt the growth of c14dm‾ nearly completely after the one-hour exposure (Fig 8C). CCCp treatment was able to rescue the mutants in this wash out experiment (Fig 8B and 8C), suggesting that the high ΔΨm and/or mitochondrial ROS are responsible the hypersensitivity of c14dm‾ to pentamidine.

Fig 8 — (A) Log phase promastigotes were inoculated in M199 media containing various concentrations of PEN. Culture densities were determined after 48 hours and the PEN concentrations required to inhibit growth by 90%, 50%, and 25% were determined (as EC90, EC50 and EC25 respectively) using cells grown in the absence of PEN as controls. “WT + ITZ” represent WT parasites grown in the presence of 0.5 μM of ITZ. (B-C) Log phase promastigotes were inoculated in M199 media containing 2.5 μM (B) or 25 μM (C) of PEN for one hour, followed two washes with PBS. Cells were then re-inoculated in M199 media and culture densities were determined every 24 hours. Some *c14dm¯* mutants were also incubated in the presence of 75 μM of CCCp along with PEN (for one hour). Parasites originally inoculated in the absence of PEN were included as controls. Error bars represent standard deviations from three or four independent experiments.

Discussion

Our previous work has revealed significant plasma membrane defects in c14dm‾ mutants including increased membrane fluidity and failure to maintain detergent resistant membrane fractions [7]. While predominantly located in the plasma membrane, sterols are also present in substantial amounts in lipid droplets and intracellular organelle membranes [20, 58]. A more thorough characterization of the c14dm‾ mutants would reveal new insight into the roles of sterol metabolism in Leishmania and provide new leads to exploit the vulnerability caused by the accumulation of abnormal sterols. In this study, we found a 2–5 fold increase in the ΔΨm of c14dm‾ relative to WT and add-back parasites and similar results were observed in ITZ-treated WT parasites (Fig 1 and S2 Fig). We also detected a modest swelling of mitochondria in c14dm‾ mutants (Fig 1B and S1A Fig). These findings are consistent with the previously reported hyperpolarization of mitochondrial membrane in Leishmania mexicana following treatment with triazole compounds [59].

In mammalian cells, elevated ΔΨm may cause electron flow in the reverse direction through the ETC leading to electron leakage and superoxide accumulation in the mitochondrial matrix [37, 39]. By flow cytometry and fluorescence microscopy, we observed significantly higher levels of superoxide in the mitochondria of c14dm‾ mutants and ITZ-treated WT parasites in comparison to control cells (Fig 2 and S2 and S3 Figs). Under the nutrient-replete condition, a 3-5-fold increase was detected in c14dm‾ mutants. Under the nutrient-limiting condition, the difference became 10-12-fold (Fig 2B). Meanwhile, the cytosolic ROS level in c14dm‾ mutants was only about 2-fold higher than WT and add-back parasites, suggesting that the majority of accumulated ROS was sequestered in the mitochondria under ambient temperature (Fig 2F and S3 Fig). In agreement with this notion, the mitochondrial membrane integrity in c14dm‾ appeared to be intact based on the intracellular distribution of cytochrome c (S1B Fig). It is not clear why inactivation of C14DM leads to elevated levels of ΔΨm and mitochondrial ROS. The expression and activity of SODs were not compromised in c14dm‾ mutants (Fig 3), suggesting that ROS production was higher in the absence of C14DM. In mammalian cells and yeast, mitochondria can synthesize phosphatidylglycerol (PG), cardiolipin (CL), phosphatidic acid, and a portion of phosphatidylethanolamine (PE, through the decarboxylation of phosphatidylserine) [19, 60, 61]. Other lipids including phosphatidylcholine, sphingolipids and sterols have to be imported. Changes in mitochondrial lipidome, especially the abundance of CL and PE, can lead to ETC defects [62, 63]. It is therefore possible that defects in sterol synthesis at the ER alter the lipid composition of mitochondrial membrane in c14dm‾ mutants. Such membrane modification may negatively affect the functions of ETC including the terminal F0-F1 complex, leading to the accumulation of proton motive force (ΔΨm), leakage of electron and partial reduction of oxygen.

In comparison to WT and add-back parasites, c14dm‾ mutants showed significant impairment in oxygen consumption yet produced higher than WT-level of ATP in a glucose-free buffer (Fig 4). Similarly, the cellular ATP content in c14dm‾ mutants was 30–40% higher than WT and add-back parasites when cells were incubated in the presence of glucose (Fig 4). These results suggest that without C14DM, Leishmania parasites downregulate respiration and rely more on glycolysis to generate energy. The mechanism of this energetics switch remains to be determined. In many eukaryotes, the heterotrimeric AMP-activated protein kinase (AMPK) complex is a key regulator of cellular energy metabolism [64–66]. Future investigation will examine if AMPK activation in response to C14DM ablation is responsible for reduced respiration and increased glucose consumption via glycolysis.

Consistent with the increased dependency on glucose as an energy source, c14dm‾ mutants were extremely sensitive to glucose starvation (Fig 5). This hypersensitivity may arise from the lack of energy production after glucose is depleted via glycolysis and the rapid increase in ROS due to respiration deficiency (Figs 2 and 5). An alternative and not mutually exclusive explanation is that the accumulation of aberrant sterol intermediates in c14dm‾ mutants compromised the progression of autophagy, a possibility currently under investigation. Interestingly, supplementation of either glucose or glycerol at concentrations above 100 μM completely rescued the survival of c14dm‾ mutants (Fig 5 and S4 Fig). While glycerol could be converted into glucose, it is unlikely to account for the rescue as the energy required for gluconeogenesis exceeds that generated from glycolysis. Instead, it is possible that Leishmania parasites metabolize glycerol into glycerol-3-phosphate (by glycerol kinase) and then dihydroxyacetone phosphate (by the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase) which can be incorporated into the glycolysis pathway, as described in T. brucei [50, 51]. We also observed increased uptake of glucose and glycerol by c14dm‾ mutants (Fig 6), a possible adaptive response to their high demand for carbon sources. Future research will focus on effects on sterol synthesis on the permeability of plasma membrane to probe the mechanism of this phenotype.

Next, we examined the involvement of mitochondrial defects in the previously reported heat sensitivity exhibited by c14dm‾ mutants. Despite the high mitochondrial ROS, c14dm‾ parasites were fully viable at ambient temperature in the presence of glucose or glycerol. However, within 3 hours of exposure at 37 °C, the ΔΨm in c14dm‾ mutants showed signs of collapse, along with a significant increase in cytosolic ROS level and the release of cytochrome c to the cytosol (Fig 7). One possibility is that similar to the effect on plasma membrane, the accumulation of aberrant sterol intermediates compromises the mitochondrial membrane integrity at high temperature in c14dm‾ parasites. Although the presence of a canonical apoptotic pathway in Leishmania is debatable, earlier studies have suggested the involvement of mitochondrial contents such as cytochrome c in causing cell death [32, 67]. In addition, treatment with antioxidant GSH could partially rescue the heat induced cell death c14dm‾ mutants, and the ROS generating agent antimycin A made WT L. major vulnerable to heat (Fig 7). Together, these findings suggest that ROS accumulation is partially responsible for the heat sensitivity in c14dm‾ parasites.

Finally, we also observed heightened sensitivity of c14dm‾ to pentamidine, in both the 48-hour growth inhibition assay and the one-hour exposure assay (Fig 8). Previous studies on the amphotericin B resistant lines in L. mexicana have identified mutations in sterol biosynthesis and hypersensitivity to pentamidine to be a common feature among these parasites [68, 69]. Our results here are in line with those previous reports as c14dm‾ mutants are highly resistant to amphotericin B due to their lack of ergostane-based sterols [7]. It is possible that the elevated ΔΨm and/or mitochondrial ROS cause accumulation of pentamidine in the mitochondria of c14dm‾, making them more susceptible to this drug than WT parasites [55, 56].

In summary, we have shown that deletion or inhibition of C14DM leads to significant alterations in the mitochondria highlighted by increases in ΔΨm and ROS levels, along with deficiency in respiration. These defects make c14dm‾ extremely vulnerable to glucose restriction and contribute to their hypersensitivity to heat. Thus, in addition to its stabilizing effects on the plasma membrane, sterol synthesis plays important roles in the regulation of cellular energetics, redox homeostasis and parasite fitness under stress. Analysis of the dynamics of ER-mitochondria transport, the mitochondrial lipid composition, and the spatial distributions and activities of ETC complexes in c14dm‾ would shed light on the mechanistic aspect of how endogenous sterols influence mitochondria in Leishmania. Furthermore, the hypersensitivity of c14dm‾ to pentamidine raises the possibility of using azole-pentamidine or amphotericin B-pentamidine combinations against leishmaniasis. Future studies may also explore the application of glycolysis inhibitors or ROS inducers in conjugation with sterol biosynthesis inhibitors.

Materials and methods

Materials

Tetramethylrhodamine ethyl ester (TMRE), rhodamine 123, Mitotracker CMXRos, MitoSox Red, and dihydroethidium (DHE) were purchased from Thermo Fisher Scientific. Itraconazole (ITZ) was purchased from LKT Laboratories. Antimycin A was from ENZO Life Sciences. The MitoXpress oxygen consumption assay kit was purchased from Luxcel Biosciences. For ATP analysis, the CellTiter-Glo luminescent assay kit was purchased from Promega. All other chemicals were purchased from VWR International or Sigma Aldrich Inc. unless otherwise specified.

Leishmania culture

L. major LV39 clone 5 WT (Rho/SU/59/P), c14dm‾ (C14DM-null mutant), smt‾, c14dm‾/+C14DM (episomal add-back), and smt‾/+SMT80 promastigotes were cultivated at 27 °C in complete M199 media (with 10% fetal bovine serum and additional supplements, pH 7.4) [70]. Leishmania mexicana (MNYC/BZ/62/M379) axenic amastigotes were cultured using an amastigote medium based on the Drosophila Schneider’s medium supplemented with 20% fetal bovine serum and 0.0015% hemin (pH 5.5) in vented flasks in a humidified 32 °C/5% CO₂ incubator [71]. Culture density and cell viability were determined by hemacytometer counting and flow cytometry after propidium iodide (PI) staining, respectively as previously described [72]. In this study, log phase promastigotes referred to replicative parasites at 2.0–6.0 x 10⁶ cells/ml, and stationary phase promastigotes referred to non-replicative parasites at densities higher than 2.0 x 10⁷ cells/ml.

Determination of mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level

ΔΨm was determined by flow cytometry after staining with TMRE or rhodamine 123 as previously described [73]. Briefly, promastigotes were resuspended in complete M199, serum-free M199, phosphate buffered saline (PBS, pH 7.2) containing 5.5 mM of glucose, or PBS alone at 2.0 x 10⁶ cells/ml; TMRE was added to a final concentration of 100 nM; after incubation at 27 °C for 15 min, the mean fluorescence intensity (MFI) was determined using an Attune NxT Flow Cytometer. Control samples include WT and c14dm‾ parasites pretreated with 75 μM of carbonyl cyanide m-chlorophenyl hydrazone (CCCp) for 15 min and WT parasites grown in the presence of 0.5 μM of ITZ for 48 hours.

Mitochondrial superoxide accumulation was determined as described previously with minor modifications [33]. Promastigotes were resuspended in complete M199, serum-free M199, PBS containing 5.5 mM of glucose, or PBS alone at 1.0 x 10⁷ cells/ml and labeled with 5 μM of MitoSOX red at 27 °C. After 25 min, MFI was measured by flow cytometry as described above. To examine the level of cytosolic ROS, parasites were labeled with 5 μM of DHE for 30 min at 27 °C followed by flow cytometry analysis. Parasites pretreated with 5 μM of antimycin A, 75 μM of CCCp, or 100 μM of H₂O₂ for 15 min were used in these assays as controls. Such treatments did not affect parasite viability based on PI-staining (<5% PI positive). C14dm‾ mutants grown in the presence of 1 mM or 5 mM of glutathione (GSH) for 48 hours were also included. In all flow cytometry experiments, 20,000 events were acquired for each sample.

Determination of superoxide dismutase (SOD) activity

SOD activity in whole cell extracts was measured as described previously for L. amazonensis [74]. Briefly, 2 x 10⁸ promastigotes were harvested, washed twice with PBS and resuspended in a hypotonic buffer (5 mM of Tris–HCl, 0.1 mM of EDTA, and 1 x complete EDTA-free protease inhibitor cocktail, pH 7.8) at a final concentration of 5 x 10⁸ cells/ml. To prepare lysates, cells were subjected to three freeze-thaw cycles alternating between liquid nitrogen and a 37 °C water bath. Cell lysis was confirmed by microscopy. Lysates were clarified by centrifugation (12,000 g for 30 min at 4 °C) and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). SOD activity in whole cell extracts was measured using the EnzyChrome superoxide dismutase assay kit (BioAssay Systems) according to the manufacturer’s protocol.

Digitonin fractionation and Western blot

Crude mitochondria-enriched fractions were generated by digitonin fractionation as described previously [75]. Briefly, log phase promastigotes were incubated for 10 min on ice in 20 mM of Tris-HCl (pH 7.5), 0.6 M of sorbitol, 2 mM of EDTA and 0.025% (w/v) of digitonin at 1 x 10⁸ cells/ml at room temperature. As a positive control for mitochondrial membrane disruption, WT parasites were treated with 0.035% (w/v) of digitonin at 37 °C. After centrifugation (8000 g, for 30 min at 4 °C), the mitochondria-enriched pellet was separated from the supernatant (enriched for cytosolic proteins). Fractions of equal cell equivalents were subjected to SDS-PAGE and immunoblotting. Primary antibodies included rabbit-anti-T. brucei cytochrome c (1:500 dilution) [32], rabbit-anti-L. amazonensis SODA and SODB (1:500), rabbit-anti-L. major ISCL (1:500) [34], and rabbit-anti-Leishmania HSP83 (1:5000) antisera [7]. The goat-anti-rabbit IgG conjugated with HRP (1:5000) was used as secondary antibody.

Measurement of oxygen consumption and ATP production

Oxygen consumption was determined using the MitoXpress Intracellular Oxygen Assay kit (Luxcel Biosciences, Cork, Ireland) as described previously [45]. Briefly, log phase parasites were washed once with PBS and resuspended in DMEM or a mitochondrial respiration buffer (5.5 mM of sodium pyruvate, 5.5 mM of 2-deoxy-D-glucose in HBSS, pH 7.2) at 2.0 x 10⁷ cells/ ml. For each sample, 100 μl of parasite suspension (corresponding to 2.0 x 10⁶ cells) was added in triplicates to a 96 black-well microtiter plate. WT parasites pre-treated with 10 μM of antimycin A or 75 μM of CCCp were used as controls. 10 μl of MitoXpress (1 μM) was added to each well. A layer of mineral oil (100 μl/well) was added over the cells to prevent diffusion of atmospheric oxygen. MitoXpress signals were measured at 90 s intervals for 25–60 min using excitation/emission wavelengths of 380 nm/650 nm in a BioTek synergy 4 fluorescence microplate reader. For experiments in DMEM, we could not continue beyond 25 min when the readings from WT cells reached the saturation point. Wells containing buffer only and buffer + MitoXpress were included as blanks. For each cell line, the relative OCR was calculated by subtracting the 0 min reading from the 15 min reading.

To measure ATP production, log phase parasites were washed once with PBS and resuspended in an assay buffer containing 21 mM of HEPES, 0.7 mM of Na₂HPO4, and 137 mM of NaCl (pH 7.2) in the presence or absence of glucose (5.5 mM), sodium pyruvate (5.5 mM), 2-deoxy-D-glucose (5.5 mM) or sodium azide (20 mM) at 4.0 x 10⁶ cells/ml. After incubation at 27 °C for one hour, 25 μl of suspension (corresponding to 1.0 x 10⁵ cells) were transferred to a 96 black-well plate, mixed with equal volume of the CellTiter-Glo reagent and incubated for 10 min in the dark. Bioluminescence was measured using a DTX880 microplate reader as described [23]. For each experiment, an ATP standard curve was prepared by diluting pure ATP stock in the assay buffer.

Microscopy

To stain mitochondria, live parasites were resuspended in DMEM at 1.0 × 10⁶ cells/ml and labeled with 100 nM of Mitotracker CMXRos or TMRE. After incubation at 27 °C (30 min for Mitotracker and 15 min for TRME), parasites were washed once with PBS, fixed and processed for fluorescence microscopy as previously described [15]. MitoSox Red labeling (5 μM for 25 min at 27 °C) of live parasites was performed similarly without fixation using an Olympus BX51 Upright Fluorescence Microscope. DNA was stained with DAPI (2.5 μg/ml). Transmission electron microscopy of log and stationary phase promastigotes was performed as previously described [76].

Starvation and heat stress assays

To measure heat sensitivity, log phase promastigotes were incubated in complete M199 media for 0–24 h at either 27 °C or 37 °C/5% CO₂. Effects of antimycin A (10 μM) and GSH (1 mM or 5 mM) on heat sensitivity were also monitored. For starvation response, log phase promastigotes were washed once with HBSS and resuspended in HBSS supplemented with glucose (5.5 mM), 2-deoxy-D-glucose (5.5 mM), aspartate (5.5 mM), sodium pyruvate (5.5 mM), or glycerol (0.1 mM) at 2.5 x 10⁶ cells/ml and incubated for 0–24 h at 27 °C. In these stress assays, culture densities and percentages of dead cells were determined over time as previously described [72].

Glucose and glycerol uptake assays

Uptake of glucose and glycerol were monitored as previously described with minor modifications [53, 77]. For glucose incorporation, log phase promastigotes were washed twice with PBS and resuspended at 5.0 x 10⁷ cells/ml in PBS in the presence of 100 μM of D-[U-¹⁴C] glucose (250 mCi/mmol, PerkinElmer Inc.). The reaction mixture (200 μl each in triplicates) was immediately loaded onto 100 μl of dibutyl phthalate in 1.5 ml Eppendorf tubes. Incubation was performed at room temperature and terminated at the indicated times by pelleting cells through the oil layer through centrifugation (16,000 g, 1 min). Tubes were then flash frozen with liquid nitrogen and the bottoms (containing cell pellets) were clipped off and submerged in scintillation fluid containing 1% SDS for 24 h, followed by scintillation counting. To measure glycerol uptake, log phase promastigotes were washed twice in a transport buffer (33 mM of HEPES, 98 mM of NaCl, 4.6 mM of KCl, 0.55 mM of CaCl₂, 0.07 mM of MgSO₄, 5.8 mM of Na₂PO4, 0.3 mM of NaHCO₃, and 14 mM of glucose, pH 7.3) and resuspended at 1.0 x 10⁸ cells/ml in the same buffer. Uptake was measured at room temperature by mixing 100 μl of cells with 100 μl of transport buffer containing 8 μM of [¹⁴C] glycerol (0.125 μCi each in triplicates, PerkinElmer Inc.). Incorporation of radioactivity was determined as described above.

Susceptibility to pentamidine

To determine the effective concentration of pentamidine to inhibit parasite growth by 90%, 50% and 25% (EC90, EC50 and EC25), log phase promastigotes were inoculated in complete M199 media at 2.0 × 10⁵ cells/ml in 0–25 μM of pentamidine. Culture densities were measured after 48 hours of incubation in 24-well plates. EC90, EC50 and EC25 were determined using cells grown in the absence of pentamidine as controls. To assess the leishmanicidal activity of pentamidine in short time exposure, log phase promastigotes were inoculated in complete M199 media containing 0, 2.5 μM, or 25 μM of pentamidine. For c14dm‾, half of them were also treated with 75 μM of CCCp. After one hour, cells were washed twice with PBS and re-inoculated in complete M199 media at 2.0 × 10⁵ cells/ml. Culture densities were then determined daily for six days.

Statistical analysis

Unless specified otherwise, assay values in all figures are averaged from three to five independent repeats and error bars represent standard deviations. Differences were assessed by the Student’s t test (for two groups) or one-way ANOVA (for three or more groups) using the Sigmaplot 13.0 software (Systat Software Inc, San Jose, CA). P values indicating statistical significance were grouped in all figures (***: p < 0.001, **: p < 0.01, *: p < 0.05).

Supporting information

S1 Fig. C14dm‾ promastigotes show increased mitochondrial surface area but maintain mitochondrial membrane integrity at ambient temperature.

(A) After Mitotracker staining (Fig 1A), the average mitochondrial surface areas in log phase promastigotes of WT, c14dm¯, and c14dm¯/+C14DM were determined using Image J (~200 cells were analyzed for each parasite line, au: arbitrary unit). (B) Log phase promastigotes were lysed with digitonin at 37 °C or room temperature (RT) and mitochondria enriched membrane fractions (Mem.) were separated from cytosolic fractions (Sup.) as described in Materials and Methods. Western blots were performed using antibodies against cytochrome c, ISCL (a mitochondrial membrane protein), and HSP83 (a cytosolic protein).

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Click here for additional data file.^{(208.4KB, pdf)}

S2 Fig. C14dm‾ and smt‾ mutants show increased ΔΨm and mitochondrial ROS.

Log phase and stationary phase (day 1-day 4) promastigotes were resuspended in PBS and labeled with 5 μg/ml of rhodamine 123 for 15 min (A) or with 5 μM of MitoSox Red for 25 min (B) at room temperature. MFIs were determined by flow cytometry. Error bars represent standard deviations from three independent experiments.

(PDF)

Click here for additional data file.^{(94.6KB, pdf)}

S3 Fig. C14dm‾ mutants accumulate ROS in the mitochondria.

Log phase promastigotes were labeled with 5 μM of MitoSox Red for 25 min (A) or 5 μM of DHE for 30 min (B) and MFIs were determined by flow cytometry. Effects of AA (5 μM) and H₂O₂ (100 μM) on WT parasites were also monitored. Error bars represent standard deviations from three experiments.

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Click here for additional data file.^{(285.7KB, pdf)}

S4 Fig. 100 μM of glucose or glycerol is sufficient to rescue c14dm‾ mutants in PBS.

Log phase promastigotes were incubated in PBS in the absence or presence of glucose (100 μM) or glycerol (100 μM) and percentages of dead cells were determined by flow cytometry at the indicated time points. Error bars represent standard deviations from three experiments.

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Click here for additional data file.^{(12.7KB, pdf)}

S5 Fig. ITZ-treated WT parasites are hypersensitive to glucose depletion.

L. major WT promastigotes were cultivated in solvent alone (0.1% w/v of DMSO) or 0.2 μM of ITZ for 48 hours. Cells were then incubated in HBSS and percentages of dead cells were determined by flow cytometry at the indicated time points. Error bars represent standard deviations from three experiments.

(PDF)

Click here for additional data file.^{(5.9KB, pdf)}

S6 Fig. C14dm‾ mutants rescued by glucose or glycerol show less severe mitochondrial defects.

Log phase promastigotes were incubated in PBS in the absence or presence of glucose (100 μM) or glycerol (100 μM) for 24 hours. ΔΨm (A) and mitochondrial ROS level (B) were determined by flow cytometry. Control cells represent cells analyzed at the beginning of incubation. †: no viable c14dm‾ mutants were available after 24 hours. Error bars represent standard deviations from three experiments.

(PDF)

Click here for additional data file.^{(262.9KB, pdf)}

Acknowledgments

We thank Dr. Norma Andrews (University of Maryland College Park) for providing us the rabbit anti-L. amazonensis SODA and SODB antisera, Dr. André Schneider (University of Bern) for the kind gift of rabbit anti-T. brucei cytochrome c antibody, Dr. Wandy Beatty (Washington University in St. Louis) for transmission electron microscopy, and Dr. Catherine Wakeman (Texas Tech University) for usage on the BioTek synergy 4 fluorescence microplate reader.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by a grant from the National Institutes of Health (AI099380 for KZ). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1008810.r001

Decision Letter 0

David Horn, Michael P Barrett

4 Mar 2020

Dear Dr Zhang,

Thank you very much for submitting your manuscript "Sterol 14alpha-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

All referees considered this to be a solid piece of experimental work. However, views on acceptability for publication in Plos Pathogens were divided. Reviewer 1, in particular, felt that the gain in knowledge was incremental given that others have already identified roles of sterols in mitochondrial membrane composition and the current work is an additional piece of phenotypic analysis of the mutant line first published in Plos Pathogens in 2014. However, as I have an interest in this field, I was particularly taken with the observation that the C14DM mutant has a substantially increased mitochondrial membrane potential. We have developed a series of amphotericin B resistant lines (L. mexicana) (PMID: 30716073 & PMID: 28622334). They have mutations in different sterol pathway enzymes, converging on a loss of ergostane type sterols. We find hypersenstivity to pentamidine a common feature in these parasites (and others unpublished). Since pentamidine's activity has some dependence on mitochondrial membrane potential and accumulation into that organelle (PMID: 12435669), the findings here offer a potentially satisfying explanation to this observation. You have previously shown the mutant is resistant to Amphotericin B. It would be useful to include data here on sensitivity to pentamidine to which they are likely to be hypersensitive). This would be a simple experiment.

It would be further interesting to see if you could work out how long parasites need to be exposed to pentamidine (in wash out experiments) before they commit to death. If, as is true for African trypanosomes, fairly short exposures are needed (does dependent) it would be possible to set up experiments to see if transiently diminishing mitochondrial potential with CCCP could alter time dependence of pentamidine exposure to commit to death. Similar work was done with T. brucei as a possible marker on how to design experiments (PMID: 8103625).

If you can place your work in a more translational context of chemotherapeutic relevance I feel the work's significance will be greatly enhanced.

A few additional comments I'd make, in addition to those of the other reviewers:

Line 14 - mitochondrial not mitochondria

Line 68 - can a comment on amphotericin B activity in amastigotes be included given their apparent low level of ergostanes?

Line 109 "quite unique" is not really correct, either in the meaning of the word "unique" not in biological relevance. Lots of organisms have a single mitochondrion, so just drop the "quite unique"

Line 140: add "at least with regard to cytochrome c localisation between "integrity" and "under" (other aspects of the membrane's integrity weren't looked at)

Line 327: also in considering more pharmacological relevance in a revised manuscript - could triazole/pentamidine combinations be considered (assuming you find pentamidine hypersensitivity)

REFERENCES: Please check the format of each against Journal requirements. Note that reference 2. uses abbreviated journal title name, others use whole title, so check for consistency.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael P. Barrett, PhD

Guest Editor

PLOS Pathogens

David Horn

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

All referees considered this to be a solid piece of experimental work. However, views on acceptability for publication in Plos Pathogens were divided. Reviewer 1, in particular, felt that the gain in knowledge was incremental given that others have already identified roles of sterols in mitochondrial membrane composition and the current work is an additional piece of phenotypic analysis of the mutant line first published in Plos Pathogens in 2014. However, as I have an interest in this field I was particularly taken with the observation that the C14DM mutant has a substantially increased mitochondrial membrane potential. We have developed a series of amphotericin B resistant lines (L. mexicana) (PMID: 30716073 & PMID: 28622334). They have mutations in different sterol pathway enzymes, converging on a loss of ergostane type sterols. We find hypersenstivity to pentamidine a common feature in these parasites (and others unpublished). Since pentamidine's activity has some dependence on mitochondrial membrane potential and accumulation into that organelle (PMID: 12435669), the findings here offer a potentially satisfying explanation to this observation. You have previously shown the mutant is resistant to Amphotericin B. It would be useful to include data here on sensitivity to pentamidine to which they are likely to be hypersensitive). This would be a simple experiment.

If you can place your work in a more translational context of chemotherapeutic relevance I feel the work's significance will be greatly enhanced.

A few additional comments I'd make, in addition to those of the other reviewers:

Line 14 - mitochondrial not mitochondria

Line 68 - can a comment on amphotericin B activity in amastigotes be included given their apparent low level of ergostanes?

Line 109 "quite unique" is not really corerct, either in the meaning of the word "unique" not in biological relevance. Lots of organisms have a single mitochondrion, so just drop the "quite unique"

Line 140: add "at least with regard to cytochrome c localisation between "integrity" and "under" (other aspects of the membrane's integrity weren't looked at)

Line 327: also in considering more pharmacological relevance in a revised manuscript - could triazole/pentamidine combinations be considered (assuming you find pentamidine hypersensitivity)

REFERENCES: Please check the format of each against Journal requirements. Note that reference 2. uses abbreviated journal title name, others use whole title, so check for consistency.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This article by Mukherjee et al. investigates the role of the sterol biosynthetic enzyme C14-�-demethylase (C14DM) in mitochondrial function in promastigotes of Leishmania major. This enzyme is of interest, because it is the target of azole compounds that have efficacy as antifungals and have been investigated as potential anti-leishmanial drugs. In previous studies, the authors have generated the c14dm- null mutant and shown that plasma membrane fluidity is decreased in this mutant, that it is susceptible to increased temperature, and that its virulence in mice is greatly reduced. Of interest, these effects are probably due to accumulation of C14-methylated sterol precursors when the demethylase is knocked out or inhibited rather than to the absence of the mature sterol. The current study focuses on the effects of the c14dm- mutation, and of treatment with the C14DM inhibitor itraconazole (ITZ), on the mitochondrion. Although most sterol end products in eukaryotic cells (e.g., cholesterol or ergosterol) are present in the plasma membrane or vesicles that traffic to the PM, sterols are also present in organellar membranes including the mitochondrion, albeit at lower levels.

Indeed, the authors find major alterations in promastigote mitochondria in the c14dm- mutant and in parasites treated with ITZ. Mitochondria increase in size, the mitochondrial membrane potential is significantly increased, and levels of reactive oxygen species (ROS) increase by 10-15-fold in the mitochondrion, probably due to leakage of electrons from the mitochondrial electron transport chain due to alterations in the inner mitochondrial membrane. Furthermore, oxygen consumption is reduced in the c14dm- mutants, and the parasites appear to switch to greater reliance upon glycolysis for generation of cellular ATP. The promastigotes thus become highly sensitive to glucose restriction, but supplementation of the medium with either glucose or glycerol protects the parasites against loss of viability. Furthermore, shifting temperature from 27° to 37° also induces mitochondrial alterations in these mutants, including reduced membrane potential, leakage of cytochrome oxidase out of the mitochondrion, and increased cytosolic ROS, leading to cell death. Overall, these studies implicate a critical role for sterols in maintenance of mitochondrial function in these parasites and imply that azoles may function, at least in part, by impairing mitochondrial function.

This is a well-executed study that employs a variety of assays to assess mitochondrial function in wild type, c14dm- mutant, and ITZ-treated promastigotes. Appropriate scientific rigor is applied, with multiple replicates and statistical analysis of data. The paper reports a substantial body of work, and the studies should be of interest to the molecular parasitology community. The major new material is the elucidation of effects of blocked sterol biosynthesis on mitochondria, as opposed to earlier studies by this group that focused upon the plasma membrane, the principal site of sterol accumulation. Nonetheless, the novelty is probably not exceptional. The c14dm- mutant has been investigated previously and shown to have significant deficiencies, including on virulence. Sterols have been known to partition into intracellular organelles for some time, as indicated in various review articles published over more than a decade. Previous studies on trypanosomatids, reviewed by these authors on pages 5-6 of this manuscript, have implicated the mitochondrion in impairments induced by azoles, although they have not been as detailed or definitive as the current work. Hence, this is a strong study focused on a pathway of interest for possible drug development, but perhaps not of exceptional novelty or interest.

Reviewer #2: In this manuscript, Mukherjee et al. have explored the impact of genetic or pharmacological ablation of sterol 14�-demethylase (c14dm-) activity on the function and integrity of the mitochondrion in Leishmania major. Previous studies have demonstrated a link between disruption of sterol biosynthesis and the mitochondrion in both Leishmania and Trypanosoma, however, these studies were observational and lacked the systematic evaluation offered in this manuscript. The authors, through a meticulous set of experiments, that were appropriately controlled and which offered clear outcomes to testable hypotheses, have demonstrated that disruption of C14DM leads to the interruption of energy metabolism in the mitochondrion, making these cells highly dependent on glycolysis to meet their energy needs. The dysfunction in the mitochondrion appears to be linked to an impairment in ATP synthesis via ATP synthase, which leads to an increase in mitochondrial membrane potential and ROS production – the likely cause of the increased sensitivity of these c14dm- cells to stress.

The quality of the work in this paper is exceptionally high, the results unambiguous, and the work reaffirms C14DM, and indeed the sterol biosynthetic pathway, as an important drug target in these parasites worthy of further exploration. The work expands our current knowledge of the mechanisms of action of the azole drugs and, since it is highly likely it can be extrapolated to other trypanosomatids, should be of broad interest to the field. I have no suggested major revisions, and only a few minor comments to improve the overall manuscript.

Reviewer #3: This manuscript builds on the authors’ prior work on endogenous sterol synthesis in Leishmania major and the implications for drug design. The manuscript is clearly written and cites the existing literature well. A major strength of work from this group (including this manuscript) is the use of genetic knockout mutants (c14dm-/- and smt-/-) to understand parasite biology instead of the sole use of chemical inhibitors of these enzymes that have potential for off target impacts. The authors state that further characterization of sterol synthesis in the clinically relevant stages of Leishmania would provide interesting and potentially different insights. It is a point well made by the authors but may complicate some of the stated significance (Line 19). Some of the conclusions with regard to “adaptive” responses are overstated and not in line with the data.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: None suggested

Reviewer #3: As written, some of the conclusions regarding the relative roles of glycolysis and mitochondrial respiration in the c14dm-/- mutants vs WT parasites are a bit misleading (Fig 4).

Figure 4A: The graph shows that the c14dm-/- mutant exhibits reduced mitochondrial respiration as compared to the WT and add-back parasites. But what is presented are not the basal respiration rates for each line and therefore not a fair initial comparison. In this experiment, OCR was measured in the presence of 2-DG (line 478) to block glucose utilization – and because there is a bigger effect on the mutant, it appears as if respiration in the mutant line is impaired under basal conditions, which may or may not be the case. Therefore, a figure should be included to show basal respiration rate (OCR) measured in log-phase c14dm-/- mutants as compared to WT and add-back parasites incubated with a full complement of substrates. This data would determine if the mitochondrial defects observed (e.g. Fig1/2) result in defects in respiration. Based on the buffer composition described for Fig. 4 the OCR data appear to be oxygen consumption solely from pyruvate. These conditions, and the inferences gained need to be clarified in the text.

The authors should include the raw MitoXpress data for these experiments - linear plots as supplemental data. Also, the OCR value should be normalized to parasite number or protein in each sample.

Secondly, based on results presented in Fig 4, the authors emphasize increased dependence of the c14dm-/- mutant on glycolysis which might be expected because of the ‘mitochondrial defects’ observed in this mutant. However, as shown in Fig. 4B, most of the ATP production in the c14dm-/- mutant comes from mitochondrial respiration, not glycolysis. While glycolysis might contribute a bit more to the ATP produced by the mutant than the WT, it appears to be a minor contribution. Moreover, the authors do not include data to confirm the presumptive contribution of glycolysis to the residual ATP produced by the mutant in the presence of sodium azide. Data should be included to show that this signal is completely ablated if an excess of 2-DG is added to block glycolysis.

It is unclear why the buffer used in Fig. 4B (line 486) contains 2-DG. The figure legend does not state that 2-DG is in the buffer and may confuse readers. How is ATP production is expected to be altered in the absence of 2-DG in the buffer?

Also, in Fig 4B, in the absence of an exogenous carbon source (for 1 hr), there was no difference in the amount of ATP produced by the c14dm-/- mutants, WT and add-back. What is the major contributor to this basal ATP production – glycolysis, mitochondrial respiration? Does sodium azide or 2-DG treatment differentially impact the mutants and WT parasites? What does the pattern look like when parasites have access to a mixture of usable carbons (and absence of 2-DG in the buffer)? This information should be included and results interpreted in context.

When glucose is the only available exogenous substrate, the c14dm-/- parasites produce more ATP than WT parasites (there is no significant difference between mutant and add-back parasites) and the mutants take up more glucose and glycerol than the WT or add-back parasites. These data support the idea that the mutants might rely more heavily on glucose than other substrates to fuel energy production. Exogenous pyruvate or aspartate fail to rescue mutants from glucose starvation. In this scenario, is it possible to rule out impaired plasma membrane or mitochondrial permeability to other potential TCA substrates in the c14dm-/- mutants?

Along the same line, in some experiments, pyruvate is included in the parasite incubation buffer – presumably to fuel the TCA cycle and mitochondrial respiration. It doesn't seem like the inclusion of pyruvate makes a difference in the ability of WT parasites to generate ATP. The amount of ATP produced by all parasite lines in HBSS alone (Fig 4B, first set of bars) and HBSS + pyruvate present (Fig. 4C, first set of bars) appears to be similar. Why doesn’t ATP production increase in the presence of exogenous pyruvate and what is the effect on OCR?

At 4 hr in the absence of glucose (Fig4C) – but in the presence of pyruvate - the c14dm-/- mutants start to lose the capacity for ATP synthesis. Given that mitochondrial respiration is the main source of ATP in the c14dm-/- mutants (Fig. 4B), exogenous pyruvate should sustain ATP production at a higher level in the mutant if it is being utilized. Is ATP production lost at a higher rate in the mutant in the absence of exogenous pyruvate?

Has it been demonstrated that pyruvate is transported across the parasite plasma membrane so that it can be utilized by the mitochondria? In order to draw conclusions about substrate utilization and mitochondrial function, it is important to demonstrate that these important assumptions of the assay (e.g. substrate transport over the plasma membrane) are verified. Some of these data can be explain through substrate permeability differences between parasites.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Minor Points.

I would recommend changing the order of Figs. 5A and 5B, as the latter panel is introduced first in the manuscript.

Reviewer #2: • The error bars on Fig. 4B are very faint and need to be made more visible.

• The method used for assessing cell death by propidium iodide staining, though standard, is not detailed or referenced in the methods.

• There are few grammatical/typographical errors throughout the manuscript that need to be addressed prior to publication.

• Not a revision, but a question for the authors consideration. I’m curious if the increased uptake of glycerol is related to the enhanced activity of AQP1 and if so, how that might affect the sensitivity of the c14dm- cells to antimony drugs. Is something that is known or that has been tested?

Reviewer #3: Line 66-68. What is the evidence in [7] that scavenging predominates and there is a downregulation of ergosterol synthesis? If this is true what is the cause of the attenuated growth in vivo of the mutants (i.e. would there be a reduction in 14-methylated sterols?)? In ref 7 the statement “Clearly, some of the cholesterol was not directly associated with amastigotes instead from mouse cells” suggests that the extent/importance of cholesterol scavenging is not entirely settled with regard to amastigotes.

Fig 3B and line 210-211. Without a loading control or quantification the claim of “similar” seems to be a fair characterization but “slightly higher” may be a stretch.

Line 216: The statement “..outside of SOD deficiency.” might be interpreted, as SOD is sufficient to control ROS (which the data suggest it may not be). Would it be fair to state that parasite SODs appear unaffected but are apparently unable to alleviate excess ROS production?

Line 447 “Such treatments did not affect parasite viability based on PI-staining.” It is unclear if PI was used for all of the flow experiments to verify the integrity of the parasites. If this is the case it would be worth mentioning in the results section to support the conclusions when treatments like CCCP lead to a negative result (i.e. Fig 2A/B). This would further strengthen the authors conclusions as opposed to any results being due to alterations of general viability. Additionally (but unlikely under the stated treatment times) parasite death leading to lysis will not lead to a detectable event (PI pos or negative) to establish viability. Am I correct in assuming that parasites are not PI positive and the number of events (i.e. parasites) is consistent between treatment groups?

A major contribution of the generation of the smt-/- line (used in the 2018 publication) is the ability to compare phenotypes to the c14dm-/- line. This comparison (as noted by the authors) can help delineate if a given phenotype is caused by generation of 14-methylated sterols or absence on an endogenously synthesized sterol. In Figure 5A the smt-/- is used to support the notion that 14-methylated sterols contribute to the dependence on glucose. However, to support the notion that loss of mitochondrial membrane potential in the c14dm-/- is the cause of this glucose dependence it would be preferrable to see a side by side TMRE values (i.e. smt-/- and c14dm-/- ).

Lines 196-199 reference the 2018 publication as “not as profound” but a direct comparison/fold change would be more illustrative.

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PLoS Pathog. 2020 Aug 20;16(8):e1008810. doi: 10.1371/journal.ppat.1008810.r002

Author response to Decision Letter 0

1 Jul 2020

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PLoS Pathog. doi: 10.1371/journal.ppat.1008810.r003

Decision Letter 1

David Horn, Michael P Barrett

14 Jul 2020

Dear Dr. Zhang,

We are pleased to inform you that your manuscript 'Sterol 14-alpha-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please also address Reviewer #1 minor suggestions for corrections in the text (see below).

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Michael Barrett

Guest Editor

PLOS Pathogens

David Horn

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

The authors have made substantial changes to the manuscript including new experiments that have added to the potential translational impact of the work as well as responding to each of the queries raised by referees and addressing those where appropriate. All in all the work is good and represents an important breakthrough in our understanding of the roles played by sterols in Leishmania biology and how this can impact on chemotherapy.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This revised manuscript describes the critical role of the sterol biosynthetic enzyme C14DM in mitochondrial function in Leishmania major. The authors have addressed the comments of the 3 reviewers as well as suggestions made by the Associate Editor. Their responses have been effective and have enhanced the significance of the study. In particular, addressing the increased sensitivity of c14dm null mutant to the clinically employed drug pentamidine underscores the significance of this basic genetic study to drug treatment. The observation that either c14dm null mutants or treatment with inhibitory triazole drugs increases the efficacy of pentamidine raises the potential for combination drug therapy that would improve regimens involving delivery of pentamidine.

While this reviewer was somewhat concerned about the novelty of the original submission, this issue is addressed both by new experiments and by the recognition that the current study does indeed highlight in new ways the role of sterols in the mitochondrial function of these parasites. Thus inhibition of sterol biosynthesis has traditionally focused on the effects on the plasma membrane, where the majority of sterols reside. The current detailed study shows that there are important biological consequences to either inhibiting this enzyme or knocking out the C14DM gene that operate at the level of the mitochondrion rather than the plasma membrane. It is known from previous work on various systems that sterols are present in the inner mitochondrial membrane and that compounds that inhibit sterol biosynthesis have morphological and biochemical effects on mitochondria. However the current work provides much more mechanistic detail than was available before, showing an increase in the mitochondrial membrane potential and in mitochondrial ROS and increased sensitivity to stresses such as nutrient deprivation and heat following deletion of the C14DM gene. These detrimental effects on the mitochondrion are relevant to pharmacological properties of C14DM inhibitors, and the resulting increased sensitivity to pentamidine is of added pharmacological importance.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: None suggested.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: I have a few minor suggestions for corrections in the text.

1. Line 69 – Change ‘residue’ to ‘residual’.

2. Line 108 – Change ‘play’ to ‘plays’.

3. Change – ‘2-deoxy-G-glucose’ to ‘2-deoxy-D-glucose’ in lines 228, 250, 272.

4. Line 359 – Change ‘folds’ to ‘fold’.

5. Line 392 – Change ‘Mechanism’ to ‘The mechanism’.

**********

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Reviewer #1: No

PLoS Pathog. doi: 10.1371/journal.ppat.1008810.r004

Acceptance letter

David Horn, Michael P Barrett

4 Aug 2020

Dear Dr. Zhang,

We are delighted to inform you that your manuscript, "Sterol 14-alpha-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. C14dm‾ promastigotes show increased mitochondrial surface area but maintain mitochondrial membrane integrity at ambient temperature.

(PDF)

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S2 Fig. C14dm‾ and smt‾ mutants show increased ΔΨm and mitochondrial ROS.

(PDF)

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S3 Fig. C14dm‾ mutants accumulate ROS in the mitochondria.

(PDF)

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S4 Fig. 100 μM of glucose or glycerol is sufficient to rescue c14dm‾ mutants in PBS.

(PDF)

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S5 Fig. ITZ-treated WT parasites are hypersensitive to glucose depletion.

(PDF)

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S6 Fig. C14dm‾ mutants rescued by glucose or glycerol show less severe mitochondrial defects.

(PDF)

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

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

Sumit Mukherjee

Samrat Moitra

Wei Xu

Veronica Hernandez

Kai Zhang

Roles

Abstract

Author summary

Introduction

Results

C14dm‾ promastigotes show increased mitochondrial membrane potential (ΔΨm) and altered mitochondrial morphology

Fig 1. C14dm‾ promastigotes show high mitochondrial membrane potential (ΔΨm).

Fig 2. Inactivation of C14DM leads to ROS accumulation in the mitochondria.

Inactivation of C14DM leads to ΔΨm-dependent accumulation of ROS in the mitochondria

Examination of superoxide dismutase (SOD) expression in c14dm‾ mutants

Fig 3. Increased superoxide dismutase (SOD) activity in c14dm‾ parasites.

C14dm‾ mutants show reduced cellular respiration and increased glycolytic ATP production

Fig 4. C14dm‾ parasites show compromised cellular respiration.

Glucose starvation has deleterious effect on c14dm‾ parasites

Fig 5. C14dm‾ parasites are hypersensitive to starvation.

C14dm‾ parasites show increased uptake of glucose and glycerol

Fig 6. C14dm‾ parasites show increased uptake of glucose and glycerol.

ROS accumulation is partially responsible for the heat sensitivity in c14dm‾ mutants

Fig 7. ROS accumulation contributes to heat sensitivity in c14dm‾ mutants.

C14dm‾ mutants exhibit increased sensitivity to pentamidine

Fig 8. C14dm‾ parasites show increased sensitivity to pentamidine (PEN).

Discussion

Materials and methods

Materials

Leishmania culture

Determination of mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level

Determination of superoxide dismutase (SOD) activity

Digitonin fractionation and Western blot

Measurement of oxygen consumption and ATP production

Microscopy

Starvation and heat stress assays

Glucose and glycerol uptake assays

Susceptibility to pentamidine

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

David Horn

Michael P Barrett

Roles

Author response to Decision Letter 0

Decision Letter 1

David Horn

Michael P Barrett

Roles

Acceptance letter

David Horn

Michael P Barrett

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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