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

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


INTRODUCTION 4 7
Leishmaniasis is the second most deadly parasitic disease after malaria with more than 12 4 8 million people infected worldwide (1). The causative agents belong to a group of trypanosomatid 4 1 3 robust surface labeling at the exposure time of 100 ms (Fig. 7G). In contrast, signals from lso -2 5 0 mutants were only detectable at a longer exposure time showing significant intracellular staining 2 5 1 (Fig. 7H). Therefore, the expression of LPG was clearly altered in lso -. Meanwhile, these 2 5 2 mutants showed similar level of GP63 as WT and add-back parasites (Fig. 7C-D and F). chains, and an oligosaccharide cap (20,46). The lack of reactivity to WIC79.3 antibody in lso -2 5 7 could reflect a loss or modification of side chains that branch off the phosphoglycan backbone backbone (49,50). To probe the LPG structure in lso -, we first carried out a western blot analysis 2 6 0 using the CA7AE monoclonal antibody, which recognizes the unsubstituted (bare) Gal (β1,4)- was detected from L. major WT or lsoparasites with the CA7AE antibody, suggesting that their 2 6 5 LPG backbones had side chain modifications (Fig. 8A).

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To explore the carbohydrate composition of LPG side chain from lsomutants, we 2 6 7 purified LPG from WT, lso -, and lso -/+LSO promastigotes as previously described (53). The  Next the LPG samples were subjected to fluorophore-assisted carbohydrate poly-galactosylated residues on the side chains (47). Notably, lsomutants had a similar profile 2 7 7 as LV39 WT for the intermediate side chains (G5-G11), but their short side chains (G3-G4) were 2 7 8 much reduced (Fig. 8C). As expected, L. major FV1 parasites had more short side chains (G3-G4)   and lso -/+LSO promastigotes consistent with the galactose-and mannose-rich type II GIPLs (56, 2 8 5 57) (Fig. 8D). This profile was distinct from the type I GIPLs in L. donovani that is highly 2 8 6 enriched in mannose (57) (Fig. 8D). Together these data suggest that LSO deletion does not 2 8 7 change the carbohydrate profile of GIPLs but reduces the abundance of short (Gal1-2) side 2 8 8 chains on the LPG backbone in L. major. Previous reports indicate that inhibition of sterol biosynthesis can lead to compromised 2 9 1 mitochondrial functions in trypanosomatids (18,(58)(59)(60). In S. cerevisiae, LSO/Erg3 is not 2 9 2 required for viability in media containing ergosterol but mutants fail to grow on non-fermentable 2 9 3 substrates such as glycerol and ethanol, suggesting that this enzyme is needed for respiration (31, 2 9 4 61). To assess the role of LSO in mitochondrial functions in L. major, we first examined the 2 9 5 1 5 mitochondrial membrane potential (ΔΨ m ) after labeling cells with tetramethylrhodamine ethyl 2 9 6 ester (TMRE) (62). Compared to WT and add-back parasites, lsomutants had 30-50% higher 2 9 7 ΔΨ m in the early stationary stage but did not show significant difference in log phase or late log 2 9 8 phase (Fig. S4A). To measure the production of mitochondrial ROS, we labeled cells using a 2 9 9 mitochondria-specific ROS indicator MitoSox Red. As shown in Fig. S4B, lsomutants had 3 0 0 slightly higher fluorescence signal than the WT and lso -/+LSO parasites (the difference was not 3 0 1 statistically significant except for log phase), suggesting a modest accumulation of ROS in their 3 0 2 mitochondria. Next, we used the MitoXpress probe to examine oxygen consumption rate (63) by glucose. Under this condition, lsoshowed a slightly lower oxygen consumption rate than WT 3 0 5 and add-back parasites although the difference was not statistically significant (Fig. S4C).

0 6
Together, these data indicate that LSO deletion has minor effects on the mitochondrial functions 3 0 7 in L. major.  To study the role of LSO in L. major virulence, metacyclics were isolated from stationary 3 1 0 phase cultures and used to infect BALB/c mice in the footpads. Parasite virulence was assessed 3 1 1 by measuring the development of footpad lesions over time. Compared to WT and lso -/+LSO 3 1 2 parasites, mice infected by lsomutants showed a 2-4 weeks delay in lesion progression ( Fig.   3 1 3 9A), which was consistent with the lower parasite numbers in the infected footpads at weeks 6 3 1 4 and 14 post infection (Fig. 9B). To explore the virulence of amastigotes, we isolated amastigotes 3 1 5 from promastigote-infected footpads and used them to infect naive BALB/c mice. As shown in /+LSO amastigotes but the difference was less pronounced than metacyclics. These findings 3 1 8 suggest that LSO is important for L. major promastigotes to grow and cause disease in mice. In this study, we characterized the gene encoding LSO (sterol C5-desaturase), a sterol 3 2 2 biosynthetic enzyme, in the protozoan parasite L. major. LSO catalyzes the formation of a 3 2 3 double bond between C5 and C6 in the B ring of sterol intermediates (Fig. S1). L. major LSO-3 2 4 null mutants (lso -) were devoid of ergosterol or 5-dehydroepisterol (abundant in WT parasites). While the difference appears to be minor in chemical composition, lso -3 2 7 mutants were 2-4 times more resistant to Amp B than WT and lso -/+LSO ( Fig. 4A and Table 1).

2 8
These mutants were fully replicative in culture during the log phase with minor mitochondrial deletion also led to hypersensitivity to acidic pH (Fig. 5). These observations are largely in  It is interesting that LSO activity appears to contribute to the binding affinity between (plants) (15,(23)(24)(25)(26). Without this double bond, the A ring could twist/rotate more freely from the 3 3 6 B ring, potentially making the sterol core less flat and reducing its binding capacity to Amp B 3 3 7 (Fig. S1). Such a change in the sterol core conformation could also increase the gap between 3 3 8 sterol and phospholipid, making the membrane less stable. This is consistent with the increased 3 3 9 sensitivity of lsomutants to heat and Triton X-100 ( Fig. 4C and Fig. 5C).

4 0
Compared to WT and add-back parasites, lsomutants had a lower intracellular pH and 3 4 1 the difference became more pronounced when cells were cultivated in a pH 5.0 medium ( Table   3  4  2 2). This finding is consistent with their reduced capacity to survive and replicate under the acidic not only in the plasma membrane but also in the membrane of intracellular organelles.

4 6
Acidocalcisomes are membrane-enclosed storage organelles involved in osmoregulation, 3 4 7 phosphate metabolism, calcium homeostasis, and intracellular pH maintenance in protozoan pyrophosphatase, was lower in lsoduring the stationary phase (Fig. 6). Since VP1 transports 3 5 0 protons from the cytosol into acidocalcisomes (using pyrophosphate hydrolysis as the energy 3 5 1 source), the reduced VP1 expression may lead to a more acidic intracellular pH and slower 3 5 2 recovery of intracellular pH under acidic conditions (42).

5 3
Similar to the C14DM-null mutants, the cellular level of LPG in lsoappeared to be  (Fig. 8C). Together, these data imply that the low WIC79.3 reactivity in lsois not due to a total loss of LPG synthesis like several previously characterized LPG-synthetic mutants (49, These flanking sequences were digested and ligated into the cloning vector pUC18. Genes and/or sequencing. Oligonucleotides used in this study are summarized in Table S1.  in lso -/+LSO or lso -/+LSO-GFP parasites respectively. at 180 °C, held for 2 min, followed by 10 °C/min increase until 300 °C, and held for 15 min. To 4 2 8 confirm that the unknow GC peak retention time matched that of the episterol standard, we also 4 2 9 used a second temperature program started at 80 °C for 2 min, ramped to 260 °C at 50 °C/min,  For LSO-GFP localization, lso -/+LSO-GFP parasites were labeled with rabbit anti-T. (1:1000) for 20 min. Localizations of LPG was determined as previously described (19). To label growth under the acidic condition was determined using an acidic M199 medium (same as the 4 5 0 complete M199 medium except that the pH was adjusted to 5.0 with hydrochloric acid).

5 1
To assess cell viability under stress, mid log phase promastigotes were incubated in iodide.

5 6
To determine sensitivity to drugs, log phase promastigotes were inoculated in complete densities from drug-treated cells to cells grown in absence of drugs (18).

6 0
To determine sensitivity to detergent, log phase promastigotes were inoculated in Acidocalcisome isolation and analysis of short chain and long chain polyphosphate.

7 7
Acidocalcisome fractions were isolated from log phase and stationary phase chain polyphosphate in acidocalcisome fractions were determined as previously described (74). To determine LPG and GP63 expression, promastigotes were washed once in PBS and 4 8 2 resuspended at 5.0 × 10 7 cells/ml in 1 x SDS sample buffer. Samples were boiled for 5 min and (1:1000) followed by a goat anti-rabbit IgG-HRP (1:2000). To confirm LPG purification, 5 μ g of 4 8 9 purified LPG isolated from each strain was subjected to immunoblotting as described above  in 0.1 M acetic acid/0.1 M NaCl, and applied to a column of phenyl-Sepharose (2 mL), 4 9 7 equilibrated in the same buffer. LPG and GIPLs were eluted using solvent E (77).

9 8
To prepare LPG repeat units, the LPG samples were depolymerized by mild acid mM Tris-HCl, pH 9.0 (1 U/ml, 16 h, 37 ). After enzymatic treatment, the repeat units were and desalted as described above (57). with 8-aminonaphthalene-1,3,6-trisulfate and subjected to FACE analysis and the gel was  into an acidic medium (pH 5.0) at 1 x 10 5 cells/ml and culture densities were determined daily. Relative levels of VP1 staining were determined from 100 metacyclic-like promastigotes for stained with Hoechst. Exposure times for the FITC channel: 100 ms for G and 300 ms for H.

3 2
Scale bar: 10 µm. cell lysates were processed for Western blot with mAb CA7AE or anti-α-tubulin antibody. Log  Experiments were performed twice and results from one representative set were shown. WT and add-backs.  were resuspended in a respiration buffer (HBSS + 5 mM 2-deoxyglucose + 5 mM sodium . In addition to the perfect match of the retention time of peak 2' with the episterol standard, the full scan EI mass spectra (70 eV) plotted from peak 2' (E) and the episterol standard (F) are also identical, confirming that peak 2' is episterol.