Ethics statement

Animal studies were performed following CPCSEA guidelines and approved by the Institutional Animal Ethics Committee (IEAC, (JNU/IAEC/2020/19)) of Jawaharlal Nehru University. Swiss mice were obtained from the Central Laboratory Animal Resources, JNU, New Delhi, and maintained under standard conditions.

In silico search for SphK1 like protein in Leishmania donovani

An in-silico search was conducted in the Kinetoplastid database TriTrypDB (www.tritrypdb.org) to identify any homologous protein similar to prokaryotic SphK1 in Leishmania donovani [25]. Sequence alignments and identity computations were conducted using ClustalW [26]. Homologues of SphK1 from L. donovani, along with other prokaryotes and eukaryotes were analyzed using the Molecular Evolutionary Genetics Analysis (MEGAX) tool [27]. The evolutionary trajectory was determined using the Neighbor-Joining method. The bootstrap consensus tree was generated from replicates and the evolutionary distances were calculated using the Poisson correction method. Pymol was utilized to align the two structures and compute the RMSD post-alignment [28].

In-silico docking studies

The protein structure of hSphK1 (Uniprot ID- Q9NYA1), a protein derived from H. sapiens, was retrieved from the protein data bank [29,30]. The Leishmania major Sphingosine Kinase A sequence, as previously characterized by Zhang et al. [31], was used as a query to identify the corresponding orthologue in the L. donovani genome via a BLASTp search of the TriTrypDB database [32]. Phyre2 web server was used for homology 3D protein modelling of the LdSphK1 protein and Procheck sever was used for Ramachandran plot analysis of the model [33,34]. The stereochemical quality of the resulting model was then comprehensively validated using the MolProbity server [35,36]. The Swiss PDB viewer and ChemBio Draw ultra 3D software were used to optimize the structure of proteins and ligands [37,38]. The process of molecular docking between molecule PF-543 and hSphK1 was conducted using Autodock version 4.2 and Cygwin terminal version 3.1 software [39,40]. The residues located in the vicinity of the binding pocket were selected to create a grid (with a spacing of 0.375) centred at coordinates X-51.488, Y-53.406, Z- -1.49 for hSphK1 and X-4.021, Y- -21.535, Z- 15.254 for LdSphK1 respectively. This grid was used to facilitate the binding of the ligand. The docking data were further analyzed and visualized using the PLIP, Ligplot+ version 2.2, Discovery Studio version 19.1.0, and Pymol version 2.3.2 software tools [28,41–43].

Homologs of SphK1 from L. donovani and other representative kinetoplastids and mammals were retrieved from TriTrypDB and NCBI, respectively. The amino acid sequences were aligned using ClustalW. The phylogenetic tree was reconstructed using the Maximum Likelihood (ML) method in MEGA XII [44]. The best-fit substitution model was first determined by MEGA’s model selection tool, which identified the Jones-Taylor-Thornton (JTT) model with a Gamma distribution (+G), a proportion of invariable sites (+I), and observed amino acid frequencies (+F) as the optimal model based on the Bayesian Information Criterion (BIC). The tree was inferred using the Subtree-Pruning-Regrafting (SPR) heuristic method. The reliability of the tree topology was assessed by bootstrap analysis with 1000 replicates.

Cloning and purification of recombinant LdSphK1

Nucleotide sequence encoding full length sequence of L. donovani SphK1 (1–2808 bp) was selected for recombinant protein generation as a fusion protein with an N-terminal 6x- Histidine-tag using the vector pET-28 a(+) vector. DNA fragment for the gene cloning was PCR amplified from genomic DNA of L. donovani using the following primer pair: LdSphk1_BamHI_FP 5’-GATGGGATCCATGCACTCTTTCACCTCCAA-3’ and LdSphk1_XhoI_RP 5’-ATTCTCGAGCTACCCACTGCGCACGAGCT-3’ with Phusion High-Fidelity DNA Polymerase (Thermo Scientific, US). The amplified DNA fragment was purified with QIAquick Gel Extraction Kit (Qiagen) using the manufacturer’s protocol. Purified LdSphk1 insert and the expression vector pET-28 a(+) were digested with BamHI/XhoI restriction enzymes (New England Biolabs, UK), and were ligated overnight at 16°C using T4 DNA ligase (New England Biolabs, UK). The ligation mix was transformed into E. coli DH5-α competent cells (E.Coli DH5-α cells were obtained from ThermoFisher Scientific, USA Catalog number C0112) and positive clones were screened by colony PCR followed by confirmation of the cloned plasmid with BamHI/XhoI restriction digestion and further transformed in Rossetta E. coli (Rossetta E. coli were obtained from Sigma-Aldrich, USA with Catalog number 71397) expression strain for LdSphk1 recombinant protein expression. For recombinant protein induction, the cells were induced with 1mM IPTG for 4 hours at 37°C in Terrific broth. Purification of recombinant protein was achieved by affinity chromatography.

The rLdSphK1 protein was eluted after addition of 150mM imidazole solution. The eluted fractions were analysed by running on 12% SDS PAGE and the fractions containing pure protein were pooled and concentrated using a centricon device, followed by buffer exchange with PBS. Since most of the protein was localized in the inclusion bodies, we purified the protein from inclusion bodies under denaturation conditions. Briefly, inclusion bodies were obtained after sonication and subsequent centrifugation at 13,000 rpm and solubilized in urea buffer composed of 8M urea, 20mM Tris, 250mM NaCl with pH 8.0 and subsequently incubated with Ni-NTA beads (Qiagen, Germany) overnight for binding. The beads were then loaded in gravity flow column (Qiagen, Germany). Further, LdSphK1 protein bound with the beads was then eluted by 10mM, 25mM, 50mM, 100mM, 250mM and 500mM imidazole solutions prepared in urea buffer. Eluted fractions containing pure protein were pooled and refolded by dialyzing against refolding buffer composed of 100mM Tris, 20% Glycerol, 250mM L-Arg, 1mM EDTA, 1mM GSG and 0.5mM GSSG with pH 8.0. Refolded protein was reconstituted in PBS (pH 7.4) and concentrated using centricon tubes with 10kDa cut-off (Merck, Germany).

Purification of recombinant humanSphK1

CDS encoding full-length SphK-1 (1155 bp) (Gene ID-8877) was amplified using the following primers:

NheI_FP: 5′-AATGCTAGCATGGATCCAGCGGGCGGCCCC-3′ and

XhoI_RP: 5′-TGGCTCGAGCTATAAGGGCTCTTCTGGCGGTG-3′.

The amplified DNA fragment was cloned between NheI and XhoI restriction sites of the pET-28a (+) expression vector, and the recombinant plasmid was transformed into E. coli strain BL21 (ʎDE3) gold [45]. The rhSphK1 protein was eluted after addition of 100mM, 250mM and 500mM imidazole solution. Overexpression of 6 × His-SphK1 (rSphK1) was induced with 1mM IPTG (Sigma-Aldrich) at an optical density (OD600) of 0.6, for 4h at 37°C. The protein was purified using Ni-NTA agarose resin (Qiagen). Briefly, Rosetta (DE3) competent cells, derivatives of E. coli BL21 obtained from Sigma-Aldrich (Catalog No. 71397), were used to enhance rhSphK1 expression by compensating for rare codon usage in E. coli. Bacterial cells were harvested by centrifugation at 6000 × g for 15 minutes and lysed by sonication in lysis buffer containing 50 mM Na₂HPO₄ (pH 7.4), 200 mM NaCl, 500 μg/mL lysozyme (Sigma-Aldrich), and 1mM Phenylmethylsulfonyl Fluoride (PMSF; ThermoFisher Scientific). The bacterial lysate was centrifuged at 10,000 × g for 20 min, and the supernatant obtained was loaded onto a Nickel-Nitrilotriacetic Acid (Ni-NTA) agarose resin (Qiagen)-packed column along with 10 mM imidazole, for 4h at 4°C with mild agitation. The protein was eluted with a continuous imidazole gradient of 25mM, 50mM, 100mM, 250mM, and 500mM. The protein purification was validated by 12% SDS-PAGE, followed by immunoblotting with anti-His tag antibody (raised in Swiss mice (obtained from the Central Laboratory Animal Resources, Jawaharlal Nehru University, New Delhi) using standard protocol approved by IAEC, JNU).

Microscale thermophoresis (MST)

To validate the biophysical interaction between rLdSphK1 or rHuman SphK1 with PF-543 and analyze the binding and kinetics, Microscale Thermophoresis (MST) was performed in NanoTemper Monolith NT.115 instrument. Briefly, 20μM of rLdSphK1 or rhSphK1 in HEPES-NaCl buffer, pH 7.4 was labelled with 30μM Lysine reactive dye (5ul diluted in 200ul rLdSphK1 or rhSphK1 protein) using NanoTemper’s Protein Labelling Kit RED-NHS (L001, NanoTemper technologies, Germany) and incubated in the dark for 30 minutes at RT. Following incubation, the labelled rLdSphK1 or rhSphK1 protein along with the buffer was passed through an equilibrated column (provided in the kit). Fractions of the labelled protein were eluted followed by fluorescence count. Fluorescence counts ranging between 250–450 were taken. 5 µM PF-543, diluted in HEPES/0.05% Tween-20, was titrated against the constant concentration of the labelled rLdSphK1 or rhSphK1 protein. Pre-mixed samples were incubated for 15 minutes before centrifugation at 8000 rpm for 10 minutes. The samples were filled into the capillaries treated with standards (K002 Monolith NT.115) and thermophoretic mobility was determined. All experiments were performed at RT, at 40% MST power and 20% LED power. Data evaluation was performed with the Monolith software (Nano Temper, Munich, German). Dissociation constants (K_D) were derived from concentration-dependent thermophoretic signal changes. It is emphasized that MST-derived K_D values are context-dependent and reflective of experimental conditions, and do not replace previously reported enzymatic IC₅₀ values for mammalian SphK1 obtained using optimized biochemical assays [45].

NBD-Sph assay

A fluorescent sphingosine analog, omega (7-nitro-2–1, 3-benzoxadiazol-4-yl [2S,3R,4E]-2-amino octadec-4-ene-1,3-diol [NBD–Sphingosine (NBD-Sph); Avanti Polar Lipids]) was used as a substrate. Conversion of NBD-Sph to NBD-S1P was evaluated, where NBD-SIP served as the measurable end product of sphingolipid metabolism. Towards this, rLdSphK1 protein was incubated with 10μM NBD-Sph, at 37°C for 60 min. Following incubation, the reaction was terminated by addition of RIPA lysis buffer and lipids were extracted using a modified chloroform–methanol extraction protocol. Briefly, the reaction mixture was thoroughly mixed with 260μL methanol and 400μL chloroform:methanol (1:1) followed by addition of 16μL of 7 M NH4OH, 400μL chloroform, and 300μL of 1.5 M KCl. The mixture was centrifuged at 17,000 × g for 5 min to allow phase separation.

A 100μL aliquot of the upper aqueous phase containing NBD-S1P was transferred to a black 96-well flat-bottom plate (Corning). Fluorescence intensity was measured at excitation/emission wavelength of 485 nm/530 nm using Varioskan LUX Multimode Microplate Reader (ThermoFisher Scientific). The fluorescence intensity directly correlated with NBD-S1P formation.

Biochemical characterization of purified rLdSphK and rHumanSphK

The inhibitory effect of PF-543 on the rLdSphK1 catalytic activity was evaluated in a dose-dependent manner (0.01–5µM). The recombinant protein (200 ng) was pre-incubated with different concentrations of PF-543 (0.01μM to 5μM) for 45 min., followed by initiating the reaction in a buffer containing 0.05% Triton X-100, 1mM ATP (saturating concentration), 2% DMSO and 10μM NBD Sphingosine. The fluorescence intensity of NBD-S1P was measured as described above. The results represented a maximum inhibition at 5μM of PF-543, while the protein concentration was kept constant at 200ng.

For kinetic analysis, varying concentrations of NBD-Sphingosine (2–20µM) were incubated with 200ng rLdSphK1 in the presence of 200µM ATP, a concentration approximating reported Km values for ATP in SphK1, to allow accurate determination of kinetic parameters. Readings were taken real-time after every 5 sec for 15–20 min at an excitation/emission wavelength of 490/530 nm. Standard curve of SIP was plotted. The enzymatic activities were calculated by plotting fluorescence/time vs substrate concentration followed by plotting of fluorescence/pmoles (velocity) vs substrate concentration. Finally 1/Vmax vs 1/[S] was plotted to calculate Km and Vmax according to Lineweaver-Burk plots at 37°C (Lineweaver and Burk, Science Direct, 2011).

Leishmania cell culture

Leishmania donovani Bob promastigotes (LdBob strain/MHOM/SD/62/1SCL2D, originally obtained from Dr Stephen Beverley (Washington University, St. Louis, MO) were cultured at 26°C in M199 medium (Sigma-Aldrich, USA), supplemented with 100 units/ml penicillin (Sigma-Aldrich, USA), 100µg/ml streptomycin (Sigma-Aldrich, USA) and 10% heat-inactivated fetal bovine serum (FBS; Biowest). Whole cell lysate of promastigote was prepared by freeze–thaw cycles as described previously [46].

For axenic amastigotes, the axenically cultured forms grew optimally at a temperature of 32–33°C in a growth media with pH of 5.4. The axenic amastigotes were prepared according to the standard protocol [47–49] Briefly, the late-log promastigotes were adapted in an acidic media (RPMI-1640/25 mM 2-(N-morpholino) ethane sulfonic acid (MES)/pH 5.5), at 26°C. These parasites were then grown in RPMI-1640/MES/pH 5.5 at 37°C with 5% CO2, mimicking the acidic and increased temperature conditions of the macrophage phagolysosomal compartment. Axenic amastigotes were microscopically examined to confirm their conversion from promastigotes to amastigotes as per the previous report [47–49].

Transient transfection of the parasite Leishmania

Leishmania donovani Bob strain (LdBob strain/MHOM/SD/62/1SCL2D) promastigotes were transfected with pXG-GFP-SphKa OE plasmid which harboured the neomycin resistance cassette using electroporation technique as described previously [50] with a Bio-Rad Gene Pulser device and cultured at 26°C in M199 medium (Gibco, Thermofisher scientific USA), supplemented with 100 units/ml penicillin (Sigma-Aldrich, USA), 100µg/ml streptomycin (Sigma-Aldrich, USA) and 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA). A final concentration of 250μg/mL of G418 (Sigma-Aldrich, USA) was used to select transfected parasites.

THP-1 cell culture and infection

THP-1 cells, an acute monocytic leukaemia-derived human cell line (American Type Culture Collection, Rockville, MD), were maintained in RPMI-1640 (Sigma-Aldrich, USA) medium supplemented with 10% heat-inactivated FBS (Biowest, UK), 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C and 5% CO₂. Cells (10⁶ cells/well) were treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, USA) for 48h to induce differentiation into macrophage-like-cells before infection. Cells were washed once with phosphate-buffered saline (PBS) and incubated in RPMI medium (Sigma-Aldrich, USA), 10% heat-inactivated FBS, 100 units/ml penicillin and 100μg/ml streptomycin, before infection. To carry out in vitro infection assays, late stationary phase promastigotes (WT) were used at a ratio of 20 parasites per macrophage. Leishmania-infected macrophages were incubated at 37°C in a 5% CO₂-air atmosphere for 4h to allow the establishment of infection and proliferation of intra-macrophage parasites. The cells were then washed five times with PBS to remove non-adherent extracellular parasites. After that, the cells were incubated in RPMI medium at 37°C in a 5% CO₂-air atmosphere for 48h.

Infectivity assay

THP-1 cells (1 × 10⁶ cells/well), treated with 50 ng/ml of PMA (Sigma-Aldrich, USA) were seeded on glass coverslips in 6-well plates for 48h. They were infected and simultaneously treated with inhibitors as described above, and the intracellular parasite load (mean number of amastigotes per macrophage) was visualized by Giemsa staining as described previously by Pawar et al [51] and expressed as a percentage of the blank controls without drug. At least 10 fields were counted manually for each condition to determine the average number of parasites per macrophage.

qRT-PCR for gene expression analysis

Total RNA from infected macrophages was isolated using the TRIZOL reagent (Sigma-Aldrich, USA) and its concentration was determined by Nanodrop (Thermo Fischer, USA). cDNA was prepared from one microgram of RNase-free DNase treated total RNA using first-strand cDNA Synthesis Kit (Thermo Fischer Scientific, USA), as per manufacturer’s instructions, using random hexamer primers. The resulting cDNA was analyzed by quantitative real-time (qRT-PCR) RT-PCR (Applied Biosystems, 7500 Fast Real-Time PCR System, CA, USA) with gene-specific primers using PowerUp SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA). The details of the primers (sequences and annealing temperatures) are given in S1 Table. Thermal profile for the real-time PCR was amplification at 50°C for 2 min followed by 40 cycles at 95°C for 15 sec, 60°C for 1 min and 72°C for 20 sec. Melting curves were generated along with the mean C_T values and confirmed the generation of a specific PCR product. Amplification of RNU6AP (RNA, U6 small nuclear 1; THP-1 cells) was used as internal control for normalization. The results were expressed as fold change of control (Uninfected samples (RNU6AP)) using the 2^-ΔΔCTmethod. All samples were run in triplicates, including a no-template (negative) control for all primers used.

Western blotting

Western blot analysis was done as described previously by Darlyuk et al., 2009 [52]. Briefly, protein was isolated from Leishmania infected macrophages by resuspending cell lysates in RIPA buffer. Before lysis, adherent cells were placed on ice and washed with PBS. Macrophages were scraped in the presence of RIPA lysis buffer containing 1% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8), 10 mM 1,10-phenanthroline and phosphatase and protease inhibitors (Roche). After incubation, lysates were centrifuged for 15 min to remove insoluble matter.The proteins in the lysates were quantified, 80μg of the lysate was boiled (95˚C) for 5 min in SDS sample buffer and was subjected to electrophoresis on a 10% SDS-polyacrylamide gel. Proteins were then transferred onto nitrocellulose (NC) membrane using an electrophoretic transfer cell (Bio-Rad Laboratories, USA) at RT. The membrane was washed with 1 × TBST solution three times and blocked with 5% BSA for 2h at RT. The blocked membrane was washed again in TBST solution three times and incubated overnight at 4 °C with primary monoclonal antibody against Caspase-9 (Cell Signaling Technology, #9508) diluted in TBST containing 5% BSA. The blots were subsequently incubated with HRP-conjugated secondary antibody (1:3000 dilution) prepared in 5% BSA in TBST for 2h at RT. Enhanced chemiluminescence reaction was used for the detection of the blot. The results were expressed as fold change and quantitated by using AlphaEaseFC image analysis software (Alpha Innotech). The data were expressed as mean ± SD of three independent experiments, and the representative image of one experiment is shown.

Localization of LdSphK1 in L. donovani promastigotes

The amino acid sequence of LdSphK1 was analyzed for the presence of transmembrane domains using the DeepTMHMM server. Further, for detection of SphK1 localization in parasites, 5*10⁶ log phase Leishmania donovani promastigotes were plated on poly-L-Lysine coated glass coverslips followed by treatment with 500nM PF-543 diluted in incomplete M199 medium for 6h at 22°C in dark. After 6h of treatment, the cells were washed with 1XPBS, fixed (PBS, 4% formaldehyde, 30 min) and permeabilized (PBS, 0.5% Triton X-100, 5 min). Slides were blocked (1% BSA, 1XPBS, overnight at 4°C or for 30 minutes on rocker, RT). Next day blocked cells were incubated with mice raised anti-SphK1 primary antibody (1:500) for 2 hours at RT. The cells were then washed three times with blocking buffer followed by incubation with Alexa 488 conjugated secondary antibody for 1 hour at RT. The coverslips were mounted on glass slides with antifade DAPI solution for visualization under a confocal laser scanning microscope (Olympus FluoView FV1000 with objective lenses PLAPON ×60 O, NA-1.42) at an excitation wavelength of 556 nm. Images were processed via NIS-Elements software version 4.50. The mean fluorescence intensities were plotted using GraphPad Prism version 8.0. The experiment was performed thrice.

Checkerboard assay

Checkerboard assays were put to evaluate the effects of the combination of Amphotericin B (anti-fungal agent) and PF-543 (Sphingosine Kinase inhibitor) against the Leishmania parasite. For this, log-stage promastigotes (42–44h) maintained in M199 media were dispensed in a 96-well plate. Amphotericin B was added vertically at different concentration ranges while PF-543 was added horizontally at different drug concentration ranges in 8*8 format. As a result, the checkerboard consists of columns and rows in which each of the well along the x-axis contains drug Amphotericin B at different concentrations (12.5nM, 30nM, 50nM and 100nM) and that along the y-axis contains PF-543 at different concentrations (150nM, 250nM, 500nM and 2µM). The plate was incubated at 25°C in a humidified chamber for 48h. The fractional inhibitory concentration (FIC index = FIC A + FIC B, where FIC A is the IC₅₀ of drug (A) in combination/IC₅₀ of drug A alone, and FIC B is the IC₅₀ of drug B in combination/IC₅₀ of drug B alone) of each drug was calculated and plotted as an isobologram. A straight diagonal line with an FIC index equal to 1 indicates an additive effect between drug A and drug B, a concave graph below the diagonal with an FIC index of less than 1 indicates a synergistic effect, and a convex curve above the diagonal with an FIC index of more than 0 indicates antagonism [53–55].

Estimation of reduced (GSH) and oxidized (GSSG) glutathione levels

The estimation of glutathione levels in reduced and oxidized forms was performed in accordance with the previously documented protocol [56]. Briefly, Leishmania infected macrophages were incubated with varying concentrations of SphK inhibitors for 24h and lysate was prepared. For GSH estimation, the lysate was added to an assay mixture containing 0.1 mM sodium phosphate buffer (pH 8) and o-phthalaldehyde (1 mg/ml in methanol, Sigma Aldrich, USA) and incubated at RT for 15 min. GSH level was estimated by monitoring fluorescence change at 365Exc/430Emi using a microplate reader. GSSG was also estimated using the same method, however, the lysate was incubated first with N-ethylmaleimide (Sigma Aldrich, USA) in the dark for 5 min, to block the GSH to GSSG oxidation. Results plotted as redox index (GSH/GSSG) ratio are the mean of duplicates.

Animal studies

Swiss mice were housed under standard conditions of food, temperature (25 ± 3 °C), relative humidity (55 ± 10%) and illumination (12h light/dark cycles) obtained from the Central Laboratory Animal Resources, Jawaharlal Nehru University, Delhi.

For experimental assays, mice were divided into the following groups (n = 3 per group): Uninfected control group, Infected untreated control group, Infected + PF-543 (10 mg/kg), Infected + Amphotericin B (2 mg/kg), Infected + Combination therapy (PF-543 2 mg/kg + Amphotericin B 0.4 mg/kg). Mice in treatment groups were infected with L. donovani 24h prior to drug administration. Here-in, 10mg/Kg of PF-543 and 2mg/Kg of Amphotericin B were used as per the reported literature (Neil MacRitchie et al. Cell Signal; 2016 [57], EM Moore and D N Lockwood. J Glob Infect Dis; 2010, [6]). All drugs were administered intraperitoneally (i.p.) once daily for three consecutive days. PF-543 monotherapy: 10 mg/kg/day, Amphotericin B monotherapy: 2 mg/kg/day, Combination therapy: 2 mg/kg/day PF-543 + 0.4 mg/kg/day Amphotericin B. Drug doses were prepared freshly in 1 × PBS and administered in a final volume of 100 µL per 30 g mouse. Briefly, to prepare 10mg/Kg/day of PF-543, 0.3mg of PF-543 powder was dissolved in 1ml of 1XPBS (5.9mM). 100µl of prepared PF-543 in 1XPBS suspension was then given to each mouse weighing 30gm. To prepare 2mg/Kg/day of Amphotericin B, 0.06mg of Amphotericin B powder was dissolved in 1ml of 1XPBS (650µM). Further 100µl of this prepared Amphotericin B in 1XPBS suspension was then given to each mouse weighing 30gm. For constituting the synergistic effect of two drug partners; 2mg/Kg (1mM) of PF-543 and 0.4mg/Kg (0.12µM) of Amphotericin B were prepared in 1XPBS and given to a mouse weighing 30gm. To prepare 2mg/Kg/day of PF-543; 0.06mg of PF-543 was dissolved in 1ml of 1XPBS (1mM). Further, 100µl of this prepared PF-543 suspension in 1XPBS was given to each mouse weighing 30gm. To prepare 0.4mg/Kg/day of Amphotericin B; 0.012mg of Amphotericin B powder was dissolved in 1ml of 1XPBS (0.12µM). Further, 100µl of this prepared Amphotericin B suspension was given to each mouse weighing 30gm. All treated mice were monitored for adverse effects (body weight, behaviour, and survival) throughout the treatment period. No significant toxicity was observed at the administered doses, consistent with published reports of the tolerability of both compounds in mouse models. Further, evaluation of the immunomodulatory potential was done using cytokine profiling in Leishmania infected Swiss mice treated with PF-543 and Amphotericin B alone and simultaneously followed by determination of parasite infectivity using in-vitro parameters. To evaluate the parasitaemia and expression of inflammatory markers in infected mice upon drug administration, spleen was harvested from each group of mice and qRT-PCR analysis was performed using primers specific for inflammatory markers; TNF-α and IL-10 and parasite specific kinetoplast minicircle gene; JW in PF-543 and Amphotericin B treated infected mice in respect to the untreated Swiss mice.

Statistical analysis

Fold-expression (qRT-PCR and densitometric analysis), NBD-Sph and intracellular parasite burden were represented as mean ±SD. Each experiment was repeated three times in separate sets. Statistical differences were determined using Student’s unpaired 2-tailed t-test. All statistics were performed using GraphPad Prism Version 5.0 (GraphPad Software, USA). p ≤ 0.05 was considered significant [* (P < 0.01 to 0.05), ** (P < 0.001), *** (P < 0.0001), ns (P ≥ 0.05)].

1. Inhibition of SphK1 by PF-543 selectively impairs Leishmania donovani growth and reduces intracellular parasite load

The activity of Leishmania-specific sphingosine kinase-1 (LdSphK1) was assessed in promastigotes following exposure to three known Sphingosine Kinase inhibitors: PF-543 (500 nM), DMS (500µM), and SKI-5C (100µM) for 48 h (IC₅₀ values represented in Fig 1A and S1–S2 Figs). To investigate the impact of SphK inhibitors; such as PF-543, DMS and SKI-5C on catalytic activity of SphK1 expressed by Leishmania promastigotes (LdSphK1), we performed fluorometric assay based estimation of enzymatic activity using 7-nitro-2–1,3-benzoxadiazol-4-yl (NBD)-tagged sphingosine (NBD-sphingosine). The results demonstrated significant ~60% reduction of NBD-S1P levels in PF-543-treated Leishmania promastigotes compared to the untreated control (Fig 1A). This observation validated the presence of an enzymatically active form of SphK1 in Leishmania donovani and confirmed that PF-543 effectively targets its catalytic activity.

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Fig 1. Inhibition of SphK1 by PF-543 selectively impairs Leishmania donovani growth and reduces intracellular parasite load.

(A) Estimation of LdSphK-1-mediated generation of NBD-SIP levels against SphK inhibitors in L. donovani promastigotes. L. donovani Bob promastigotes were cultured in 25 cm² flasks followed by treatment with SphK inhibitors; PF-543, DMS and SKI-5C for 48h. Inhibition of SphK1 using these SphK inhibitors cause decreased S1P levels. SphK inhibitor treated promastigotes were resuspended in buffer containing fatty acid-free BSA (0.1% (w/v)) followed by resuspension in buffer containing BSA (1% (w/v)) and incubated with NBD-sphingosine (10μM) for 45 min at 37°C. Promastigotes incorporate NBD-sphingosine (NBD-Sph), which are phosphorylated by Sphingosine Kinase (SphK1) to NBD-S1P. Bar graph depicts ELISA-based S1P quantification in promastigotes treated with inhibitors. (B) Effect of SphK1 inhibitor; PF-543 in Leishmania spp and host macrophages. To evaluate the IC₅₀ for PF543, approximately 6 × 10³ THP-1 and 5 × 10⁴ Ld Bob cells were seeded in each well of 96-well flat bottom plates and supplemented with RPMI and M199 media containing varying concentration of PF-543 (200µl/well) in each well. For intracellular amastigotes, 1 × 10⁶ THP-1 cells, treated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) were seeded on glass coverslip in a 6-well plate for 48h. They were infected with late log-phase L. donovani promastigotes at 20:1 MOI and simultaneously treated with PF-543. The cells were further incubated for 2 days at 37°C and 5% CO₂. To determine, the intracellular parasite burden (mean number of amastigotes per macrophage) were microscopically assessed using Giemsa staining. For axenic amastigotes, the axenically cultured forms grew optimally at a temperature of 32-33°C in a growth media with pH of 5.4. The IC₅₀ were found to be 450.7nM, 17.54μM, 45μM and 90μM for Leishmania donovani promastigotes, intracellular amastigotes axenic amastigotes and THP-1 macrophages respectively. Each experiment was done in triplicates and repeated thrice. (C) Effect of SphK1 inhibition on parasite infectivity in THP-1 macrophages. THP-1 cells grown in RPMI medium were treated with SphK1 inhibitor; PF-543 for 24h followed by infection with L. donovani promastigotes at an MOI of 20:1 for 48h. (i) After 48h of infection, Giemsa staining was performed to assess the parasite burden. THP-1 cells were fixed, Giemsa stained and amastigotes were counted visually under 100X using light microscope (Scale bar - 20 µm). Virulence capacity was determined by calculating infectivity (upper panel) and parasitemia (lower panel). Untreated infected THP-1 cells were used as control. (ii) Total RNA was enriched using TRIzol. Infectivity was validated using qRT-PCR with primer specific marker JW as a molecular indicator. Expression of JW mRNA in infected cells inferred parasite load and represented as percentage infectivity. Data analysis was performed using the 2^-ΔΔCTmethod. The results are representative of three independent experiments. Statistical significance was quantified using the unpaired t-test. The results signify mean ± S.D with n = 3, *P < 0.05.

https://doi.org/10.1371/journal.pntd.0013102.g001

To evaluate the anti-parasitic efficacy and host cytotoxicity profile of PF-543, dose–response analyses were carried out in different biological systems: L. donovani promastigotes (LdBob strain/MHOM/SD/62/1SCL2D), axenic amastigotes, intracellular amastigotes, and host THP-1 macrophages (an acute monocytic leukaemia-derived human cell line, American Type Culture Collection, Rockville, MD). Cytotoxicity was quantified using the MTT assay after 48h of treatment (Fig 1B). It was found to be most effective against the promastigote stage of Leishmania donovani (IC₅₀ = 450.7nM), moderately effective against intracellular amastigotes (IC₅₀ = 17.54μM), and axenic amastigotes (IC₅₀ = 45μM), while demonstrating the least potency and cytotoxicity against host THP-1 macrophages (IC₅₀ = 90μM) (Fig 1B). These findings suggest that PF-543 possesses potent, stage-specific anti-leishmanial activity, with marked selectivity toward the parasite over host cells. PF-543 demonstrated moderate inhibition of promastigote SphK1 activity and exhibited micromolar-range IC₅₀ values against infective parasite stages, including intracellular amastigotes (>10µM), with modest selectivity (~2-fold) relative to host cells (Fig 1B). Importantly, cytotoxicity towards host THP-1 macrophages remained higher (~90µM) than the intracellular amastigote IC₅₀ (~17µM), supporting a therapeutic window favouring host-mediated parasite control rather than direct parasiticidal activity reported. These findings indicate dual targeting potency towards Leishmania and host.

The pronounced susceptibility of extracellular promastigotes compared to intracellular amastigotes aligns with earlier studies demonstrating similar stage-dependent sensitivity differences across Leishmania species [58–60]. The reduced efficacy in the intracellular stage likely reflects the protective environment provided by the parasitophorous vacuole within host macrophages, which can limit drug access and potency.

To further discern whether the observed anti-parasitic effect was specific to SphK1 inhibition, the role of SphK2 was examined using ABC294640, a selective SphK2 inhibitor. Interestingly, ABC294640 exhibited comparatively higher cytotoxicity towards host THP-1 macrophages (IC₅₀ = 60μM) and intracellular amastigotes (IC₅₀ = 69.7μM) (S2 Fig), underscoring that PF-543 selectively targets the SphK1 pathway with a more favourable therapeutic window.

Collectively, these findings establish PF-543 as a potent and selective inhibitor of Leishmania donovani SphK1, effectively impairing parasite growth while exhibiting limited host cell toxicity. This highlights SphK1 as a promising molecular target for anti-leishmanial drug development.

To further evaluate the effect of PF-543 on the intracellular survival of L. donovani, THP-1 cells were pre-treated with PF-543 (90µM) for 24h prior to infection with L. donovani at an MOI of 20:1. The intracellular parasite burden was assessed 48h post-infection using Giemsa staining and RT-PCR. Untreated- uninfected macrophages (UT-Ld) served as experimental controls whereas untreated, infected macrophages (UT + Ld) represented the baseline infection group. The UT + Ld group exhibited an infection rate of approximately 80%, confirming efficient infection under experimental conditions. In contrast, PF-543-treated infected macrophages (PF-543 + Ld) showed a marked reduction in intracellular infection, with only ~60% of macrophages harbouring amastigotes (Fig 1Ci). Upon comparing the clearance of parasitic load in PF-543 treated infected THP-1 cells (PF543 + Ld) with untreated infected THP-1 (UN + Ld), ~ 40% reduction in the number of parasites (amastigotes/macrophage) was detected per infected macrophage (Fig 1Cii).

Collectively, these findings support a dual-acting mode of action, wherein PF-543 induces moderate inhibition of parasite SphK1 while simultaneously modulating host cellular pathways that restrict intracellular parasite survival.

2. Expression and Purification of the recombinant Leishmania SphK1 (rLdSphK1) protein and its Functional Characterization in presence and absence of SphK1 inhibitor- PF-543

LdSphk1 (1–2808 bp Nucleotide sequence, S3A Fig) was successfully cloned in pET-28 a(+) vector with N-terminal 6x-Histidine-tag (S3B Fig) and recombinant protein was purified using affinity based purification method (S3C Fig). For this, we went for expression and purification of the recombinant LdSphK1 (rLdSphK1) protein and observed a single band corresponding to ~102 kDa on SDS PAGE (Fig 2A) corresponding to Leishmania specific SphK1. This confirmed the availability of purified enzyme suitable for functional and structural analyses.

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Fig 2. Purification, subcellular localization, and functional characterization of recombinant rLdSphK1 in Leishmania donovani in the presence and absence of the SphK1 inhibitor PF-543.

(A) Concentrated rLdSphK1 protein. Gel image showing a purified band of 102 kDa recombinant LdSphK1 (B) The subcellular localization of recombinant LdSphK1 was investigated in promastigotes. (i) DeepTMHMM results predicted globular location with no transmembrane domains with a probability of 1. (ii) Panel A: DIC at 60X. Panel B: DAPI. Panel C: anti-LdSphK1 antibody detected using FITC (green)-conjugated secondary antibody. Panel D: merged micrographs (Scale bar - 20 µm). LdSphK1 expression represented in the green channel. Co-localization shown as the merged image. (C) In silico docking of the rLdSphK1 in presence of PF-543. In-silico ligand-substrate interaction was done using Autodock 1.5.7rc1 and Cygwin terminal. The in silico docking generated the complex of rLdSphK1-PF-543 and represented as surface model highlighted with residues involved in the interaction using Chimera, Ligplus, Discovery Studio v19.1.0, and PyMOL v2.3.2 software. (D) Biophysical interaction and functional characterization of the rLdSphK1 (20 µM) in presence of PF-543. MST analysis confirms the interaction of rLdSphK1 with PF-543 and ABC294640. Dose-response curves of rLdSphK1–PF-543 showed K_D of 29.3 µM and no interaction between rLdSphK1 and ABC294640. (E) Analysis of functional characterization of the LdSphK1 recombinant protein. (i) Time kinetics of activity of purified rLdSphK1 and NBD-S1P-based fluorometric assay showed increase in NBD-SIP levels under the influence of varying concentrations (20ng - 500ng) of rLdSphK1 protein. (ii) The inset shows inhibition of NBD-SIP levels in the presence of different concentrations of PF-543 (0.01µM - 5µM) using 200ng of recombinant LdSphK1 in view of sharp increase of NBD-S1P levels at 200ng of rLdSphK1 protein observed in previous experiment.

https://doi.org/10.1371/journal.pntd.0013102.g002

To investigate the cellular location of rLdSphK1, a DeepTMHMM-based in silico topology analysis was performed. The results predicted, with high confidence that LdSphK1 is a globular, intracellular protein with zero transmembrane domains, confirming its soluble, intracellular nature. This finding aligns with our immunofluorescence data and further supports previous reports [15] describing cytoplasmic and membrane-associated localization of SphK1 in eukaryotic systems. Consistent with these observations, subcellular localization of rLdSphK1 was investigated in L. donovani Bob cells using confocal microscopy to validate the in-house–generated anti-rLdSphK1 antibody. Immunostaining of L. donovani revealed predominant cytoplasmic and peripheral membranous localization of rLdSphK1 (Fig 2Bii), in agreement with its predicted non-integral membrane nature.

Owing to the fact of the recent development in structural biology, notably with the awarding of the 2024 Nobel Prize in Chemistry to Demis Hassabis and John Jumper for their pioneering work on the AlphaFold model, our analysis also incorporated AlphaFold-predicted structures for comparative modelling where-in both AlphaFold and a 3D homology model of LdSphK1 were generated. The 3D homology model of LdSphK1 was validated by its stereochemical statistics, featuring a MolProbity score of 1.74 and 81.99% of residues in favoured Ramachandran regions, which were superior to the AlphaFold model (S4A and S4B Fig). Crucially, we assessed the local quality of the binding pocket predicted by both models. While the key interacting residues from our docking simulation (e.g., ARG589, GLU586, etc.) were confirmed to be in Ramachandran-favoured regions in our homology model, the overall environment of the binding site is of significantly higher quality. The near-perfect clash score of 0.29 in our model compared to 2.07 for the AlphaFold model indicates a much more physically realistic and geometrically accurate binding pocket, which is essential for reliable docking (S4A and S4B Fig). Given that our homology model demonstrates a superior MolProbity score and a virtually clash-free binding site, we concluded it provides a more reliable foundation for our docking analysis.

With a validated structural model, docking analysis of the LdSphK1 protein with the inhibitor PF-543 was performed. The optimal conformations of the docked compounds were chosen based on their minimum free binding energy to the binding domain. Upon analysis of this pocket, it was discovered that there is a single hydrogen bond between residue ARG589 and ligand, with a distance of 2.03 Å. In addition, GLU586, ARG591, ARG596, LEU632, and ASP636 established hydrophobic contacts within the pocket. The binding of PF-543 to the protein is facilitated by these interactions, which result in a minimum binding energy of -7.93 kcal/mol (Fig 2C). The analysis of these results indicates that PF-543 creates a stable structure when bound to the LdSphK1 binding pocket.

Our analysis revealed a significant and exploitable difference between the parasite and human enzymes. The crystal structure shows that PF-543 is anchored in the human SphK1 active site primarily via hydrogen bonds to ASP264 [55]. Our docking model for LdSphK1, however, predicts that the key anchoring interaction is a hydrogen bond with Arg589 (Fig 2C). Notably, the binding mode of PF-543 in LdSphK1 is distinct from human SphK1, further establishing the specificity of this interaction.

To validate the biophysical interaction between purified rLdSphK1 and PF-543 and analyze the binding and kinetics, Microscale Thermophoresis (MST) was performed in NanoTemper Monolith NT.115 instrument. Herein, labelled rLdSphK1 (20 µM) was titrated against varying concentrations of PF-543 (500nM, 1µM, 2.5µM, 5µM, 10µM, 20µM). The dose response analysis for rLdSphK1-PF-543 binding revealed dissociation constant, K_D to be 29.3 µM, suggesting measurable and specific binding of rLdSphK1 with PF-543 (Fig 2D). Additionally, we also evaluated interaction between rLdSphK1 and ABC294640 as a negative experimental control where no such binding between the two could be detected, underscoring the specificity of PF-543 for rLdSphK1.

To evaluate the functional competence of rLdSphK1, the time course kinetics for rLdSphK1 was done using NBD-Sphingosine based fluorometric assay for detection of NBD-S1P levels Across the tested enzyme range (20ng to 500ng), a pronounced increase in NBD-S1P levels was observed as rLdSphK1 concentration increased from 200ng to 500ng (Fig 2Ei), following which saturation in activity was detected. In addition, the inhibition of rLdSphK1 catalytic activity was also evaluated in a dose dependent manner with PF-543’s concentration ranging from 0.01µM to 5µM. The results represented IC₅₀ close to 500nM (used for experimentation) and a maximum inhibition at 5µM of PF-543, while the protein concentration was kept constant at 200ng (Fig 2Eii), further corroborating effective target engagement.

To detect Km and Vmax values, varying concentration of NBD Sphingosine (2–10µM) was taken along with 200ng of rLdSphK1. The results confirmed 10.48μM and 10.91μM/sec as Km and Vmax respectively, according to Lineweaver-Burk plots, while D-erythro-sphingosine is used as the substrate for LdSphK1 (S3D Fig), confirming robust enzyme activity under these conditions. Our in silico docking, MST, and enzymatic inhibition data are internally consistent, all supporting specific binding to parasite SphK1.

3. Expression and Purification of the recombinant human SphK1 (rhSphK1) protein and its Functional Characterization in presence and absence of SphK1 inhibitor- PF-543

To evaluate the similarities and evolutionary divergence between Leishmania and human SphK1, we first performed amino acid sequence alignment of Ld_SphK1 and human_SphK1 using the MEGAX program (version 10.2.2). The analysis revealed considerable agreement, with 26% sequence identity over 58% query coverage (S5A Fig). A phylogenetic tree was also constructed to assess the evolutionary relationship between Leishmania SphK1 and its orthologs. The resulting tree shows a clear separation between the kinetoplastid and mammalian enzymes, which form distinct, highly supported clades. Within the kinetoplastids, all Leishmania species form a monophyletic group, demonstrating the conservation of this enzyme across the genus and its divergence from the host enzymes (S5B Fig). Further, we conducted 3D structural alignment of the two protein structures with Pymol software. We observed substantial alignment between the two proteins, with an RMSD of 0.585Å (S5C Fig), demonstrating notable conservation of key structural features despite low overall sequence identity with notable correspondence between the alpha helices and beta sheets of the human (blue) and Leishmania (yellow) DAGKc domains (S5C Fig).

Building on this, the humanSphk1 gene was successfully cloned in pET-28 a(+) expression vector, and the recombinant plasmid was transformed into E. coli strain BL21 (ʎDE3) gold. Later, we went for expression and purification of the recombinant humanSphK1 (rhumanSphK1) protein (S6A Fig). A single band corresponding to ~50 kDa was observed on SDS PAGE (Fig 3A) corresponding to recombinant human specific SphK1.

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Fig 3. (A) Purification of Recombinant rhSphK1 from Supernatant.

Gel image showing a concentrated rhSphK1 protein band of 50kDa. (B) In silico docking of the rhumanSphK1 in presence of PF-543. In silico ligand-substrate interaction was done using Autodock 1.5.7rc1 and Cygwin terminal. The in silico bound complex of rhSphK1-PF-543 was represented as surface model highlighted with residues involved in the interaction using Chimera, Ligplus, Discovery Studio v19.1.0, and PyMOL v2.3.2 software. The list of residues involved in hydrophobic interaction and hydrogen bond formation were highlighted in the model. (C) Functional characterization of the rhSphK1 in presence of PF-543. MST analysis confirms the biophysical interaction of rhSphK1 with (i) PF-543 and (ii) ABC294640, which was used as a control. Dose-response curves of PF-543 (20µM) and ABC294640 showed K_D of 34.3µM and 70.6µM respectively. (D) Analysis of catalytic activity of the rhSphK1 using NBD-S1P-based fluorometric assay. Time kinetics of activity of purified rhSphK1 demonstrated increase in NBD-SIP levels in presence of 200ng of rhSphK1 protein.

https://doi.org/10.1371/journal.pntd.0013102.g003

To elucidate the potential for pharmacological targeting of hSphK1, in-silico docking studies were performed between the protein and the SphK1 inhibitor PF-543. Upon examination of this pocket, it was discovered that it contains two residues, namely HIS156 and THR157, which are linked together by hydrogen bonds. The hydrogen link between them has a distance of 3.44 Å, while the other hydrogen bond has a distance of 2.53 Å. Similarly, GLU93, LEU161, VAL227, GLN230, GLN231, and PRO240 established hydrophobic interactions within the pocket. The binding of PF-543 to the protein is facilitated by these interactions, resulting in a minimum binding energy of -8.59 kcal/mol (Fig 3B). The analysis of these results indicates that PF-543 creates a stable structure when bound to the hSphK1 binding pocket. These data inferred that rhSphK1 and PF-543 are possible interacting partners having favourable binding energy.

To corroborate these findings experimentally, biophysical binding assays (MST) was performed in NanoTemper Monolith NT.115 instrument. Herein, labeled rhSphK1 (20 µM) was titrated against varying concentrations of PF-543 and the SphK2 inhibitor ABC294640 as a control. Dose–response analysis revealed dissociation constants (K_D) of 34.3 µM for rhSphK1–PF-543 and 70.6 µM for rhSphK1–ABC294640, confirming specific and preferential binding (Fig 3C). Here, rhSphK1 MST experiments were performed under identical buffer and labeling conditions as rLdSphK1 to enable direct comparison, not to re-define canonical affinity. We clearly state that experimental MST-derived K_D values are method- and context-dependent and do not replace previously published enzymatic IC₅₀ values (reported in nanomolar range).

Functional activity of the recombinant protein was assessed using NBD-sphingosine–based fluorometric detection of NBD-S1P formation. Kinetic analysis represented a steep increment in generation of NBD-S1P level over time with 200 ng rhSphK1 (Fig 3D) [45, as standardized for rLdSphK1], following which saturation in activity was detected. To detect Km and Vmax values, varying concentration of NBD Sphingosine (2–10 µM) was taken along with 200 ng of rhSphK1. The results confirmed 9.56μM and 9.13μM/sec as Km and Vmax respectively with D-erythro-sphingosine is used as the substrate for rhSphK1 (S6B Fig).

Together, these results demonstrate the successful expression and purification of functional recombinant human SphK1, demonstrate its evolutionary and structural conservation with the Leishmania enzyme, and confirm robust inhibitor binding and enzymatic activity under defined experimental conditions.

4. Effect of PF-543 on cytotoxicity and infectivity of L. donovani SphK1 overexpressor (LdSphKa) promastigotes

To verify transient genetic manipulation, Leishmania donovani promastigotes were successfully transfected with the pXG-GFP-SphKa construct (Fig 4A), in which the SphK1 gene was fused at the 3′ end of GFP to enable fusion protein detection. Confocal microscopy revealed a distinct population of GFP-positive parasites, confirming transgene expression, with a subset of GFP-negative cells and untransfected controls included to validate specificity (Fig 4B). This robust GFP signal established the basis for subsequent functional studies.

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Fig 4. Effect of PF-543 on cytotoxicity and infectivity of L. donovani SphK1 overexpressor promastigote cell line.

(A) Vector map showing the construct designed for pXG plasmid containing SphKa gene and GFP for the overexpression of the gene through transfection in LdBob cells. (B) Confocal microscopy images confirming successful integration of the plasmid. Integration was verified by the presence of GFP fluorescence signal in the transfected parasites. (C) (i) Estimation of LdSphK-1-mediated generation of NBD-SIP levels against PF-543 in L. donovani SphKa promastigotes. L. donovani bob promastigotes were cultured in 25 cm² flasks followed by treatment with SphK1 inhibitor; PF-543 for 48h. Inhibition of SphK1 using PF-543 had no significant effect on S1P levels. SphK inhibitor treated SphKa promastigotes were resuspended in buffer containing fatty acid-free BSA (0.1% (w/v)) followed by resuspension in buffer containing BSA (1% (w/v)) and incubated with NBD-sphingosine (10μM) for 45 min at 37°C. Promastigotes incorporate NBD-sphingosine (NBD-Sph), which are phosphorylated by Sphingosine Kinase (SphK1) to NBD-S1P. Bar graph depicts ELISA-based S1P quantification in SphKa promastigotes treated with PF-543 using LdBob as control. (ii) Comparative analysis of the half maximal inhibitory concentration of PF543 against the wild type LdBob and LdSphka OE line. (D) Effect of SphK1 inhibitor; PF-543 in Leishmania overexpressor spp. (i) Effect of L. donovani SphK1 overexpression on intracellular parasite clearance. THP-1 macrophages were cultured in six-well plates in the presence or absence of L. donovani (LdBob) infection (MOI, 20:1) for 6h. Further, THP-1 cells were infected with L. donovani SphK1 overexpressor promastigotes at various MOIs (10:1, 20:1, 30:1, 50:1). Infected THP-1 were washed to remove non-internalized parasites. Total RNA was enriched using TRIzol and the resulting cDNA was subjected to real-time PCR analysis using primers specific for infectivity (JW) genes in infected macrophages. RNU6A was used as a housekeeping gene. The results are expressed as fold-change of uninfected THP-1 cells. Statistical significance was quantified using the unpaired t-test. The data is a representation of mean ± SD from three independent experiments *, p < 0.05; ***, p < 0.001. (ii) GFP-based confirmation of SphK1 overexpression in L. donovani promastigotes. Detection of the presence of GFP encoded SphKa and Bob plasmids in Leishmania donovani and Leishmania overexpressor parasites. L. donovani bob and L. donovani overexpressor promastigotes were cultured in 25 cm² flasks followed by confocal microscopy.

https://doi.org/10.1371/journal.pntd.0013102.g004

The functional activity of Leishmania-specific SphK1 in over-expressor promastigotes (LdSphKa) was confirmed using fluorometric quantification of NBD-tagged sphingosine conversion. Treatment with generic SphK inhibitors (PF-543, DMS, SKI-5C) resulted in a marked reduction in NBD-S1P fluorescence intensity compared to untreated LdBob promastigotes, demonstrating effective inhibition of SphK catalytic activity across all tested compounds and doses (500nM PF-543; 100 µM SKI-5C; 500 µM DMS). Notably, LdSphKa-overexpressing parasites displayed elevated baseline NBD-S1P levels relative to LdBob controls, directly supporting both the overexpression and functional activity of the enzyme (Fig 4Ci). These data establish that LdSphK1 overexpression leads to enhanced SphK1 enzymatic output in Leishmania donovani.

Further dose-response inhibition studies demonstrated that each inhibitor showed comparable suppression of SphK1 activity in LdSphKa lines. These findings are mechanistically explained by the greater absolute SphK1 expression in LdSphKa compared to LdBob, resulting in negligible changes in activity for LdBob at inhibitor concentrations used. Thus, these data confirm both the inhibitor specificity and the physiological relevance of SphK1 overexpression in mediating enzymatic output.

Given the robust inhibition by PF-543, its potency was analyzed in greater detail. MTT cytotoxicity assays revealed that PF-543 elicited an IC₅₀ of 36.04µM against LdSphKa promastigotes (Fig 4C ii), which was higher than observed for wild-type L. donovani promastigotes, indicating a degree of resistance conferred by SphK1 overexpression, likely through modulation of downstream lipid signaling pathways.

The functional consequences of SphK1 overexpression on parasite infectivity were assessed through infection of THP-1 cells at multiple MOIs (10:1, 20:1, 30:1, 50:1), with intracellular amastigote loads quantified by RT-PCR 48h post-infection. LdSphKa-infected THP-1 cells exhibited pronounced infection rates (approximately 86% at MOI 10:1 versus ~80% at MOI 20:1 for LdBob controls), with higher MOIs further supporting a rapid-growth, high-infectivity phenotype (Fig 4D i). Strong GFP fluorescence in LdSphKa promastigotes and absence in LdBob controls (Fig 4D ii) further confirmed correct genetic targeting and specificity.

5. Decrease in Leishmania glutathione levels mediates ceramide-induced parasite death

Ceramide, a central sphingolipid metabolite, lies upstream of SphK1 in the sphingolipid biosynthetic pathway and is antagonized by Sphingosine-1-phosphate (S1P), an established pro-survival lipid mediator. During Leishmania infection, elevated ceramide production disrupts host membrane lipid rafts, lowers cholesterol, and impairs CD40-mediated signaling, ultimately weakening ERK1/2 activation and antigen presentation, contributing to parasite survival and disease progression [56].

To interrogate the role of ceramide in parasite-induced cytotoxicity, the ceramide analog DL-threo-PPMP, a known inhibitor of glucosylceramide synthase, was employed. Dose-response experiments revealed that exogenous DL-threo-PPMP (20μM) exerts a potent cytotoxic effect on THP-1 macrophages, as indicated by reduced cell survival (IC₅₀ = 19.53μM, Fig 5A). In contrast, the isomeric control DL-erythro-PPMP displayed significantly lower potency (IC₅₀ = 116μM, Fig 5B), validating the isomer-specific action of DL-threo-PPMP and supporting its selection for subsequent studies.

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Fig 5. (A) Determination of in vitro cytotoxicty of DL PPMP threo and DL PPMP erythro.

DLPPMP threo and DLPPMP erythro at varying concentrations (2µM, 5µM, 10µM, 20µM and 50µM) were incubated with THP-1 cells seeded 6 × 10³ in a 96-well plate for 48h. The cytotoxicity was analysed using colorimetric estimation of formazan crystals formed by the conversion of MTT by live cells. IC₅₀ determined by plotting the non-regression linear curve was 19.53µM and 116µM for DLPPMP threo and erythro respectively. (B) Estimation of Caspase 9 levels upon PF-543 treatment in Leishmania infected macrophages. (i) THP-1 macrophages were cultured in six-well plates in the presence of L. donovani infection (MOI, 20:1) for 6h. Infected THP-1 were washed to remove non-internalized parasites and treated with PF-543 (20μM) or DL-threo-PPMP; glucosylceramide synthase inhibitor (20μM) or SIP (10μM) for next 48h. Caspase-9 levels were analysed using cell lysates for Western blot. (ii) Band intensities were quantified by ImageJ software and were plotted in GraphPad Prism. Data from one of three experiments is shown.

https://doi.org/10.1371/journal.pntd.0013102.g005

Next, THP-1 cells infected with L. donovani (MOI 20:1) were exposed to DL-threo-PPMP. Subsequent biochemical analysis demonstrated that PPMP treatment led to a marked decrease in the GSH/GSSG ratio (indicative of oxidative stress, S7 Fig) and a pronounced induction of apoptosis, evidenced by elevated Caspase 9 expression (Fig 5C i). These findings confirm that ceramide accumulation drives parasite-induced host cell death through glutathione depletion and apoptotic signalling.

To dissect the functional interplay between ceramide and S1P, infected macrophages were co-treated with both DL-threo-PPMP and S1P (10μM, a non-toxic concentration for parasites). Remarkably, S1P supplementation abolished ceramide-induced cytotoxicity, restoring GSH/GSSG ratios and suppressing Caspase 9 activation (Fig 5Cii). Correlation analysis further revealed a strong negative association between glutathione levels and apoptotic signalling (Pearson r = –0.78, p = 0.05), supporting a direct mechanistic link.

Collectively, these data demonstrate that S1P acts as a downstream physiological antagonist to ceramide-induced stress and apoptosis in Leishmania-infected macrophages, highlighting the pivotal role of glutathione balance and the ceramide-SphK1-S1P axis in regulating host-parasite interactions and cell fate decisions.

6. Elucidation of inflammatory responses and infectivity post-PF-543 treatment in Leishmania infected macrophages

Cytokines play a crucial role in mediating T-cell responses and host defense mechanisms in macrophages. LPS potently activates macrophages and cytokine signalling by TLR4 stimulation, while Leishmania parasites suppress LPS-induced host inflammatory cytokine responses [57]. To probe the effect of SphK1 pathway inhibition on host inflammatory balance, we assessed cytokine profiles following PF-543 treatment of LPS-stimulated, Leishmania-infected macrophages. We found that PF-543 post-treatment in infected macrophages reduced the levels of anti-inflammatory cytokine IL-10 (~80%) as confirmed by RT-PCR and ELISA while the level of pro-inflammatory cytokines IL32γ, IL-12 and TNF-α remain unaltered (Fig 6Ai, and 6Aii). We further evaluated the parasite load in presence of PF-543 using qRT-PCR based on estimation of JW (kDNA) mRNA levels in PF-543 treated and untreated infected cells. The results suggested PF-543 post-treatment leads to decrease (_~50%) in parasite load in infected macrophages (Fig 6B), thereby indicating the role of PF-543 in reducing the parasite survival.

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Fig 6. (A(i), B) Effect of PF-543 on the expression of inflammatory molecules and clearance of intracellular parasite load.

THP-1 macrophages were cultured in six-well plates in the presence or absence of L. donovani infection (MOI, 20:1) for 6h. Infected THP-1 were washed to remove non-internalized parasites and treated with PF-543 for next 48h. Total RNA was enriched using TRIzol and the resulting cDNA was subjected to real-time PCR analysis using primers specific for inflammatory (IL32γ, IL-12, IL-10 and TNF-α) and infectivity (JW) genes in PF-543 treated and untreated infected macrophages. RNU6A was used as a housekeeping gene. The results are expressed as fold-change of uninfected THP-1 cells. Statistical significance was quantified using the unpaired t-test. The data is a representation of mean ± SD from three independent experiments *, p < 0.05; ***, p < 0.001. (A (ii)) Graph represents cytokine levels measured by sandwich ELISA. Statistical significance was quantified using the unpaired t-test with Welch’s correction, in LPS-stimulated, Ld-infected and PF-543 treated macrophages.

https://doi.org/10.1371/journal.pntd.0013102.g006

Together, these findings highlight the key role of the SphK1/S1P axis in regulating anti-inflammatory cytokine production and parasite persistence within macrophages, and demonstrate that pharmacological inhibition with PF-543 has both immunomodulatory and anti-parasitic effects in the context of Leishmania infection.

7. Evaluation of antileishmanial activity of PF-543 and Amphotericin B combined formulation against L. donovani promastigotes and intracellular amastigotes

Amphotericin B (Amph-B) remains a cornerstone of visceral leishmaniasis treatment [58] due to its ergosterol-binding mechanism, which disrupts protozoan membrane integrity and induces cell death [59]. To explore the therapeutic potential of combining PF-543, a SphK1 inhibitor, with Amph-B, in vitro anti-leishmanial assays were conducted in L. donovani promastigotes using the MTT assay. The in-vitro synergistic activity of PF-543 along with Amphotericin B was represented by an isobologram. Upon analysis of antileishmanial activity, Amphotericin B showed an IC₅₀ of 30nM and PF-543 showed IC₅₀ of 500nM alone (as already shown in Fig 1B) however upon administering both PF-543 and Amphotericin B in combination, synergistic impact was prominent, and the IC₅₀ was observed to be ~ 12.5nM and 150nM for Amphotericin B and PF-543 respectively, as depicted by the isobologram micrographs in promastigotes (Fig 7A).

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Fig 7. (A-B). Evaluation of PF-543 and Amphotericin B formulation on cytotoxicity or metabolic viability of Leishmania promastigotes and intracellular amastigotes.

(A) To determine the combinatorial impact of PF-543 and Amphotericin B formulation, promastigotes of L. donovani were seeded in 96-well plates (3 × 10⁶ parasite/mL) in exponential growth phase at increasing concentrations (12.5nM–500nM) of Amphotericin B and (150nM- 2000nM) of PF-543 compounds and maintained at 26°C. After 48h of incubation, the IC₅₀ was determined by MTT assay. The absorbance was measured in a MultiskanEX photometric plate reader for microplates at 540 nm. Data were obtained from three independent experiments performed in triplicate. The fractional inhibitory concentration index (FIC) was evaluated to determine the synergistic activity of lead compounds; PF-543 with Amph-B. Finally, FIC < 0.5 represented strong synergistic activity between two drugs. While FIC > 4 is was considered as the antagonistic activity. FIC values were obtained from three independent experiments. (B) For validating the antileishmanial activity by PF-543 and Amphotericin B formulation on intracellular amastigotes, qPCR-based analysis was performed for all the treatments. For the same, THP-1 cells were seeded at a concentration of 8 × 10⁵ cells/mL in 96-well plates and incubated for 24h with PMA (10ng/mL) supplemented with RPMI 1640. Following 24h priming with PMA, the culture medium was removed, and the cells were infected with L. donovani followed by treatment with the formulations having concentrations kept similar as used previously for promastigotes at 37°C and 5% CO₂. Total RNA was extracted, and the resulting cDNA was subjected to real-time PCR analysis using primers specific for kinetoplast minicircle DNA (kDNA)/JW of L. donovani. This primer does not match to human genome, thus directly indicates the intracellular parasite load against the treatments. Data analysis was performed using the 2^-ΔΔCTmethod.Values are mean ± S.D (n = 3). The results are representative of three independent experiments performed in triplicates.

https://doi.org/10.1371/journal.pntd.0013102.g007

For validating the anti-leishmanial activity by PF-543 and Amphotericin B formulation on intracellular amastigotes, qPCR-based analysis was performed for all the treatments. Here-in the synergistic treatment of Amphotericin B (12.5nM) and PF-543 (150nM) formulation to intracellular amastigotes reduced the parasite growth by 60%. Whereas, treatment with PF-543 or Amphotericin B alone decreased the parasite burden by 30%. Further reduction of 40% and 60% in parasite growth was observed with usage of higher dosage of PF-543 alone (250nM, 500nM and 2000nM), while these doses in combination with Amphotericin B (12.5nM + 150nM, 12.5nM + 250nM, 12.5nM + 500nM, 12.5nM + 2000nM) showed higher reduction of parasite growth to ~80–90% (Fig 7B). These data clearly suggested that PF-543 along with Amphotericin B can be a promising potent formulation to be tested.

8. Elucidation of inflammatory responses and infectivity post-PF-543 treatment in Leishmania-infected Swiss mice

PF-543 is a potent and selective sphingosine kinase 1 (SphK1) inhibitor that has shown notable anti-cancer activity in preclinical studies by blocking SphK1-mediated signaling pathways, leading to the induction of apoptosis and growth inhibition in various cancer cell lines. It is also reported to exhibit low systemic toxicity at experimentally determined doses in mice (typical half-life in mice is approximately 1–1.2 hours). Amphotericin B, by contrast, is a well-established anti-leishmanial agent approved for clinical use. Both were selected for the present study due to established safety and efficacy profiles in vivo.

To investigate the anti-leishmanial efficacy and immunomodulatory impact of PF-543 in a murine model, Swiss mice were infected with L. donovani and subsequently treated intraperitoneally with PF-543 (10 mg/kg), Amphotericin B (2 mg/kg), or a combination regimen (PF-543 2 mg/kg + Amph-B 0.4 mg/kg) for three consecutive days. Dose selection was based on prior reports and pilot tolerability studies, ensuring all regimens were well tolerated without adverse effects or significant weight loss. These criteria align with established clinical protocols for Amphotericin B and emerging preclinical data for SphK1 inhibitors.

To evaluate the parasitaemia and expression of inflammatory markers in infected mice upon drug administration, spleen was harvested from each group of mice and qRT-PCR analysis was performed using primers specific for inflammatory markers; mTNF-α and mIL-10 (S1 Table) and parasite specific kinetoplast minicircle gene; JW in PF-543 and Amph-B treated infected mice in respect to the untreated Swiss mice.

In concordance with the published studies, here-in, upon analysis, treatments with either Amphotericin B or PF-543 alone in infected mice resulted in a significant decrease in IL-10 expression (~60%), whereas increased TNF-α expression was observed (1.5-2 folds) (Fig 8A). Further, this effect on inflammatory markers was more pronounced upon the synergistic treatment of Amphotericin B and PF-543 formulation to Leishmania infected Swiss mice (~90% decrease in IL-10 & 3 folds increase in TNF-α) (Fig 8A). Amplification of RNU6AP (RNA, U6 small nuclear 1; THP-1 cells) was used as an internal control for normalization.

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Fig 8. Expression of JW and inflammatory markers upon PF-543 treatment in Leishmania-infected Swiss mice.

Evaluation of the (A) immunomodulatory potential and (B) infectivity of PF-543 and Amph-B in Leishmania-infected Swiss mice was performed. 10mg/Kg of PF-543 and 2mg/Kg of Amphotericin B alone were given for individual treatment in Leishmania infected mice. For constituting the synergistic effect of two drug partners; 2mg/Kg (1mM) of PF-543 and 0.4mg/Kg (0.12µM) of Amphotericin B were prepared in 1XPBS and given together to a mouse weighing 30gm. To evaluate the parasitaemia and expression of inflammatory markers in infected mice upon drug administration, spleen was harvested from each group of mice and qRT-PCR analysis was performed using primers specific for inflammatory markers IL-10 and TNFα and parasite specific kinetoplast minicircle gene, JW in PF-543 and Amph-B treated infected mice in respect to the untreated Swiss mice. RNU6A (RNA, U6 small nuclear 1; THP-1 cells) was used as a housekeeping gene. The results were expressed as normalized fold-change of respective control. Statistical significance was quantified using the unpaired t-test. The data is a representation of mean ± SD from three independent experiments *, p < 0.05; ***, p < 0.001.

https://doi.org/10.1371/journal.pntd.0013102.g008

Most importantly, parasite load was also observed to be reduced upon treatment with PF-543 or Amphotericin B alone in infected mice (Fig 8B) where ~33% and ~80% reduction in parasite growth was observed respectively. Further, this down-regulation in parasite burden was drastically reduced to >90% (as demarcated by reduction in JW transcript) upon synergistic treatment of PF-543 and Amphotericin B formulation (Fig 8B).

Collectively, these in vivo results affirm that combination therapy with PF-543 and Amphotericin B leads to enhanced protective immune responses, substantially reduced regulatory cytokine (IL-10) levels, increased pro-inflammatory milieu (TNF-α), and synergistic clearance of L. donovani in an experimental mouse model. This highlights the promise of SphK1-targeted interventions as adjuncts in anti-leishmanial therapy.