1. Cell Biology
  2. Microbiology and Infectious Disease

Scrutinized lipid utilization disrupts Amphotericin-B responsiveness in clinical isolates of Leishmania donovani

  1. Supratim Pradhan
  2. Dhruba Dhar
  3. Debolina Manna
  4. Shubhangi Chakraborty
  5. Arkapriya Bhattacharyya
  6. Khushi Chauhan
  7. Rimi Mukherjee
  8. Abhik Sen
  9. Krishna Pandey
  10. Soumen Das
  11. Budhaditya Mukherjee  Is a corresponding author
  1. School of Medical Science and Technology, Indian Institute of Technology Kharagpur, India
  2. ICMR-Rajendra Memorial Research Institute of Medical Sciences, India
https://doi.org/10.7554/eLife.102857.3
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Cite this article
  1. Supratim Pradhan
  2. Dhruba Dhar
  3. Debolina Manna
  4. Shubhangi Chakraborty
  5. Arkapriya Bhattacharyya
  6. Khushi Chauhan
  7. Rimi Mukherjee
  8. Abhik Sen
  9. Krishna Pandey
  10. Soumen Das
  11. Budhaditya Mukherjee
(2025)
Scrutinized lipid utilization disrupts Amphotericin-B responsiveness in clinical isolates of Leishmania donovani
eLife 14:RP102857.
https://doi.org/10.7554/eLife.102857.3
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11 figures, 4 videos, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Severe dyslipidemia is linked with higher intracellular proliferation of LD-R amastigotes.

(A) Serum lipid profile of 28 days LD-infected Balb/c mice (N = 5). Equal number of sorted metacyclics promastigotes of two representative LD-S (LD-S1, LD-S2) and LD-R (LD-R1, LD-R2) strains were used to perform independent infection. (B) Number of attached LDs on Kupffer cell (KC) surface 4 hrs p.i, data represent (N = 3) for two LD-S and two LD-R strains. (C) (i) Representative Giemsa stain images of LD-S1 and LD-R1-infected KC at 4, 24, and 48 hr p.i. Black arrow represents LD amastigotes. N denotes host nucleus. Scale bar: 20 μM. Giemsa Images are represented in gray scale to clearly represent LD nucleus (black arrow). (C) (ii) Intracellular amastigote count for 4, 24, and 48 hr p.i. Each dot represents count from 100 infected KC (N = 6). (D) (i) LD-infected-KC imaged to visualize the parasitophorous vacuole (PV) at 4 and 24 hr p.i. PV marked by ATP6V0D2 (magenta), KC and LD nucleus (blue), Scale bar: 5 μm. Yellow arrow represents LD amastigotes. In the merged image, white dotted line marks KC’s periphery, and host nucleus is represented with N. (D) (ii) PV counts measured from 30 infected KCs. (D) (iii) Confocal 3D reconstruction illustrating the spatial distribution of PVs in Kupffer cells (KCs) infected for 24 hr. ATP6V0D2, a lysosomal vacuolar ATPase subunit, is visualized in magenta, while the nucleus is depicted in cyan. The final panel highlights PV structural grooves outlined in red solid lines, with intracellular Leishmania donovani (LD) amastigotes indicated by white arrows. Higher magnification of (D) further emphasizes the increased abundance of PVs in LD-R infected cells, suggesting enhanced intracellular replication and adaptation mechanisms of drug-resistant strains. Scale bar: 5 µM. Both yellow and magenta solid line box represents the same area of the image. *** signifies p-value <0.0001, * signifies p-value <0.05, n.s., non-significant.

Figure 1—figure supplement 1
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Kupffer cells (KCs) as an in vitro model for LD-infection.

(A) Flow cytometry representing % Metacyclics LD: (i) LD-S (AG83, DD8) and (ii) LD-R (BHU814, BHU575) based on FSC and SSC. Left, small box with low FSC and SSC gate for metacyclic parasites and right panel with high FSC and SSC gated procyclic parasites. (B) Flow cytometry of F4/80-PE-positive KC from the non-parenchymal cell population isolate from murine liver. (C) Representative Giemsa-stained images LD-infected-PEC, 4 and 24 hr p.i. for (i) LD-S and (ii) LD-R strains. Giemsa images are represented in gray scale to clearly represent LD nucleus (black arrow). Scale bar: 20 μM. (C) (iii) Intracellular amastigote count for 4 and 24 hr p.i. Each dot represents count from 100 infected KC. N = 6. (D) Amastigote count from Giemsa-stained image of KC-infected with LD amastigotes, isolated from the spleen of 28 days LD-infected mice. Each dot represents mean from 100 infected KCs. N = 6. *** signifies p-value <0.0001, ** signifies p-value <0.001, n.s., non-significant.

Figure 2
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LD-R-infection results in a higher organ parasite load in hypercholesterolemic Apoe−/− mice.

(A) (i) Representative Giemsa images of LD-infected-KCs cultured in high and low lipid media. Black arrow represents LD amastigotes; N represents host nucleus. Scale bar: 20 μM. (A) (ii) Graph representing number of intracellular LD amastigotes in Kupffer cells (KCs) with LD infection performed either in high, low, or lipid-free media (N = 6). (B) (i) Giemsa images of LD-infected-KCs isolated from wild-type BL/6 and Apoe−/− mice. Scale bar: 20 μm, N representing host nucleus. Giemsa images are represented in gray scale to clearly represent LD nucleus (black arrow). (B) (ii) Amastigote count from 100 LD-infected-KCs (N = 6) as in (B) (i). Data are presented as mean ± SEM. (C) (i) Schematic representation of in vivo LD-infection in BL/6 and Apoe−/− mice performed with EGFP-LD-S or EGFP-LD-R. (C) (ii) Flow cytometry representing GFP-positive splenocytes isolated from 11 days EGFP-LD infected BL/6 and Apoe−/− mice. The black box indicates % of GFP-positive splenocytes (LD-infected). (C) (iii) Graph representing data from six infected mice as in (C) (ii) are presented. *** signifies p-value <0.0001, ** signifies p-value <0.001, * signifies p-value <0.05, n.s., non-significant.

Figure 3 with 2 supplements
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Endocytosed-low-density lipoprotein (LDL) is the primary lipid source for intracellular LD-R proliferation.

(A) (i) Live cell images of LDL-endocytosis in 24 hr post infected Kupffer cells (KCs) with LDL (red), LD (green), and the nucleus (blue). Images were acquired directly from 96-well glass bottom plate and bright field was merged with reduced brightness and increased contrast identically for all experimental conditions. Scale bar: 40 μm. Yellow arrow represents intracellular LD. (A) (ii) Graph representing fluorescence plate reader-based quantification of (A) (i). (B) (i) Co-localization of LDL with the LD-amastigotes in infected KCs 24 hr p.i. Merged image showing LDL (magenta), LD (green), nucleus (blue), white dotted line in the merged image demarcates infected KC for co-localization analysis. Scale bar: 10 μm. Yellow arrow represents LD nucleus and N represents host nucleus. (B) (ii) Intensity plot representing co-localized signals from (B) (i), marked with gray transparent box. (C) (i) Confocal image showing the isolated LD-S and LD-R-PV, stained with LAMP1 (magenta) and LD (green). Scale bar: 2 µm. (C) (ii) Western blot analysis validated the purification of parasitophorous vacuole (PV) fractions, indicated by LAMP-1 positivity, while the presence of LD parasites within the PV was confirmed by KMP-11 detection. (C) (iii) Western blot of cellular fraction without PV showing minimal presence of LAMP1. (D) (i) Gas chromatography–mass spectrometry (GC–MS) lipid profile of isolated PVs from LD-infected-KCs. Retention time 27.73 corresponding to the cholesterol and 28.56 corresponding to fatty acids are represented with * for LD-S-PV and with black arrowheads for LD-R-PV. (D) (ii) Relative abundance of cholesterol and fatty acids of (D) (i). (E) (i) Representative confocal Raman spectroscopy image of uninfected or LD-infected-KCs 24 hr p.i., illustrating distinct lipid-related signal distribution patterns marked with pseudo-colors. Yellow dotted circle in the left most panel demarcates the host cell nucleus while white arrow marking intracellular LD nucleus. In merged panel, white dotted line demarcates cells periphery. The right most panel shows dot plot representation of lipid distribution, where each color corresponds to different lipid-related peaks with respect to blue (B) indicating the nucleus. (E) (ii) Comparative Raman spectra from LD-S and LD-R-infected-KCs. Lipid-related peaks are demarcated with shaded box, representing 1. 540–560 cm−1 (cholesterol), 2. 1080–1090 cm−1 (phospholipids), 3. 1270–1280 cm−1 (triglycerides), 4. 1300–1340 cm−1 (Amide-III bond), 5. 1440–1453 cm−1 (fatty acids and triglycerides), and 6. 1650–1660 cm−1 (Amide-I bond). *** signifies p-value <0.0001, ** signifies p-value <0.001, * signifies p-value <0.05, n.s., non-significant.

Figure 3—source data 1

Source data for western blots shown in Figure 3Cii and iii.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig3-data1-v1.zip
Figure 3—source data 2

Source data for western blots shown in Figure 3Cii and iii, with relevant bands labelled.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig3-data2-v1.zip
Figure 3—figure supplement 1
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LD-S and LD-R quenches host membrane cholesterol during initial entry.

(A) (i) Kupffer cells (KCs) were pre-incubated with NBD-cholesterol for 16 hr. Subsequently, KCs were infected with RFP expressing LD-S or LD-R for 4 hr or left uninfected and imaged. NBD-cholesterol was excited at 488 nm, and LDs are presented with magenta pseudo color. White arrow indicates the NBD-cholesterol within LD while yellow arrow represents LD nucleus. Scale bar: 5 μm. (A) (ii) Co-localization analysis of LD (magenta), and NBD-cholesterol (green) from (A) (i), marked in gray transparent box. (B) (i) Representative flow cytometry-based analysis of NBD in LD-amastigotes isolated from LD-infected-KCs 2 hr after infection pre-incubated with NBD cholesterol. Black box representing % NBD-positive population. (B) (ii) Graphical representation of MFI for three independent experiments as in (B) (i). n.s., non-significant.

Figure 3—figure supplement 2
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LD-R-PV have higher accumulation of cholesterol and fatty acids.

(A) Detection of parasitophorous vacuole (PV) cholesterol, isolated from LD-S or LD-R-infected-KC. Data represented as μg of protein/ μg of cholesterol (N = 6). (B) Thin layer chromatography (TLC) profile of isolated lipids from 24 hr LD-infected-PVs with cholesterol standard kept as control. (C) Individual Raman spectra from six independent LD-infected-KCs (i) LD-S-infected and (ii) LD-R-infected. Lipid-related peaks are demarcated within the spectra, including those at 1.540–560 cm−1 (cholesterol), 2. 1080–1090 cm−1 (phospholipids), 3. 1270–1280 cm−1 (triglycerides), 4. 1300–1340 cm−1 (Amide-(II) I bond), 5. 1440–1453 cm−1 (fatty acids and triglycerides), and 6. 1650–1660 cm−1 (Amide-I bond). (D) Area under the curve, and shift are calculated and plotted for each experimental condition as represented in (C) (i) and (ii). ** signifies p-value <0.001.

Figure 4 with 1 supplement
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Increased low-density lipoprotein (LDL) uptake in LD-R-infected-MΦs through receptor-independent fluid-phase endocytosis.

(A) Immunofluorescence images of LDLr expression in LD-infected-KCs. Scale bar: 40 μm. Enlarged view of A showing infected KC with low LDLr expression (yellow arrow) compared to neighboring uninfected Kupffer cell (KC; white arrow) in the merged image. (B) (i) Cofilin and phosphorylated-Cofilin expression by western blot in LD-infected-KCs. (B) (ii) Graphical representation of comparative expression of Cofilin and phosphorylated-Cofilin with respect to β-actin for B (i). (C) (i) Structural illumination microscopy (SIM) images showing the Cofilin Distribution in LD-infected-KCs. Yellow arrow represents intracellular LD, white dotted line in the merged image demarcates cell boundary. Scale bar: 5 μm. (C) (ii) Color spectrum representing Cofilin Distribution pattern of (C) (i) with green representing maximum and red representing minimum Distribution. (D) (i) Confocal images representing Filamentous actin (F-actin) protrusions in LD-infected-KCs. Scale bar: 20 μm, with yellow arrow representing LD amastigotes. Individual experimental condition is represented in pseudo-color, with uniform high contrast to clearly represent the F-actin protrusions. (D) (ii) Graph representing Cofilin and F-actin Distribution in KCs infected with LD or left uninfected. (E) Still images from live cell video microscopy representing co-localization of LD-R with LDL in presence or absence of Latrunculin-A (LAT-A). Scale bar: 60 μm. Yellow arrow represents LD-infected-KCs with LDL (red). (F) Graphical representation (N = 6) of amastigote count in 100 LD-R-infected-KCs treated with either LAT-A, or Cytochalasin (CYT-D) or left untreated. Data are presented as mean ± SEM. *** signifies p-value <0.0001 ** signifies p-value <0.001, * signifies p-value <0.05, n.s., non-significant.

Figure 4—source data 1

Source data for western blots shown in Figure 4Bi.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig4-data1-v1.zip
Figure 4—source data 2

Source data for western blots shown in Figure 4Bi, with relevant bands labelled.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig4-data2-v1.zip
Figure 4—figure supplement 1
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Latrunculin-A restricts extracellular low-density lipoprotein (LDL) influx inhibiting LD-R amastigote proliferation.

(A) Western blot comparing of LDLr expression between uninfected, LD-S, and LD-R-infected-KC 24 hr p.i. (B) (i) Expression of LDLr determined by immunofluorescence in PECs treated with LDLr siRNA and scrambled siRNA (Sc). Scale bar: 10 μm. (B) (ii) Western blot confirming LDLr knockdown upon SiRNA treatment. Scrambled RNA (ScRNA) was used as a negative control, while Small Interfering RNA (SiRNA) specifically targeted LDLr transcripts, TR-1 and TR-2 represent independent experimental trials. β-Actin was used as an endogenous loading control for western blot normalization. (C) Graph representing number of LD-amastigotes in LDLr knockdown per 100 peritoneal exudate cells (PECs) for (B) (i) (N = 6). Data: mean ± SD. (D) (i) Cofilin and phosphorylated-Cofilin expression by western blot in LD-infected-PEC. (D) (ii) Graphical representation of comparative expression of abundance of Cofilin and phosphorylated-Cofilin with respect to β-actin for (D) (i). (E) Giemsa-stained images of Latrunculin-A (LAT-A) and Cytochalasin-D (CYT-D) treated LD-infected-KCs in gray scale. LD nucleus represented with black arrow and host nucleus is represented as N. Scale bar: 20 μm.* signifies p-value <0.05, n.s., non-significant.

Figure 4—figure supplement 1—source data 1

Source data for western blots shown in Figure 4—figure supplement 1A, Bi, Di.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 1—source data 2

Source data for western blots shown in Figure 4—figure supplement 1A, Bi, Di, with relevant bands labelled.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig4-figsupp1-data2-v1.zip
Figure 5
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Endocytosed-low-density lipoprotein (LDL) fuses with LD-R-PV to provide cholesterol to LD-R amastigotes.

(A) (i) Confocal images of representing LDL-vesicles, with LAMP1-positive lysosomal vesicles in LD-infected-KCs 4 hr p.i. Yellow arrow represents LD nucleus, white dotted box marks the region further cropped and zoomed to show fusion of lysosomal vesicles with LDL and LD-R. Scale bar: 10 μm. (A) (ii) Schematic representation showing trafficking of LDL-vesicle toward the LD-R-PV. (B) (i) Structural illumination microscopy (SIM) images representing convergence of LDL-vesicles with LD amastigotes and LAMP1 in 24 hr with LD-infected-KCs. White dotted box marks the region further cropped and zoomed to clearly show the convergence of LD amastigotes with LDL and LAMP-1 (white vesicles shown by yellow arrow). Scale bar: 5 μm. (B) (ii) Intensity plot representing of co-localization signals for (B) (i). (C) Western blot analysis showing LAMP1 expression in LD-infected-KCs from early to late hours. (C) (i), (ii) representing LAMP1 expression LD-S and LD-R-infected-KC (D) (i) Filipin staining of EGFP-LD-infected-KCs. Filipin (white) and LD (green), yellow arrow represents intracellular LD. Scale bar: 10 μm. White dotted line marked cells used to generate co-localization plot. (D) (ii) Co-localization signals of LD (EGFP) and Filipin represented by the dot plot. Pearson co-localization coefficient value (r) represented for LD-S and LD-R-infected-KC. (D) (iii) Graph representing Pearson’s coefficient for EGFP-LD-infected-KCs (N = 19). (E) Filipin staining of EGFP-LD-R-infected KCs incubated with dextran beads (magenta) revealed minimal colocalization between the beads and Filipin, while the LD-R amastigotes within same macrophage shows a significant co-localization with Filipin. Blue arrow demarking beads with limited filipin colocalization, red arrow demarking LD-R with filipin colocalization. Scale bar: 5 µM. Statistical significance in the observed differences was determined through ANOVA utilizing GraphPad Prism software (version 9.0). *** signifies p-value <0.0001.

Figure 5—source data 1

Source data for western blots shown in Figure 5Ci.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig5-data1-v1.zip
Figure 5—source data 2

Source data for western blots shown in Figure 5Ci, with relevant bands labelled.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig5-data2-v1.zip
Figure 6
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LD-R-infection suppress NPC-1 to alter cholesterol mobilization in infected MΦs.

(A) 24 hr LD-S and LD-R-infected-PECs were subjected to RNAseq analysis keeping uninfected-PECs as control. Heat map representing average expression of differentially expressed genes related to lipid metabolism between LD-S and LD-R-infected-PEC. Scale represents median-centered counts (TPM). Row Z score data represented here. Expression of RNAs (right margins) is presented as centered and ‘scaled’ [mean normalized log2(TPM + 1)]. (B) (i) Expression of SREBP2 and (B) (ii) NPC-1 in LD-infected and uninfected Kupffer cells (KCs) by western blot with β-actin as control. (C) Intracellular localization of SREBP2 in response to LD-infection in KC 24 hr p.i. White dotted line represents cell periphery with yellow arrow representing intracellular LD and N representing host nucleus. 3D image represented below with one quadrant clipped to confirm nuclear translocation of SREBP2. Scale bar: 5 μm. (D) Expression of HMGCR LD-infected and uninfected KCs by western blot with β-actin as control. (E) Immunofluorescence showing NPC-1 expression in LD-infected-KCs. White arrow representing NPC-1 expression in LD-R-infected-KCs and yellow arrow representing NPC-1 in adjacent uninfected-KCs in the merged panel. White dotted box marks regions further cropped and shown in the bottom panel with enlarged view (Zoomed). Scale bar: 20 μm. (F) (i) Total internal reflection fluorescence (TIRF) microscopic images of Laurdan stained LD-infected and uninfected KCs with two different spectra (488 and 594 nm), changes from red to green represents gel to fluid-phase transition of the host plasma membrane. Color distribution pattern corresponding to the TIRF images representing each experimental condition is presented in the right most panel. Scale bar: 5 μm. (F) (ii) Measurement of membrane cholesterol by Amplex red assay kit. Graphical representation showing total membrane cholesterol divided by total membrane protein. (G) (i) Expression of NPC-1 determined by western blot in peritoneal exudate cells (PECs) transfected with NPC-1 siRNA or scrambled (sc) siRNA. (G) (ii) Expression of NPC-1 determined by immunofluorescence in PECs transfected with NPC-1 siRNA or scrambled (sc) siRNA. Scale bar: 5 μm. (G) (iii) Giemsa-stained images of LD-infected NPC-1 knockdown-PECs with scrambled siRNA control (Sc). Scale bar: 20 μm. Images are represented in gray scale with increased contrast to represent LD nucleus (black arrow). (G) (iv) Graphical representation of number of intracellular LD-amastigotes as in (F) (ii). Results show the counting of 100 infected PEC from 6 independent replicate. * signifies p-value <0.05, n.s., non-significant.

Figure 6—source data 1

Source data for western blots shown in Figure 6Bi, ii, D, Gi.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig6-data1-v1.zip
Figure 6—source data 2

Source data for western blots shown in Figure 6Bi, ii, D, Gi, with relevant bands labelled.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig6-data2-v1.zip
Figure 7
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LD-R-infected-MΦs selectively excludes ox-LDL to suppress inflammatory response in host.

(A) Scheme representing ex vivo co-culture experiment performed by isolating T-cells from LD-infected mice with in vitro LD-infected-KCs. (B) (i) IFN-γ level in the supernatant of LD-infected-KCs co-cultured with the T-cells isolated from spleen of Balb/c mice infected with LD-S or LD-R 28 days p.i. Data represented as mean ± SD from three independent experiments. (B) (ii) IFN-γ level in the supernatant of splenocytes cultures from LD-infected-BL/6 and Apoe−/− mice stimulated with SLA (soluble Leishmania antigen) specific for LD-S or LD-R. Data are presented as mean ± SD from six independent mice. (C) (i) ox-LDL incubated LD-infected-KCs was fixed and stained with Oil-red-O and hematoxylin. LD-SHigh represents MOI (1:20) and LD-RLow represents MOI (1:5). Scale bar: 10 μm. Black arrow represents LD nucleus. (C) (ii) Graph representing % of ox-LDL-positive MΦs under different experimental conditions as represented in (C) (i). (D) (i) Confocal image showing MSR-1 in LD-infected-KCs. Yellow arrow represents LD-amastigotes while N represents host nucleus. Scale bar: 20 μm. (D) (ii) Expression of MSR1 detected by western blot of LD-infected-KC with β-actin as loading control. (E) Expression of ATP6V0D2 in LD-infected-KCs by western blot with β-actin as loading control. (F) (i) Scheme representing the time line of in vivo experiment for IFA. (F) (ii) Expression of MSR1 and neutral lipid droplet accumulation assayed by confocal microscopy from the cryo-sectioned liver of 28 days LD-infected mice. Representative image showing lipid droplet marked in red and MSR1 in green, nucleus in blue. Scale bar: 40 μm. (F) (iii) Graphical representation of % positive lipid droplets accumulation and MSR-1 expression under different experimental condition as in F (i). Data collected from N = 6 mice. *** signifies p-value <0.0001, ** signifies p-value <0.001, * signifies p-value <0.05.

Figure 7—source data 1

Source data for western blots shown in Figure 7Dii, E.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig7-data1-v1.zip
Figure 7—source data 2

Source for western blots shown in Figure 7Dii, E, with relevant bands labelled.

https://cdn.elifesciences.org/articles/102857/elife-102857-fig7-data2-v1.zip
Figure 8 with 1 supplement
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Lipid droplets accumulation around LD amastigotes is inversely related with susceptibility toward Amphotericin-B.

(A) Ex-M image showing lipid droplet assimilation in LD-R amastigotes. White dotted region further cropped and shown in enlarged panel (Zoomed). Nile red (magenta), LD (green), and nucleus (cyan). White spots represent lipid droplets assimilated in LD-R and host nucleus is represented as N. Scale bar: 5 μm. (B) Transmission electron microscopy (TEM) images showing lipid droplets around the LD-R amastigotes. Black box further magnified and represented in right panel. White * representing LD-R amastigotes, yellow * showing lipid droplets. Scale bar 2 μm. (C) Schematic representation representing experimental scheme used to determine EC50 against Amphotericin-B under different experimental conditions. (D) (i) and (ii) Determination of EC50 value against Amphotericin-B in low and high lipid media by performing infection with of two independent LD-R strains. (D) (iii) Determination of EC50 value against Amphotericin-B in Kupffer cell (KC) infected with LD-SAmp-B-R. Intracellular amastigotes were counted through Giemsa staining. (E) Confocal images showing lipid droplets accumulation around intracellular amastigotes with Aspirin (5 μM) or Amp-B (0.36 μM) or with a combination of Aspirin (5 μM) and Amp-B (0.11 μM)at 48 hr p.i. (F) Determination of amastigote load in KCs infected with two independent LD-R-isolates in different experimental conditions either untreated or treated with Aspirin (5 μM) or Amp-B (0.36 μM) or with a combination of Aspirin (5 μM) and Amp-B (0.11 μM) at 48 hr p.i. (G) Comparative lipid profile of visceral leishmaniasis (VL) patients (Cured and Relapsed) in response to Amp-B treatment along with healthy individuals from endemic region of Bihar, India. *** signifies p-value <0.0001, ** signifies p-value <0.001, * signifies p-value <0.05.

Figure 8—figure supplement 1
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Aspirin treatment inhibits lipid droplets formation and increases responsiveness of LD-R amastigotes toward Amphotericin-B.

(A) Determination of lipid droplet accumulation by Nile red staining in LD-S and LD-R-infected-KC or Kupffer cell (KC) left uninfected. Dotted line mark the cell periphery while dotted box cells were used for 3D reconstruction and represent in Video 4. Scale bar: 5 μm. (B) Effect of Aspirin (5 μM) on lipid droplets accumulation in LD-R-infected-KCs. Independent infection was performed with LD-R1 and LD-R2 strains (N = 6) and % KCs with high lipid droplets accumulation are represented. (C) Representative Giemsa stained images of LD-R-infected-KCs in different experimental conditions either untreated or treated with Aspirin (5 μM) or Amp-B (0.36 μM) or with a combination of Aspirin (5 μM) and Amp-B (0.11 μM) at 48 hr p.i. Scale bar: 20 μm. *** signifies p-value <0.0001.

Figure 9
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Scrutinized uptake of host lipids results in breakdown of Amphotericin-B responsiveness in clinical LD isolates with primary resistance to antimony.

LD-R reside within membrane-bound parasitophorous vacuoles (PVs) and actively modulate host actin cytoskeleton dynamics through cofilin-mediated depolymerization, inducing high fluid-phase endocytosis of low-density lipoprotein (LDL). The internalized LDL-containing vesicles subsequently fuse with LD-R-associated PVs, facilitating increased lipid acquisition essential for their aggressive intracellular proliferation. In these fused membranes, the vesicular cholesterol export protein NPC-1 is downregulated, effectively sequestering LDL-derived cholesterol within the PV and preventing its release into the host cytoplasm. Concurrently, LDL-derived cholesterol esters either directly or getting processed in the endoplasmic reticulum (ER) can lead to the formation of lipid droplets (as presented by ?, since it needs further investigation). Lipid droplets then accumulate in and around the LD-R-PV. This lipid droplet accumulation is a key factor in rendering LD-R-infections unresponsive to Amphotericin-B. Additionally, LD-R regulate inflammatory responses linked to oxidized lipid (ox-LDL) accumulation by downregulating macrophage scavenger receptor 1 (MSR-1). The elevated LDL-influx also leads to a shutdown of de novo cholesterol biosynthesis by restricting the nuclear translocation of sterol regulatory element-binding protein 2 (SREBP2).

Author response image 1
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Representative images of Kupffer cells infected with Leishmania donovani at 72Hrs post-infection showing a significant morphological change.

Infected cells exhibit a rounded morphology and progressive detachment. Scale bar 10µm.

Author response image 2
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qPCR Analysis of HMGCR Expression Following Leishmania donovani Infection: Quantitative PCR analysis showing the relative expression of hmgcr (3-hydroxy-3-methylglutaryl-CoA reductase) in Kupffer cells after 24 hours of Leishmania donovani (LD) infection compared to uninfected control cells.

Gene expression levels are normalized to β-actin as an internal control, and fold change is represented relative to the uninfected condition.

Videos

Video 1
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Phase contrast video representing attached LD promastigotes to peritoneal exudate cell (PEC) 6 hr p.i.

Black arrow showing flagella of attached LD parasite. 1A represents LD-S and 1B represents LD-R-infection. Scale bar: 10 μm.

Video 2
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Phase contrast video representing 72 hr LD infected Kupffer cells (KCs).

Scale bar: 20 μm.

Video 3
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Effect of Latrunculin-A (LAT-A) on low-density lipoprotein (LDL) uptake in LD-R infected peritoneal exudate cells (PECs).

(3A) In absence of LAT-A. (3B) In presence of LAT-A. LD (green), LDL (red), and nucleus (blue). Scale bar: 60 μm.

Video 4
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3D representation of lipid droplets around the LD-S and LD-R amastigotes (Figure 8—figure supplement 1B).

Scale bar: 5 μm.

Tables

Table 1
EC50 against SAG (antimonial) for the LD strains used in this study.
Strain nameEC50 value for SAG (μg/ml) in PECEC50 value for SAG (μg/ml) in KC
MHOM/IN/2009/BHU575/022.73 ± 3.723.89 ± 2.1
MHOM/IN/10/BHU814/130.3 ± 7.228.5 ± 5.3
MHOM/IN/83/AG831.52 ± 1.23.3 ± 2.2
MHOM/IN/80/DD82.1 ± 1.23.2 ± 1.9
DD8-AmpB1.8 ± 2.12.2 ± 1.3
BHU575-EGFP21.45 ± 3.222.81 ± 3.1
AG83-EGFP2.1 ± 1.42.4 ± 3.1

  1. Magenda shaded: drug unresponsive strains; green shaded: drug responsive strains.

Table 2
Lipid profile of visceral leishmaniasis (VL) patients.
Serum codeAgeSexTime of blood collectionInfection statusTreatment receivedPatient statusLDL (rg/d)HDL (rrg/dl)Cholesterol (mg/dl)Tridycerides (mg/dl)
RMRI N145FBeforeControl population from endemic zoneNANA45.350158546.5365931101.6949153177.258567
RMRI N670FBeforeControl population from endemic zoneNANA110.85547850.3762712111.8644068206.5420561
RMRI N745FBeforeControl population from endemic zoneNANA75.562078741.871186484.40677966165.4205607
RMRI N819MBeforeControl population from endemic zoneNANA83.94603862.9533123100.3389831224.9221184
RMRI N1137MBeforeControl population from endemic zoneNANA72.4549921120.914196134.2372881244.5482866
RMRI N1260MBeforeControl population from endemic zoneNANA52.6307448113.85173592.54237288180.6853583
RMRI N1330MBeforeControl population from endemic zoneNANA66.753328148.3413249138.3050847236.4485981
RMRI N1422MBeforeControl population from endemic zoneNANA47.893977849.6807571100260.7476636
RMRI N1537FBeforeControl population from endemic zoneNANA46.490491345.9059937108.8135593219.6261682
RMRI Cl20FBeforeConfirmed VL, hospitalizationAmbisome Single doseCured110.38904544.2700565112.5423729200.623053
RMRI C262FBeforeConfirmed VL, hospitalizationAmbisome Single doseCured121.73890464.5514124145.42372883018691589
RMRI C315MBeforeConfirmed VL, hospitalizationAmbisome Single doseCured76.183988836.746327795.59322034185.6697819
RMRI C431MBeforeConfirmed VL, hospitalizationAmbisome Single doseCured72.14157341.1079096116.9491525241.7445483
RMRI C552MBeforeConfirmed VL, hospitalizationAmbisome Single doseCured98.106320248.1954802117.2881356282.2429907
RMRI C618FBeforeConfirmed VL, hospitalizationAmbisome Single doseCured68.876544936.746327796.61016949200.623053
RMRI C745FBeforeConfirmed VL, hospitalizationAmbisome Single doseCured110.54452255.6101695124.4067797381.9314642
RMRI C832FBeforeConfirmed VL, hospitalizationAmbisome Single doseCured94.841292143.0706215106.1016949232.3987539
RMRI C1866MBeforeConfirmed VL, hospitalizationAmbisome Single doseCured61.413623635.983050891.86440678203.4267913
RMRI R148MBeforeConfirmed VL, hospitalizationAmbisome Single doseRelapse79.293539383.633333396.94915254262.9283489
RMRI R1649MBeforeConfirmed VL, hospitalizationAmbisome Single doseRelapse56.9047753102.27909663.05084746209.0342679
RMRI R1718MBeforeConfirmed VL, hospitalizationAmbisome Single doseRelapse59.7033708101.07966183.38983051217.1339564
VRMRI R1859MBeforeConfirmed VL, hospitalizationAmbisome Single doseRelapse63.434831594.31920984.40677966304.6728972

  1. Green shaded: healthy; yellow shaded: cured; magenta shaded: relapsed patients.

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  1. Supratim Pradhan
  2. Dhruba Dhar
  3. Debolina Manna
  4. Shubhangi Chakraborty
  5. Arkapriya Bhattacharyya
  6. Khushi Chauhan
  7. Rimi Mukherjee
  8. Abhik Sen
  9. Krishna Pandey
  10. Soumen Das
  11. Budhaditya Mukherjee
(2025)
Scrutinized lipid utilization disrupts Amphotericin-B responsiveness in clinical isolates of Leishmania donovani
eLife 14:RP102857.
https://doi.org/10.7554/eLife.102857.3