# Interplay between cholesterol, Bis(monoacylglycerol)phosphate, and parasitophorous vacuole dynamics in Leishmania infantum infection of macrophages

Clara Hennot, Marine Leroux, Remi Lambert, Fanny Boukherrouba, Marion

Guichard, Karen Gaget, Federica Calevro, Isabelle Delton, Samira

Azzouz-Maache, Celine Luquain-Costaz

► To cite this version:

Clara Hennot, Marine Leroux, Remi Lambert, Fanny Boukherrouba, Marion Guichard, et al. Interplay between cholesterol, Bis(monoacylglycerol)phosphate, and parasitophorous vacuole dynamics in Leishmania infantum infection of macrophages. Biochimie, 2026, 245, pp.8-17. 10.1016/j.biochi.2026.02.017. hal-05528508

HAL Id: hal-05528508

https://hal.science/hal-05528508v1

Submitted on 26 Feb 2026

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# Interplay between cholesterol, Bis(monoacylglycerol)phosphate, and parasitophorous vacuole dynamics in *Leishmania infantum* infection of macrophages

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Clara Hennot<sup>a</sup>, Marine Leroux<sup>a</sup>, Remi Lambert<sup>a,b</sup>, Fanny Boukherrouba<sup>a</sup>, Marion Guichard<sup>a</sup>, Karen Gaget<sup>c</sup>, Federica Calevro<sup>c</sup>, Isabelle Delton<sup>a,b</sup>, Samira Azzouz-Maache<sup>a</sup>, Celine Luquain-Costaz<sup>a,b,*</sup>

## ARTICLE INFO

**Article history:**

Received 6 October 2025

Received in revised form

27 January 2026

Accepted 20 February 2026

Available online 24 February 2026

**Keywords:**

Bis(monoacylglycerol)phosphate

Macrophages

Leishmania

Cholesterol

Parasitophorous vacuole

## ABSTRACT

*Leishmania* spp., the causative agents of leishmaniasis, are protozoan parasites displaying two life stages: promastigote in the insect vector and amastigote in host macrophages. After inoculation, the promastigote differentiates into the amastigote which multiplies within a parasitophorous vacuole formed by the fusion of the phagosome with the macrophage endolysosome. This compartment is characterized by a specific enrichment in bis(monoacylglycerol)phosphate (BMP), an atypical phospholipid that regulates endosomal dynamics and cholesterol trafficking. Host cell cholesterol is essential for parasite intracellular development. In this study, we examined the relationships between cholesterol, BMP, and the parasitophorous vacuole during the infection of J774 murine macrophages with *L. infantum*. Our results showed that cholesterol is redistributed in the vicinity of the parasite within infected cells. BMP is redistributed along with the same pattern and colocalizes with markers of the parasitophorous vacuole. Transcriptomic analyses revealed an upregulation of key genes governing cholesterol uptake and synthesis (HMGCR, SREBP2, LDLR) during infection and conversely a downregulation of ABCA1 involved in cholesterol efflux. Noteworthy, the overexpressions of HMGCR, SREBP2, LDLR were significantly attenuated by macrophage BMP enrichment. As for functional impact, BMP enrichment was associated with a significant increase of the parasite infectivity toward macrophages, assessed by infection rate and parasite load. Together, our results confirm the essentiality of macrophage cholesterol and demonstrate the involvement of BMP during *Leishmania* infection likely by facilitating parasitophorous vacuole remodeling and cholesterol trafficking.

© 2026 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

*Leishmania* spp. are protozoan trypanosomatid parasites causing cutaneous, muco-cutaneous or visceral leishmaniasis in humans and other mammals. These parasites display high morphological plasticity and clearly defined developmental forms: the promastigote in the insect vector, a phlebotomine fly, and the amastigote in mammalian host cells, primarily macrophages [1]. After transmission to the host upon insect blood meal, the promastigotes are rapidly internalized by host macrophages via phagocytosis and are sheltered within a vacuole called parasitophorous vacuole (PV). PV is essential for parasite intracellular development as it creates a protective environment against host

**Abbreviations:** ABCA1, ATP-binding cassette A1; BMP, bis(monoacylglycerol)phosphate; EEA1, early endosomes antigen 1; DOPG, dioleyl-phosphatidylglycerol; DOBMP, dioleyl-bis(monoacylglycerol)phosphate; ER, endoplasmic reticulum; HMGCR, hydroxymethylglutaryl-CoA reductase; Lamp-1, lysosome associated membrane protein 1; LE, late endosomes; LXR, liver x receptor; LDLR, low-density lipoprotein receptor; NPC, Niemann-Pick type C; NO, nitric oxide; PG, phosphatidylglycerol; PV, parasitophorous vacuole; Rab7, Ras-related protein 7; SREBP2, sterol regulatory element-binding protein-2; TBP, TATA-binding protein.

* Corresponding author. Univ Lyon, Université Claude Bernard Lyon 1, CNRS, LAGEPP UMR, 5007, Villeurbanne, France.

E-mail address: celine.costaz@insa-lyon.fr (C. Luquain-Costaz).

<footnote><sup>a</sup> Univ Lyon, Université Claude Bernard Lyon 1, CNRS, LAGEPP UMR, 5007, Villeurbanne, France</footnote>

<footnote><sup>b</sup> INSA-Lyon, Villeurbanne, France</footnote>

<footnote><sup>c</sup> INRAE, INSA Lyon, BF2I, UMR203, 69621, Villeurbanne, France</footnote>
cell immune response factors and a niche for promastigote dif-
ferentiation into amastigote and multiplication [2,3]. It also rep-
resents the place of exchange between parasites and host cells
from which *Leishmania* recovers essential nutrients [3]. Depending
on the *Leishmania* species, parasite resides in communal or indi-
vidual PV. As for its structure, PV exhibits a hybrid nature arising
from successive fusions between endolysosome from macrophage
and the phagosome that contains promastigote freshly internal-
ized by phagocytosis. Intracellular components derived from the
endocytic pathway such as Early Endosomes antigen 1 (EEA1) and
Ras-related protein 7 (Rab 7) respectively marker of the early
endosomes (EE) and the late endosomes (LE), and the lysosome-
associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2),
are found to be displayed on the PV membrane and constitute
PV markers [3,4].

A key difference between host cells and *Leishmania* parasites is the major sterol present in their membranes since *Leishmania* parasites that lack cholesterol biosynthesis enzymes, synthesize ergosterol instead. Several studies using cholesterol depletion with cyclodextrins or cholesterol lowering drugs have demonstrated that host cell cholesterol was mandatory for binding, internalization and intracellular development of the *Leishmania* parasites [5,6]. Noteworthy, it was also reported that *Leishmania* parasites hinder macrophage cholesterol trafficking causing cholesterol retention in PV and upregulation of genes involved in cholesterol synthesis [7]. We previously showed a strong lipid remodeling of parasite membranes during the intracellular transformation of the *L. donovani* and *L. infantum* promastigotes into the amastigote stage, with a marked enrichment in cholesterol at the expense of ergosterol [8], suggesting cholesterol transfers between the parasite and host cell. Surface-enhanced Raman scattering (SERS) microscopy, which uses plasmonic nanostructures to greatly amplify Raman signals for sensitive chemical imaging, enabled the assessment of cholesterol distribution in the amastigote stage of the parasite and revealed its immediate surroundings within the vacuole [9].

Bis(monoacylglycerol)phosphate (BMP) is a peculiar phospho-
lipid enriched in the endolysosomal compartment of cells. This
phospholipid is well documented for its involvement in biogenesis
and dynamics of endocytic vesicles, sorting functions of LE,
endolysosomal degradative pathway, exosome formation and
distribution of cholesterol originating from the endocytosis of low
density lipoproteins (LDL) [10–13]. It has been demonstrated that
BMP is involved in intracellular distribution of LDL-cholesterol
through interaction with the sterol transport proteins Niemann
Pick type C1 and C2 (NPC1 and C2) [14–16]. In our previous works,
we have extensively studied BMP metabolism and functions in
macrophages. We have shown that BMP plays a key role in
cholesterol trafficking and metabolism in these cells by regulating
the intracellular redistribution of LDL-cholesterol, the expression
of cholesterol homeostasis genes and the oxidative metabolism of
cholesterol, in interaction with the sterol transport protein oxy-
sterol binding protein related protein 11 (ORP 11) [17–19].
Furthermore, BMP has been reported to modulate the toll-like
receptor (TLR4) signaling pathway in relation to the regulation of
cholesterol distribution [20]. BMP has also been proposed as an
important player in viral infections [21,22].

Considering the specificities of BMP respective to its endoly-
sosomal localization, its fusogenic properties and its regulation of
cholesterol, we have investigated its possible contribution in
cholesterol trafficking and PV formation during *L. infantum* infec-
tion, as well as its impact on parasite infectivity.

## 2. Materials and methods

2.1. *Ethic statements*

An isolate of *L. infantum* human strain MON24 causing cuta-
neous leishmaniasis (CL) was used in this study. The strain is part
of the biobank of Tunis Pasteur Institute and has been provided by
Pr. Karim Aoun. Its use for research purposes has been approved by
the Review Board of Tunis Pasteur Institute. The strain was initially
isolated as part of the routine diagnostic procedure recommended
by WHO for CL samples. The strain was anonymized, archived and
cryopreserved in the biobank of the Parasitology department of
Tunis Pasteur Institute. No personal data was used.

2.2. *Cell and parasite culture*

2.2.1. Macrophage culture

The BALB/c mice macrophage cell line J774A.1 was maintained by successive passages in RPMI-1640 culture medium with 10% (v/ v) fœtal bovine serum (FBS) 2% (v/v) Penicillin/Streptomycin and 1% (v/v) Mycokill (control medium). Culture was performed at 37 °C with 5% CO2.

2.2.2. *Parasite culture*

CL strain was isolated from dermal serosity as described [23]. Primocultures of promastigotes were performed in biphasic Novy-MacNeal-Nicolle (NNN) medium at 26 °C with subculture every week for 5 consecutive weeks. Identification of isolates was performed by isoenzyme electrophoresis [24].

Promastigotes were cultured at 26 °C in Medium 199 supple-
mented with 20% (v/v) FBS and 2% (v/v) Penicillin/Streptomycin
and 1% (v/v) Mycokill. The parasites were cultured in 25-cm²
culture flasks and were harvested during their exponential growth
phase by centrifugation at 1300g for 15 min.

2.3. *Supplementation with PG liposomes*

Liposomes composed of phosphatidylglycerol (PG) were pre-
pared as previously described [25]. Macrophages were incubated
with 30 µM DOPG (dioleoyl-PG) or DPPG (dipalmitoyl-PG) lipo-
somes in RPMI-1640 culture medium for 24h. Liposomes were
maintained at the same concentration throughout the whole
experiment. Control cells (not supplemented) were cultured
simultaneously in control medium.

2.4. *Parasite infectivity*

Cells were seeded at a density of 70 000 cells/well on a 12 mm
diameter glass coverslip in 24-well plates. Cells were incubated
with parasites at a ratio of 10 parasites per cell at different times to
cover the onset of infection (2h) and intracellular development
(24h or 48h: 24h followed by additional 24h after parasite
removal). After infection, cells were washed 3 times with sterile
PBS to remove extracellular parasites, then fixed with para-
formaldehyde (PFA) 4% and stained with DAPI (4', 6-diamidino-2-
phenylindole) (Life Technologies, Paisley, UK). Coverslips were
finally washed 3 times with PBS and once with ultrapure water and
mounted on glass slide with Fluoromount mounting medium.
Slides were observed under the fluorescent microscope Leica
DM6B to count infected cells and intracellular amastigotes. For
each slide, on average 500 cells were counted. The infection rate
represents the percentage of infected cells and is defined as the
number of infected cells per 500 cells counted. The parasite load
represents the average number of amastigotes per infected cells
multiplied by the infection rate.
## 2.5. Immunostaining, fluorescence and colocalization quantification

### 2.5.1. Immunostaining

Cells were seeded at a density of 70 000 cells/well on a 12 mm diameter glass coverslip in 24-well plates. Cells were infected or not at a ratio of 10 parasites per cell for different times as indicated in the figure legends. Cells were washed 3 times with PBS and permeabilized with Triton X-100 0.1% (v/v) in PBS for 12 min at room temperature. After 3 washes with PBS, cells were blocked with Bovine Serum Albumine (BSA) 0.1% (v/v) in PBS for 30 min at room temperature. Cells were then incubated with primary antibody (1:100 v:v) for 1h at room temperature: anti-BMP, anti-Rab7, anti-EEA1 or anti-LAMP1. Monoclonal antibody against BMP (anti-LBPA antibody, 6C4) was a gift from Dr Kobayashi [26]. Antibody to LAMP1, Rab7 and EEA1 were from Sigma (Saint-Quentin Fallavier, France).

After 3 washes with PBS, cells were incubated with appropriate Alexa-488 or Alexa-546 conjugated secondary antibody (1:1000 v:v) for 1h at room temperature. Cell and parasite nuclei were then stained with DAPI. Coverslips were finally washed 3 times with PBS and once with ultrapure water and mounted on glass slide with Fluoromount mounting medium (Sigma-Aldricht, St Quentin Fallavier, France).

Free cholesterol staining was performed on PFA fixed cells by 3h incubation with Filipin III at 100 µg/mL in PBS. After 3 washes, cells and parasite nuclei were stained with SYBR™ Green I Nucleic Acid Gel Stain (1:10 000 v:v) for 5 min, washed and mounted on glass slide with Fluoromount mounting medium.

### 2.5.2. Fluorescence and colocalization quantification

Images were acquired with fluorescent microscope Leica DM6B by performing z-stacks acquisition. In addition to perform z-stacks acquisitions, fluorescent microscope Leica DM6B is equipped with Thunder™ technology which enables blur and diffusion reduction through real-time deconvolution. A mean of 30 z-stacks were performed for each image acquisition. Image analysis was performed using LAS X software and ImageJ software. Fluorescence Intensity was assessed on ImageJ software by determining the corrected total fluorescence by subtracting out background signal. Colocalization was calculated with Pearson's coefficient measured on ImageJ software using the Colocalization test Plugin.

## 2.6. Lipid quantification

Cells were seeded at a density of $1 \times 10^6$ cells/well in 6-well plates. After treatment as detailed above and indicated in the figure legends, cells were washed with sterile PBS, detached and centrifuged twice at 200g for 10 min, with sterile PBS washes between each centrifugation. Before the last centrifugation, 10 µL of cell suspensions were collected for cell counting, and cell pellets were stored at -80 °C before analysis.

For cholesterol analysis, total lipids were extracted according to the Folch method [27]. The total lipids were then derivatized to trimethylsilyl ethers by heating at 60 °C for 30 min after the addition of 200 µL of BSTFA (Supelco, Bellafonte, PA, USA) and 3.6 µg of 5alpha-cholestane as internal standard (Merck, Lyon, France). Trimethylsilyl ethers derivatives were analyzed on a gas chromatograph coupled to flame ionization detection (Trace 1310 gas chromatograph Thermo Scientific, Courtaboeuf, France). A 1 µL-aliquot was injected by an automated injector in splitless mode at 290 °C on a DB-5MS fused silica capillary column (30 m × 0.25 mm id, 0.25 µm film thickness; Agilent, Santa Clara, CA, USA). The initial oven temperature was kept at 60 °C for 1 min, and then increased at a rate of 20 °C/min to 290 °C and at 2 °C/min to a final temperature of 300 °C. The data were computed using the Chromeleon software (version 7.1.10 ES, Thermo Scientific).

For BMP analysis, cell pellets were spiked with diC14:0-BMP and diC14:0-PG as internal standards, and total lipids were extracted from cell lysates and media according to the method of Bligh and Dyer [28]. The resulting samples were dried under nitrogen flow and resuspended in methanol prior to measurement. LCMS/MS was carried out on an Agilent 1100 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) integrated with a 4000 QTrap mass spectrometer (AB SCIEX, Foster City, CA, USA) using a Shiseido CAPCELL PAK C18 MGIII Type column (5 µm, 2.0 × 50 mm) (Shiseido CO., LTD., Tokyo, Japan) as previously described [18,29]. Quantification of BMP and PG was done by relating the peak area of molecular species to the peak area of the internal standard. Quantification was assessed by total oleate-containing species.

## 2.7. qRT-PCR analyses

Cells were seeded at a density of $5 \times 10^5$ cells/well in 6-well plates. After treatment as detailed above and indicated in the figure legends, Total RNA was isolated using Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. RNA concentrations were determined with a Thermo Scientific NanoDrop One (ThermoFischer Scientific, Waltham, MA, USA). Total RNAs were treated with RQ1 RNase-Free DNase I (Promega Corporation, Madison, WI, USA), and 500 ng of total RNA reverse-transcribed with the SuperScript™ III First-Strand Synthesis System (Life Technologies, Carlsbad, CA, USA), using Oligo(dT)20 primers (Invitrogen) in order to obtain first-strand cDNA. Oligonucleotide primer sequences are listed in S3 Fig. In the negative controls, reverse transcriptase was replaced by nuclease-free water. The absence of genomic DNA contamination was checked with PCR using primers targeting the *HPRT* control gene in each sample used for the following qRT-PCR experiments. Quantitative real-time RT-PCR reactions were performed on a Bio-Rad CFX96 qPCR (Bio-Rad, Hercules, CA, USA) using a 1:5 diluted cDNAs and a LightCycler® 480 SYBR-Green I Master Mix (Roche, Basel, Switzerland) according to manufacturer's instructions. All qRT-PCR reactions were performed in technical triplicates starting from each of the three independent biological replicates.

## 2.8. Cytokines production measurement

Cells were seeded at a density of $7 \times 10^4$ cells/well in 24-well plates. After treatment as detailed above and indicated in the figure legends, culture media were collected and TNF-α, IL-6, TGF-β and IL-10 cytokines were measured using Quantikine™ ELISA (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. Absorbance was measured using a SPARK multimode microplate reader (TECAN) at 450 nm.

## 2.9. Intracellular arginase activity measurement

Cells were seeded at a density of $1 \times 10^6$ cells/well in 6-well plates. After treatment as detailed above and indicated in the figure legends, cells were washed with sterile PBS and intracellular arginase activity was measured using Arginase Activity Colorimetric Assay Kit (Sigma-Aldrich, St Quentin-Fallavier, France) according to manufacturer's instructions. Absorbance was measured using a SPARK multimode microplate reader (TECAN) at 570 nm in a kinetic mode during 30min.
## 2.10. Intracellular Nitric Oxide (NO) measurement

Cells were seeded at a density of $2.5 \times 10^4$ cells in 96-well plates. After treatment as detailed above and indicated in the figure legends, culture media were collected and NO was measured using Griess Reagent System (Promega) that measures nitrite ($NO_2^-$), a stable breakdown product of NO, according to manufacturer's instructions. Absorbance was measured using a SPARK multimode microplate reader (TECAN) at 550nm.

## 2.11. Statistical analyses

For qRT-PCR experiments, two normalization gene candidates, *TBP* (*TATA-binding protein*) and *HPRT* (*hypoxanthine-guanine phosphoribosyltransferase gene*) were tested for gene expression variation in all experimental conditions used in this work. The *HPRT* gene was employed as a normalization gene as it meets the criteria imposed by the BestKeeper software analysis [30]. The expression levels (log2) of the different genes were then normalized relative to those of the *HPRT* gene. The relative expression ratio R (and the associated standard error) of each condition was calculated using as control the mean of the non-infected cells as control. This ratio was calculated taking into account the real-time PCR efficiency of each gene (E) and the crossing point difference (DCP) of a test condition, as compared to the reference condition, and expressed in comparison to the normalization gene (*HPRT*) using the following model: $R=(E_{target})^{\Delta CP_{target}(control-sample)}/(E_{reference})^{\Delta CP_{reference}(control-sample)}$.

All experimental data are shown as means ± sd of at least three biological experiments. Statistics were performed using Jamovi 2.3.28 Software. When two groups were compared, we used a Welch *t*-test. When more than two groups were compared, we used an ANOVA with Post-hoc Tukey correction. Mixed-Model with post-hoc Bonferroni correction was used to account for

both fixed and random effects.

# 3. Results

## 3.1. Trafficking of cholesterol and BMP during L. infantum infection

To follow the distribution of macrophage cholesterol during infection with *L.infantum*, we used Filipin III, a fluorescent probe that labels free cholesterol and SYBR green to label macrophage and parasite nuclei (Fig. 1). At 2h and 24h post-infection, intra-cellular parasites were clearly visible as small green dots in a halo characteristic of individual PV. Filipin staining displayed different patterns in infected cells compared to control cells, with choles-terol distribution around parasites at 2 h and even more markedly 24 h post-infection.

Since BMP is known to regulate cholesterol trafficking in different cells including macrophages especially at the endolysosomal level, [17,18,31], we examined the localization of cholesterol relative to that of BMP during parasite infection. In non-infected macrophages, cholesterol was strongly co-localized with BMP (as expressed by Pearson's correlation coefficient of 0.85) (Fig. 2A and B), indicating a major localization in the endolysosomal compartment, as previously observed in RAW macrophages [31]. Noteworthy, similar colocalization of BMP and cholesterol was observed in macrophages after 2h and 24h infection with *L. infantum*, indicating that BMP and cholesterol are redistributed together following the same pattern.

## 3.2. Colocalization of BMP with parasitophorous vacuole markers

Since PV shares features with LE/Lysosomes that concentrate cellular BMP, we examined whether BMP localization and/or synthesis were modified in macrophages in the course of infection with *L.infantum*. Respective markers for EE (EEA1), LE (Rab7), and

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Cholesterol redistribution during macrophages infection by *Leishmania infantum*</figcaption></figure>

J774A.1 macrophages were infected by *Leishmania infantum* for 2h and 24h (MOI 10:1). Intracellular cholesterol was stained with Filipin III (100 µg/mL). Macrophages and parasites nuclei were stained with SYBR™ Green I Nucleic Acid Gel Stain. Images were acquired with fluorescent microscope Leica DM6B equipped with Thunder™ technology which enables blur and diffusion reduction through real-time deconvolution. Representative images of 3 independent experiments. White arrows indicate parasites nuclei. Scale-bar: 5 µm.
A

BMP

Filipin

SYBR Green

Merged

Colocalized pixels

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** BMP and cholesterol redistribution during macrophages infection by *Leishmania infantum*. J774A1 macrophages were infected by *Leishmania infantum* for 2h and 24h (MOI 10:1). BMP was stained with primary antibody anti-BMP and Alexa-546 conjugated secondary antibody. Intracellular cholesterol was stained with Filipin III (100 µg/mL). Macrophages and parasites nuclei were stained with SYBR™ Green I Nucleic Acid Gel Stain. Images were acquired with confocal microscope Leica SP5. White arrows indicate parasites nuclei. Scale bar: 5 µm. Representatives images of 2 independent experiments (A). Colocalization was calculated with Pearson's correlation coefficient measured on ImageJ software using the Colocalization test Plugin. Results are mean 142 ± 21 cells per condition. (B). Statistical differences were analyzed by ANOVA test with post-hoc Tukey correction.</figcaption></figure>

B

<figure><img src="image_5.png" /></figure>

lysosomes (Lamp-1) in host cells are all established markers of PV. As shown in Fig. 3, in infected cells, both EEA1 (A), Rab7 (C) and Lamp-1 (E) showed diffuse fluorescence throughout the cells in the vicinity of the parasite visualized as small blue dots by DAPI staining, consistent with the recruitment of both EEA1, Rab7 and Lamp-1 in the PV. In non-infected cells, BMP showed extensive colocalization with Rab7 (Pearson 0.71) and to a lower extent with EEA1 (Pearson 0.38) and Lamp-1 (Pearson 0.33), which is consistent with its localization primarily in LE. As shown above, BMP exhibited a more diffuse distribution by 2h post-infection and even more markedly at 24h with a tendency to accumulate near the parasites. Pearson's correlation coefficient for BMP colocalization with EEA1 and Lamp-1 were same in infected and non-infected cells, and slightly decreased with Rab7 at 24h. These results indicate that BMP distribution in infected cells follows broadly the same pattern as PV markers, suggesting its recruitment into this parasitic structure. Nonetheless, the quantity of BMP, as assessed by total oleate-containing species of BMP that are the most abundant in macrophages [17], was not different between non-infected and infected macrophages (Fig. 4A and B). Taken together, these results show that BMP initially present in LE in non-infected cells is recovered to the vicinity of the parasite and colocalizes with PV markers, without quantitative modification, suggesting only redistribution.

### 3.3. Effect of BMP enrichment on cholesterol synthesis and trafficking in infected macrophages

To further investigate the role of BMP in cholesterol trafficking and metabolism, we used our model of BMP enrichment by supplementation with the precursor (DOPG, at 30 µM) that we previously validated in RAW macrophages [17,25]. As expected, the supplementation of J774 macrophages with DOPG induced a specific 10-fold enrichment of dioleoyl-BMP (DOBMP), with only minor accumulation of the precursor DOPG (macrophages

hereinafter referred to as BMP-enriched cells) (S1 fig.).

As revealed by Filipin staining (Fig. 5A), after 24h infection, cholesterol was redistributed around parasites in BMP-enriched macrophages similar to what observed in non-enriched macrophages. Furthermore, parasite infection induced a significant and similar increase of free cholesterol content in non-enriched and BMP-enriched macrophages (Fig. 5B). Cholesterol homeostasis was further investigated by analyzing the expression of key cholesterol-related genes (Fig. 5C). The expression of cholesterol biosynthesis genes including HMG-CoA reductase (HMGCR) (2-fold) and sterol regulatory element-binding protein-2 (SREBP2) (1.4-fold) as well as that of cholesterol trafficking genes such as low-density lipoprotein receptor (LDLR) (2.3-fold) and Niemann-Pick C1 (NPC1) (1.3-fold), was significantly up-regulated in infected cells vs non-infected ones. Conversely, the expression of the cholesterol efflux gene ATP-binding cassette A1 (ABCA1) was significantly down-regulated in infected cells (0.5-fold). Noteworthy, the overexpression of HMGCR, SREBP2, LDLR genes induced by *L. infantum* infection was significantly lower in BMP-enriched cells (respectively 1.4-fold vs 2-fold, 1.0-fold vs 1.4-fold, and 1.5-fold vs 2.3-fold). The downregulation of ABCA1 and NPC1 genes was maintained in BMP-enriched macrophages as in non-enriched ones. Altogether, these results indicate that *L. infantum* infection induced cholesterol homeostasis disruption including cholesterol redistribution in the vicinity of PV and modulation of cholesterol regulating genes in control macrophages. Of interest, the molecular regulation mechanisms appeared to be minored in BMP-enriched cells.

### 3.4. Effect of BMP enrichment on parasite infectivity and macrophage response

Finally, we investigated the impact of BMP enrichment on *L. infantum* infectivity at several times post-infection. Parasite infectivity was evaluated by the infection rate and the parasite
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Redistribution of BMP in parasitophorous vacuole in infected macrophages.</figcaption></figure>

J774A.1 macrophages were infected by *Leishmania infantum* MON24 for 2h and 24h (MOI 10:1). BMP was stained with primary antibody anti-BMP and Alexa-546 conjugated secondary antibody EEA1 (A), Rab7 (C) and LAMP1 (E) were used as markers of PV with primary antibodies and appropriate Alexa-488 conjugated secondary antibody. Macrophages and parasites nuclei were stained with DAPI. Images were acquired with fluorescent microscope Leica DM6B by performing z-stacks acquisition, equipped with Thunder™ technology which enables blur and diffusion reduction through real-time deconvolution. Colocalization (white pixels) was assessed with ImageJ software. White arrows indicate parasites nuclei. Scale bar: 5 µm. Representative images of 3 independent experiments. Colocalization was calculated with Pearson's correlation coefficient measured on ImageJ software using the Colocalization test Plugin (B,D,F). Results are mean of 157 ± 13 cells per condition. Statistical differences were analyzed by ANOVA test with post-hoc Tukey correction.

<figure><img src="image_7.png" /></figure>

load as detailed in Materials and Methods section. As shown in Fig. 6A, the infection rate was significantly increased at 2h, 24h and 48h post infection by 1.4-, 1.8- and 1.8-fold in DOPG-supplemented macrophages compared to respective controls.

Parasite load was also increased in DOPG-supplemented cells by 1.6, 2.1 and 2.2-fold at 2h, 24h and 48h post infection, compared to respective controls (Fig. 6B). To confirm that these changes resulted from BMP enrichment after DOPG supplementation,
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** BMP quantification during macrophages infection by *Leishmania infantum*.</figcaption></figure>

J774A.1 macrophages were infected by *Leishmania infantum* for 2h and 24h (MOI 10:1). BMP was quantified by LC-MS/MS in macrophages. Results are means ± sd of biological triplicates for each condition. No significant differences by Welch *t*-test (A). Quantification of oleate species of BMP was done by LC-MS/MS for palmitoyl-oleoyl-BMP (POBMP), stearoyl-oleoyl-BMP (SOBMP), dioleoyl-BMP (OOBMP), oleoyl-linoleoyl-BMP (OLBMP) and oleoyl-docosahexaenoyl-BMP (ODBMP). Results are mean ± sd of biological triplicates for each condition. No significant differences with Welch *t*-test (B).

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Effect of BMP enrichment on cholesterol distribution during macrophages infection.</figcaption></figure>

J774A.1 macrophages were enriched with 30 µM DOPG liposomes and infected by *L. infantum* MON24 for 24h. Intracellular cholesterol was stained with Filipin III (100 µg/mL). Macrophages and parasites nuclei were stained with SYBR™ Green I Nucleic Acid Gel Stain. Images were acquired with fluorescent microscope Leica DM6B equipped with Thunder™ technology which enables blur and diffusion reduction through real-time deconvolution. White arrows indicate parasites nuclei (A). Quantification of cholesterol level by LC-MS. Results are mean ± SD of each condition in triplicates and 2 independent experiments (B). mRNA quantification of genes involved in cholesterol homeostasis by RT-qPCR. In the same conditions, mRNA contents from cells were analyzed for SREBP2, NPC1, LDL-R, LXR-B, ABCA1, HMG-CoA and LXR-A by RT-qPCR. Data are the mean ± sd of each condition in triplicates and qPCR triplicates. (C). Statistical differences were analyzed by Mixed-Model with post-hoc Bonferroni correction for cholesterol quantification, and ANOVA with post-hoc Tukey correction for RT-qPCR.

similar experiments were conducted with macrophages supplemented with DDPG. Indeed, as shown in Fig. S1, DPPG was poorly converted into BMP, making a good negative control. No difference in parasite infectivity was observed in DPPG-supplemented macrophages compared to controls.

The consequences of BMP enrichment were also evaluated on the inflammatory and oxidative defense responses of macrophages assessed by cytokine and NO/arginase profiles (S2 fig). The production of pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (TGF-β and IL-10) cytokines was measured at 24 h post-infection with *L.infantum*. Data indicate no significant changes in cytokine profiles in either control or BMP-enriched macrophages upon *Leishmania* infection. NO production was enhanced by infection in BMP-enriched cells whilst no change in arginase activity was observed.

Together, these results suggest that BMP facilitates parasite

infectivity, most likely by interacting with PV formation and cholesterol trafficking rather than macrophage defense responses.

## 4. Discussion

This study highlights the complex interactions between macrophage cholesterol, BMP and PV during *L. infantum* infection. Our findings indeed reveal the redistribution of cholesterol together with BMP during parasite infection and colocalization of BMP with PV markers, as well as the functional impact of BMP on cholesterol regulation and parasite infectivity.

Consistent with the study of Semini et al. [7], we showed that macrophage cholesterol was redistributed in macrophages infected by *L.infantum* with focal accumulation at the interface with the parasite. Notably, BMP shared the same pattern of distribution than cholesterol both before infection and at any time post-
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Effect of BMP on macrophages infection.
J774A.1 macrophages were enriched with 30 µM DOPG or DPPG liposomes and infected by *Leishmania infantum* for 2h, 24h and 48h (ratio10 parasites/cell). Macrophages and parasites nuclei were stained with DAPI. Slides were observed under fluorescent microscope Leica DM6B to count infected cells and intracellular amastigotes. For each slide, on average 500 cells were counted. Infection rate (A) and parasite load (B) were evaluated as described in Materials and Methods. Data are expressed as a ratio as follows: values obtained for DOPG- or DPPG-enriched macrophages versus control macrophages, and are the mean ± sd of 4 independent experiments in triplicate. Statistical differences were analyzed by ANOVA test with post-hoc Tukey correction.</figcaption></figure>

infection, suggesting that BMP might participate in the transfer of cholesterol. Analysis of BMP colocalization analyses with PV-specific markers provided further insights. PV is a dynamic compartment which exhibits many of the characteristics of phagolysosomes, thus PV membrane contains EEA1, Rab7 and Lamp-1 which concentration varies throughout the vacuole maturation process [3,4,32]. We here showed that BMP is strongly associated with Rab7 at early and late time points post-infection. These findings are consistent with the known maturation and acidification requirements of *Leishmania* PV [2]. Previously, it was reported that two other *Leishmania* species, *L.mexicana* and *L major* interfere with cholesterol transport in macrophages, leading to cholesterol retention within the PV [7,9]. Our results thus suggest that cholesterol and BMP are both recruited to the PV during infection with *L.infantum*. Our observations also suggest that cholesterol recruited to the PV mainly originates from the endolysosomal compartment in which it initially colocalizes with BMP. To date, only one study has reported the presence of BMP in the PV of *L. amazonensis* and suggested the existence of a convergence point between the endocytic and autophagosomal pathways [33]. Altogether, our findings suggest a significant role of BMP in PV formation and/or function. Despite BMP redistribution, BMP levels in infected macrophages remained comparable to controls, indicating that the process does not involve up-regulation but rather a relocation from LE/lysosomes to the vicinity of PV.

In addition, our results indicate an increase of total free cholesterol content in infected macrophage. Conflicting data regarding host cellular cholesterol levels following *Leishmania* infection have been reported. While certain studies indicate an increase in cholesterol content in infected macrophages as we observed [34–36], others suggest that *Leishmania* infection conversely leads to cholesterol depletion in host cells [37,38]. The regulation of both biosynthesis and uptake of cholesterol occurs at the gene expression level through SREBP or LXR transcription factors, which enhance the transcription of HMGCR and LDLR when ER cholesterol levels are low [36,39]. LXR also regulates the transcription of the transporters ABCA1 and ABCG1 that control cholesterol efflux pathways. In the present study, qRT-PCR analyses clearly indicate that cholesterol gene regulation was strongly disrupted upon *L. infantum* infection, which possibly reflects the parasite's need of cholesterol. Indeed, up-regulation of biosynthesis and uptake genes is indicative of a lower transfer of cholesterol from LE to ER that might result from cholesterol mobilization by the parasite/PV. Downregulation of ABC

transporters could favor intracellular cholesterol retention thus also ensuring its availability for the parasite/PV. Other studies have revealed that *Leishmania* infection modulates the expression of genes involved in cholesterol biosynthesis, uptake, and efflux, potentially facilitating parasite internalization and survival by altering host cell signaling pathways [7,34,35].

To further evaluate the function of BMP upon *L. infantum* infection, we used the model of BMP-enrichment by supplementation of DOPG liposomes that we validated in a previous study [17,25] and has been used by others since [16]. In particular, we showed that DOPG supplementation induced the specific accumulation of BMP in the late endosomal compartment. Note that as the biosynthetic pathway of BMP remains incompletely defined, targeted depletion of BMP in cells is currently not achievable. Several studies support the notion that late endosomal BMP and cholesterol are intimately linked. Kobayashi et al. (1999) first reported that perturbation of BMP in the late endosomal membrane compromised cholesterol egress from this compartment [14]. Furthermore, experimentally-induced BMP accumulation in LE was shown to correlate with cholesterol accumulation in this compartment [15,40]. In addition, BMP has been shown to stimulate the activity of lysosomal acid lipase which is essential for the degradation of cholesterol esters delivered to endolysosomes by endocytosed LDL into free cholesterol [41]. We here investigated whether BMP enrichment could interfere with macrophage cholesterol regulation in the course of infection with *L. infantum*. Our findings indicate that both cholesterol redistribution in the vicinity of PV and increase of cholesterol content were maintained after BMP enrichment. However, in BMP-enriched macrophages, HMGCR and LDLR genes were less upregulated following *L. infantum* infection compared to non-enriched cells. One possible explanation is that BMP accumulation in macrophages induced cholesterol retention in LE making it readily available/accessible for the parasite/PV, while being transported to the ER. Enhanced availability of LE-associated free cholesterol in BMP-enriched cells might thus favor intracellular development of *L. infantum*.

Consistent with this hypothesis, we showed that BMP accumulation resulted in a significant increase in the infection index from the onset of infection to 48h post infection, suggesting that BMP may favor intracellular parasite development, including multiplication in the PV and/or protection against killing by macrophages. Noteworthy, the facilitating effect of BMP enrichment was also observed upon infection with a visceral strain of *L. infantum* (data not shown), suggesting that BMP may modulate
the infectivity of various *L. infantum* strains or even various *Leishmania* species.

The inflammatory/oxidative responses of the macrophages elicited through activation of toll-like receptors (TLR) during *Leishmania* infection constitute a defense mechanism impacting on the parasite intracellular development [42,43]. Altered intracellular cholesterol distribution has been shown to modulate the inflammatory response of immune cells including macrophages [44]. Yet, no change in inflammatory response of the macrophage was observed in our infection conditions, which is consistent with our previous study reporting moderate production of ROS in this infection model [45]. The adaptive responses of the macrophage and the parasite are intricate and controversial and appear to depend on both the macrophage lineage and the *Leishmania* species/strain, and the time of infection [46,47]. These results rule out the involvement of altered defense mechanisms in the BMP facilitating effect towards the parasite infection.

## 5. Conclusion

Our findings support a model where BMP serves as a key lipid mediator in PV formation, cholesterol transfers and parasite infectivity, without directly altering total cholesterol content nor creating a pronounced inflammatory imbalance. Future studies should elucidate the role of cholesterol transport proteins and their interaction with BMP during *Leishmania* infection. Other mechanisms relying on fusogenic properties of BMP that could contribute to PV formation are also worth to investigate. Interestingly, a recent study identified the patatin-like phospholipase (*Pf*PNPLA2) as responsible for the conversion of PG into BMP in the parasite *Plasmodium falciparum* and showed that *Pf*PNPLA2 knockdown was associated with defective parasite replication and survival [48]. Moreover, it has been shown that acquisition of host cell material by *L. mexicana* can occur from the fusion of PV with BMP-positive multivesicular endosomes in infected macrophages [33]. These results are consistent with ours, also suggesting a facilitating role of BMP in parasite development in host cells. Therapeutic strategies targeting BMP pathways may provide novel avenues to disrupt PV maturation and enhance host immunity against *Leishmania*. Noteworthy, anti-BMP antibody has been developed [26] that may be used for animal studies. Furthermore, recent insights on enzymes involved in BMP synthesis [49,50] may reveal useful to develop pharmacological or genomic inhibitors of BMP and challenge its therapeutic potential.

### CRediT authorship contribution statement

**Clara Hennot:** Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation. **Marine Leroux:** Methodology, Investigation, Formal analysis. **Remi Lambert:** Software, Methodology, Investigation. **Fanny Boukherroua:** Methodology, Investigation, Formal analysis. **Marion Guichard:** Resources, Investigation. **Karen Gaget:** Methodology, Formal analysis. **Federica Calevro:** Writing – review & editing, Validation, Formal analysis. **Isabelle Delton:** Writing – review & editing, Validation, Supervision, Project administration, Formal analysis, Data curation, Conceptualization. **Samira Azzouz-Maache:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization. **Celine Luquain-Costaz:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Formal analysis, Conceptualization.

## Funding sources

This work was supported by own fundings, INSA Lyon (Institut National des Sciences Appliquées de Lyon, France) and University and University Claude Bernard Lyon.

## Declaration of competing interest

The authors have declared no conflict of interest.

## Acknowledgments

We would like to thank Karim Aoun from Tunis Pasteur Institute for providing *Leishmania* strains, the INOBIOVIR plateform, Baptiste Fourmaux from the Functional lipidomics platform (IMBL-INSA Lyon) for BMP analyses, Niyazi Acar and Lucy Martine from CSGA-INRAE Dijon for sterol analyses. We acknowledge the contribution of SFR Santé Lyon-Est (UAR3453 CNRS, US7 Inserm, UCBL) facility: CIQLE, especially Denis Ressnikoff for performing confocal analysis.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2026.02.017.

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