Triton X-100, 4’,6-diamidino-2-phenylindole dilactate (DAPI), phorbol 12-myristate 13-acetate (PMA), 1,6-Diphenyl-1,3,5-hexatriene (DPH), 1-[4-(trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), Phenylmethylsulfonyl Fluoride (PMSF), NaCl, HEPES, EDTA, TRIS and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich (St. Louis, MO). L-glutamine and penicillin/streptomycin were obtained from Gibco. Bradford reagent was purchased by Bio-Rad (Hercules, USA). Protease inhibitors were provided by Thermo Scientific (Waltham, USA). All chemicals were of the highest quality available.

We used promastigotes of L. infantum lines: (i) JPCM5 (MCAN/ES/98/LLM-877) as a reference control line; and (ii) LLM2070, LLM2165, LLM2255 and LLM2221 lines as L. infantum lines isolated from HIV positive patients with visceral leishmaniasis and TF, treated with liposomal amphotericin B and antiretroviral therapy, from the WHO Collaborating Center for Leishmaniasis, Instituto de Salud Carlos III (ISCIII) (Dr. F. Javier Moreno). The sensitivity profile of the L. infantum lines has been previously described (Perea-Martinez et al., 2022). All these L. infantum lines were grown at 28°C in RPMI 1640-modified medium (Invitrogen) supplemented with 10% hiFBS (Invitrogen), as described (Manzano et al., 2019). Human myelomonocytic THP-1 cells were grown at 37°C and 5% CO₂ in RPMI-1640 medium supplemented with 10% iFBS, 2 mM glutamate, 100 U/mL penicillin and 100 mg/mL streptomycin as described (Sanchez-Fernandez et al., 2019).

THP-1 cells (3x10⁷ cells in 175-cm² flasks or 5x10⁵ cells/well in 24-well plates) were differentiated to macrophages with 20 ng/mL of PMA treatment for 48 h followed by 24 h of culture in fresh medium. For in vitro infection, macrophage-differentiated THP-1 cells were infected with different L. infantum stationary-phase promastigotes incubated for 72 h in acid medium plus 10% hiFBS or heat-killed promastigotes (incubated for 1 h at 65°C) at a macrophage/parasite ratio of 1:10 (Perea-Martínez et al., 2022). Infected macrophages were incubated in RPMI 1640 medium plus 10% hiFBS at 37°C and 5% CO₂ for 96 h. On the other hand, to determine the percentage of infection and the average of amastigotes by cell, macrophages were infected with the same L. infantum lines and treated in parallel with identical conditions described above. For microscopy visualization, cells were fixed for 30 min at 4°C with 2.5% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 30 min. The infection level (60-80%) and mean number of intracellular parasites/cell (7-10) were detected by nuclear staining with DAPI (Invitrogen) ( Supplementary Table 1 ). Additionally, we used macrophages that had been allowed to phagocytose heat-killed parasites and uninfected macrophages as controls. In this way, we will identify changes in gene expression that represent a general result of phagocytosis and phagolysosome formation rather than being specific to Leishmania infection. The abbreviations for different infected host cell lines used are the following: (i) Hi-LJPC for THP-1 cells infected with L. infantum JPCM5 line; (ii) Hi-L2070 for THP-1 cells infected with L. infantum LLM2070 line; (iii) Hi-L2165 for THP-1 cells infected with L. infantum LLM2165 line; (iv) Hi-L2255 for THP-1 cells infected with L. infantum LLM2255 line; and (v) Hi-L2221 for THP-1 cells infected with L. infantum LLM2221 line.

Approximately 3x10⁷ L. infantum infected THP-1 cells cultured in 175-cm² flasks were washed three times with PBS and subsequently detached and broken with cold hypotonic buffer (10 mM Tris-HCl pH 7.4, 2 mM EDTA), a mixture of protease inhibitors (Thermo Scientific) and PMSF (Sigma-Aldrich). The cells were centrifuged at 12000 g for 20 min to eliminate unbroken cells and amastigotes. Then, the supernatant was collected and centrifuged at 50000 g for 60 min to discard the rest of the cellular components. The pellet containing the Leishmania depleted THP-1 plasma membrane fractions was resuspended in a buffer containing 20 mM HEPES pH 7.4 and 150 mM NaCl enriched with protease inhibitors. Protein content was quantified using Bradford assay (Bio-Rad).

In order to discard the presence of parasites in the THP-1 plasma membrane samples, and consequent validation of the technique, the absence of Leishmania membranes from the isolated fractions was checked. For that purpose, we analyzed the presence of the Leishmania surface protease GP63 in the samples. Additionally, we detected Na⁺/K⁺ ATPase, an enzyme located in the plasma membrane of cells to confirm the isolation of the infected THP-1 membrane fraction ( Figure S1 ).

Consequently, protein samples were fractionated by SDS-PAGE under standard conditions and electrotransferred onto Immobilon-P membranes (Millipore). Subsequently, membranes were cut in order to perform incubations with different antibodies on the same loaded samples. Immunodetection was performed by using a 1:5000 dilution of mouse monoclonal anti-(GP63) (Life Span BioSciences) or mouse monoclonal anti-(Na⁺/K⁺ ATPase, α1 subunit) (Sigma-Aldrich) in phosphate-buffered saline (PBS) plus 0.01% Tween 20 and 0.1% bovine serum albumin (BSA). After washing, membranes were incubated with horseradish peroxidase conjugated secondary goat anti-mouse immunoglobulin G (DAKO) using a 1:5000 dilution. Signal was detected employing the ECL chemiluminescent substrate (Pierce).

Cholesterol content in the plasma membrane fractions of THP-1 cells was measured using Amplex Red Cholesterol Assay kit (Life Technologies, Oregon, USA), following the manufacturer’s protocol. For each reaction, we used 2.5 μg protein of plasma membrane that correspond with the fraction obtained after depleting Leishmania amastigotes following the protocol described above. Briefly, reactions took place in a 96-well plate by the addition of 50 μL of assay sample and 50 μL of Amplex Red working solution. The 5 mL of Amplex Red working solution were prepared previously, containing Amplex Red reagent, horseradish peroxidase, cholesterol oxidase and cholesterol esterase in the indicated amounts in the kit. The reactions were incubated for 30 min at 37°C protected from light. Finally, the fluorescence intensity was measured at 540 nm (excitation)/590 nm (emission) using an Infinite F200 microplate reader (Tecan Austria GmbH, Austria). The cholesterol contents were determined using 0-6 µg/mL cholesterol standards (provided in the Amplex Red kit and used as reference to obtain the standard curves).

The changes in membrane fluidity of THP-1 cells infected with different clinical L. infantum isolates were measured by assessing fluorescence depolarization of the probes DPH and TMA-DPH as described (Manzano et al., 2011). Briefly, L. infantum- infected THP-1 plasma membrane samples that correspond with the fractions obtained after depleting Leishmania amastigotes following the protocol of preparation described above (0.14–0.15 mg/mL final protein concentration) in 10 mM HEPES, 145 mM NaCl, pH 7.4, buffer were incubated with DPH or TMA-DPH probes in N,N′-dimethylformamide (DMF) in the dark for 30 min at room temperature at a 1/2500 probe/protein weight ratio. The final DMF concentration in the membrane suspension was always < 0.05%. A Cary Eclipse spectrofluorometer (Agilent Technologies) was used to measure the steady-state anisotropy (r), quantifying the vertical and horizontal components of the fluorescence emission with excitation polarized vertically. The grating factor (GF) is specific for the instrument and is defined by the ratio of the fluorescence intensities with polarizers in the horizontal and vertical position (excitation and emission respectively), and both horizontal, respectively. The slit widths for both excitation and emission were 5 nm and the integration time was 1 s. The excitation wavelength for DPH and TMA-DPH was 360 nm, with emission being monitored at 430 nm. The data for each experiment were calculated as the average of 10-s anisotropy measurements at a fixed temperature of 37°C due to its physiological relevance.

The sequences of THP-1 cells infected with TF lines employed in the present manuscript were obtained from a previous RNA-seq project available at NCBI Short Read Archive (SRA) under the accession number: PRJNA781438, as described (Perea-Martínez et al., 2022). Briefly, miARma-Seq pipeline was used (Andrés-León et al., 2016) to perform sample quality control, the alignment of samples and the calculation of differentially expressed genes. Therefore, sequences were aligned by using HISAT2 (Kim et al., 2015), against the protein coding genes from the Homo sapiens Gencode version GRCh38-M26 genome-build. Gene expression values were used to compare the three replicates of THP-1 infected with L. infantum lines adjusted by control samples (uninfected cells and phagocytosis control) using edgeR (Nikolayeva and Robinson, 2014; Ritchie et al., 2015). All genes having a false discovery rate (FDR) value < 0.05 were included in the study. Log₂FC was used to evaluate the significance and the change in expression of a gene respectively between different types of samples. We considered a gene to be differentially expressed (DEGs) using the parameters Log₂FC value ≥ 0.58 or ≤ -0.58 and FDR value ≤ 0.05.

The cluster Profiler Bioconductor package (Yu et al., 2012) was used with the aim of identifying differential gene expression effects by carrying out a functional enrichment study. For this purpose, all expressed genes of the RNA-seq assay were compared obtaining the Gene Ontology (GO) terms from the Bioconductor Homo sapiens database and associated to Entrez gene identifiers in an orgDB R object through the AnnotationForge package to be used with clusterProfiler. Significant (p value ≤ 0.05) terms related to sterol pathways from the GO: biological process (BP) ontology were selected and the complete ancestor chart of the BP ontology (Binns et al., 2009) was created with QuickGO tool. The genes from the GO terms: cholesterol efflux (GO:0033344), cholesterol transport (GO:0030301), cholesterol storage (GO:0010878) and cholesterol metabolic process (GO:0008203) were selected, and heatmaps were created with R 4.2.1 program (Galili et al., 2018) using the heatmaply package (Team, 2021). In these heatmaps, we represented the log₂FC values of the genes based on the differential expression previously performed. In this way, the heatmaps allow us to cluster samples according to gene expression alteration. The measurement of dissimilarity between sets of observations was calculated using the euclidean distance. To specify the dissimilarity of the pairwise distances, the complete-linkage algorithm was used for the hierarchical clustering.

Membrane fluidity is known to affect the function of biomolecules residing within, or associated with, the membrane structure. For example, the binding of some peripheral proteins is dependent on membrane fluidity (Heimburg and Marsh, 1996). Membrane-dependent functions, such as phagocytosis and cell signaling, among others, can be regulated by the fluidity of the cell-membrane (Helmreich, 2003; Zhou et al., 2015).

Changes to plasma membrane fluidity in THP-1 cells infected with L. infantum lines from TF patients were evaluated by fluorescence anisotropy spectroscopy analysis using the fluorescent probes DPH and TMA-DPH. Since this parameter gives an estimation of the probe free rotation in the lipid bilayer, a higher anisotropy value should correspond to a decrease in the membrane fluidity. TMA-DPH is located near the surface while DPH is found deeper in the plasma membrane, and thus, each one reports the fluidity of a different region of the bilayer (Liang et al., 2004; Poojari et al., 2019). As expected, we obtained higher anisotropy values with TMA-DPH versus DPH probe ( Table 1 ), due to the fact that the surface bilayer of plasma membrane is less fluid than the internal plasma membrane, as a consequence of lower amounts of cholesterol, as previously described (Do Canto et al., 2016). As can be seen in Table 1 , the surface fluidity determined using TMA-DPH showed similar values in all samples, thus suggesting that there are no major changes at this level. However, measurements of THP-1 plasma membrane fractions fluidity using the DPH probe showed significant differences between the phagocytosis control and all the THP-1 cell samples infected with L. infantum lines from patients with TF (Hi-L2070, Hi-L2165, Hi-L2255 and Hi-L2221; Table 1 ). Indeed, the plasma membrane of these THP-1 infected cells was less fluid than in the controls (phagocytosis control and untreated cells). Additionally, we found significant differences in DPH anisotropy between THP-1 cells infected with L. infantum from TF isolates versus THP-1 cells infected with the reference L. infantum line (Hi-LJPC) ( Table 1 ).

Overall, this study showed that these Leishmania clinical isolates from patients with leishmaniasis and TF are able to modulate plasma membrane fluidity in infected host cells, which could partially explain the parasite’s ability to survive in the host macrophages, thereby contributing to TF.

Several factors could affect the fluidity of plasma membranes: (i) the length and the degree of saturation of the fatty acids that compose the lipid bilayer, (ii) temperature, and (iii) the cholesterol content of the membranes, among others (Gennis, 1989).

Considering that our data showed a significant decrease in the fluidity of the plasma membranes of Leishmania-infected THP-1 cells, we decided to analyze the cholesterol content in these samples, using the Amplex Red Cholesterol Assay kit, which is based on the oxidation of cholesterol and generation of hydrogen peroxide, with subsequent oxidation of the Amplex red reagent to produce a fluorescent compound. The results showed a significant increase in the cholesterol content of the plasma membrane fractions of THP-1 cells infected with TF Leishmania lines in comparison with the control (heat-killed phagocytosed parasites; Figure 1 ). The biggest difference was found for Hi-L2255, which generated an increase in the cholesterol concentration to 4 µg/mL. In contrast, THP-1 cells infected with the reference control line (Hi-LJPC), and non-infected cells, exhibited a significant decrease in cholesterol content ( Figure 1 ). These results are consistent with our data for plasma membrane fluidity obtained for the different Leishmania-infected host cells ( Table 1 ), as an increase in the cholesterol content of the plasma membrane fractions should lead to a decrease in the fluidity of the plasma membranes mainly affecting the hydrophobic core of the bilayer, as previously described (Gennis, 1989; Shrivastava et al., 2008).

Additionally, our results indicate that the infection of host THP-1 cells with heat-killed or TF parasites seems to increase the cholesterol content of plasma membrane fractions compared with uninfected cells, which showed the lowest cholesterol values ( Figure 1 ).

After analyzing the results obtained for plasma membrane fluidity and cholesterol content in membranes from THP-1 cells infected with the different Leishmania lines, we examined the cholesterol-related enriched pathways (GO: BP terms) by analyzing the RNA-seq data for the infected host cell lines used in this study (described in the Materials and Methods section).

These enriched terms are highlighted with a yellow background in Figure 2 , and the THP-1 cells infected with different Leishmania lines from TF in which they appear enriched are indicated by colored boxes. In addition, all the pathways related to the enriched terms are also shown ( Figure 2 ). These GO terms are shown in an ancestor chart from the most general “biological process” (top of Figure 2 ), to the more specific sterol-related term (bottom of Figure 2 ). Thus, three main differentiated terms that are related to each other can be observed: (i) “metabolic process”, (ii) “biological regulation”, and (iii) “localization” ( Figure 2 ).

According to previous studies, the increase in cholesterol in the plasma membrane fractions of cells infected with TF lines might be caused mainly by changes in storage, synthesis, and transport (Samanta et al., 2017). Consequently, we focused our analysis on genes belonging to the following enriched pathways: (i) “cholesterol storage” included in “localization”; (ii) “cholesterol metabolic process” directly related to the “metabolic process” category, and finally encompassed into “biological regulation”; (iii) “cholesterol transport”; and (iv) “cholesterol efflux”.

In order to represent the differences between genes integrated in the enriched GO terms for Leishmania-infected THP-1 cells, two heatmap plots were generated. One of these plots comprised the genes belonging to “cholesterol metabolic process” and “cholesterol storage” ( Figure 3A ), and the other grouped the genes related to “cholesterol transport” and “cholesterol efflux” ( Figure 3B ). Both heatmaps provided an overview of the distribution of the genes upregulated and downregulated in the THP-1 cells infected with the different Leishmania lines. As shown in Figure 3 , the Hi-L2221 line exhibited the greatest differences in terms of gene expression (high positive or low negative log₂FC values), whereas Hi-L2255, Hi-L2165 and Hi-L2070 were similar to each other. Hi-LJPC, in turn, presented medium log₂FC values for the vast majority of genes.

Amongst the biological processes studied, we highlighted some DEGs as key factors that could explain the high cholesterol present in the plasma membrane fractions of THP-1 cells infected with L. infantum lines from TF. Thus, NR1H3, which codes for an important transcription factor (liver X receptor, LXR) was overexpressed in all lines except Hi-LJPC. LXR regulates the uptake and efflux of cholesterol by acting directly on ABCG1 and ABCA1 (Yang et al., 2006); these genes were found to be upregulated in Hi-L2165, Hi-L2070 and Hi-L2255. APOE is also regulated by LXR, and was found to be one of the DEGs in Hi-L2165 and Hi-L2221. In the “cholesterol metabolic process” pathways, SCD, which codes for a desaturase involved in biosynthesis, was overexpressed in cells infected with all different Leishmania lines from TF. CYP27A1, LPL and MSR1 were some of the relevant DEGs found in some of the lines and included in the GO category “cholesterol storage”. Finally, we observed that the genes APOC1, LRP1, SCARB1, CD36, VLDLR, LDLR, APOA2, APOC2 and ABCB4, which were grouped in the categories related to cholesterol efflux, uptake and transport, were differentially expressed in the lines mentioned in Table 2 .