Fig 1.
Hierarchical clustering of lipid metabolism related genes that are differentially expressed in L. major infected BALB/c derived macrophages.
Analysis using dChip software of lipid related transcripts identified four distinct clusters. Cluster 1 contains genes down-regulated in both live parasites (P) and heat-killed parasites (KP)-infected cells. The cluster 3 contains genes up-regulated in P and down-regulated in KP-infected cells. Cluster 4 contains genes more heavily up-regulated in KP- infected cells. Finally, cluster 2, contains genes up-regulated during early infection times (3 and 6 h pi) in both P and KP. Each row of the cluster represents a spot on the microarray and each column a separate microarray. The BMM response was studied at five different time points (from the left to the right: T1h, T3h, T6h, T12h and T24h) in P and KP-infected cells. The left-hand column shows non-infected cells (NI) that were used as internal control.
Fig 2.
IPA analysis of Lipid related genes.
Lipid related metabolic pathways identified by Inguenuity Pathway Analysis (IPA) software as significantly altered (p < 0.05) in L. major infected BALB/c macrophages for all time points. Each bar graph shows the number of Leishmania modulated genes belonging to a given pathways and ranked by their scores. The negative log10(p-value) is plotted on the Y-axis.
Fig 3.
Time-course of LD accumulation in L. major infected BMMs.
BMMs were infected for different times with live parasites. Cells were then fixed and subjected to Bodipy 493/503 staining for lipid droplets accumulation enumeration. Each bar represents the mean plus standard error of the mean (SEM) from 100 consecutively counted macrophages from at least 4 independent experiments. Statistically significant (*, p <0.05) and (***, p <0.001) differences between control and infected groups are indicated by asterisks.
Table 1.
Prostaglandin modulated genes in L. major infected BMMs validated by qPCR.
Gene expression of prostaglandin pathway related genes in BALB/c Leishmania infected macrophages using qRT-PCR is analysed. Changes in mRNA levels are calculated using the 2−ΔΔCT method (ΔΔCt = ΔCt -ΔCt NI (t: point of the infection with P or KP) and ΔCt = Ct gene of interest- Ct of reference gene). Four reference genes were used for the qPCR normalization. The numbers presented for each time points are the average of the three biological replicates. The abbreviations NI, P and KP respectively refer to Non Infected macrophages, macrophages infected with live Parasite, macrophages infected with Killed Parasite.
Fig 4.
L. major induced LD formation in BMMs infected with live and killed parasites and in non-infected neighbouring cells
(A) LDs (green) were visualized after Bodipy staining, 24h post treatment with DsRed-L. m (red) or fluorescent latex beads (red). (B) Quantification of LD in red latex beads stimulated and L. major infected BMMs, by enumeration of LD positive cells after Bodipy 493/503 staining. (C) Enumeration of LDs after Bodipy staining in BMMs infected by live and heat-killed parasites. (D) Bar graphs show LD positive cells in L. major containing and L. major free cells. (E) Quantification of cytochalasin D effect on L. major internalization in BMMs. (F) Lipid droplets positive cells were quantified in non-infected and Leishmania infected macrophages previously treated or not with cytochalasin D. (G) Quantification of LD positive cells was performed in naïve cells exposed for 24 hours to non infected or L. major infected cells conditioned medium. Statistically significant (*, p <0.05), (**, p <0.01) and (***, p <0.001) differences between control and infected groups are indicated by asterisks. Data are representative of four independent experiments and are expressed as the mean plus standard error of the mean (SEM).
Fig 5.
LD accumulation in live Ds-red-L. major infected BMMs.
Macrophages were infected with the transgenic fluorescent parasites Ds-Red-L. major GLC94. The LDs in Lm-infected BMMs were stained with Bodipy and time-lapse sequence of the recruitment processes was investigated using live time confocal microscopy. (A) 3D images were acquired by confocal microscopy through the time-series of Z-stacks and 3D representation was performed using the opacity tool (Volocity). LDs presence was detected in the cell cytoplasm (white arrowheads), in internalized parasites (yellow arrowheads) as well as in extracellular parasites (red arrowheads). (B) Representative time-lapse series examining late-time course of LD recruitment during L. major infection. Neutral lipids in the cytoplasm (LDs) were stained with Bodipy after 24 h of parasitic infection and time-lapse photography of the cells at 30 min intervals for 18 hours was done. Images clearly show that parasites (red) are massively surrounded by LDs (green) across the acquisition time. (C) Quantification of LD accumulation coming in contact with L. major parasites during the time lapse series presented in B, was performed using ImageJ software. Statistically significant (*, p <0.05), and (***, p <0.001) differences between control (point 24h+0h) and the four different time points are indicated by asterisks. Data are representative of three independent experiments and are expressed as the mean plus standard error of the mean (SEM).
Fig 6.
LD formation is neither restricted to parasitophorous vacuoles containing L. major nor to LPG stained Leishmania parasites in infected BMMs.
BMMs were infected with WT L. major parasites for 24h. Cells were then washed to remove extracellular parasites. (A) Parasitophorous vacuoles were stained using anti-LAMP1 and nuclei were stained using Draq5. Cells were then incubated with Bodipy for Lipid Droplet localization. White arrowheads show LD out of the LAMP1 stained vacuoles while yellow arrowheads show LD localized inside the parasitophorous vacuoles. Lipid accumulation inside the parasitophorous vacuole is shown in a magnification box. (B) Quantification of lipid droplets percentage inside and outside the LAMP1 positive parasitophorous vacuole in Leishmania infected macrophages. Statistically significant (***, p <0.001) difference between the percentage of LD inside and those outside the LAMP1 stained vacuole are indicated by asterisks. Data are representative of three independent experiments and are expressed as the mean plus standard error of the mean (SEM). (C) LD localization in cell cytoplasm as well as in parasite cytoplasm is shown by a Z-stacks and 3D image rendering. Cells were subjected to anti-LPG for parasite outline, Bodipy for LD and Draq5 for nuclei staining. Different snapshots of LD localization around and in parasite cytoplasm from the 3D image rendering were pinpointed in magnification boxes.