The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging

Abstract

An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs) by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i) hosting amastigotes of either L. major or L. amazonensis and ii) loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i) entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii) the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii) the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.

Figure 1. Enlargement of L. amazonensis PVs.

(A) Immunostaining of LAMP1 for L. amazonensis PV identification in fixed samples of infected macrophages. Red color represents LAMP1 labeling, blue color represents DAPI stain, and green color represents CFSE staining (upper panel) or immunostaining with L. amazonensis-specific antibody (lower panel). Arrowheads indicate parasites sheltered within tight-fitting PVs after 2 h of infection (upper image, bar = 2 µm) and within large PVs sheltering several amastigotes after 48 h of infection (lower image, bar = 5 µm). (B) Expansion of L. amazonensis PVs recorded by time-lapse microscopy of infected macrophage cultures loaded with Lysotracker (green color). Arrowhead indicates an enlarging PV from a tight-fitting phenotype to a large, communal, Lysotracker-positive phenotype. Time-lapse acquisition started after 2 h of infection, and the time of acquisition is shown as d:hh:mm. Bar = 10 µm. (C) Multidimensional imaging of L. amazonensis PVs loaded with Lysotracker in live infected macrophages. The first row of images shows Lysotracker signals exhibited by three large PVs during the recordings. Multidimensional acquisition started after 2 h of infection, and the time of acquisition is shown as hh:mm. The second row shows the software recognition of the three PVs, represented by tridimensional objects; for each object, the software attributed an isosurface (a, b, and c), which permitted measurements of PV volume, diameter, and RFI. In the third row, isosurfaces a, b, and c, representative of each PV, display a statistic-coded color in accordance with volume measurements ranging from cyan (smaller volume) to magenta (larger volume). Bar = 10 µm. (D–E) The isosurface measurements of volume (D) and diameter (E) are shown; although isosurfaces objects a and b increased in volume and diameter, isosurface c presented a discreet decrease in its dimensions. Gray dots in the graphs indicate the time points to which images presented in C are associated. The graphs are an example of 20 macrophage multidimensional images in which PVs enlarged in size. (F) The volumes of L. amazonensis PVs were measured after 2 and 48 h of intracellular infection using multidimensional images acquired using 0.1- or 0.3-µm z-intervals, respectively. The graph shows the median PV volumes measured from three different microscopic fields for each time point (median ± confidence interval, n = 3). There is a significant (P<0.05) increase in PV volumes (Wilcoxon Signed Rank Test), and the measurements were compatible with those acquired using multidimensional images constructed from a z-interval of 1 µm.

Figure 2. Macrophage acidic vesicles decrease in number during L. amazonensis PV growth.

(A) Separated identification of acidic vesicles and Leishmania PVs using the same fluorescence channel. In the left image, isospots 1 µm in diameter were attributed by the software to Lysotracker channel voxels of multidimensional images. The software identified small vesicles surrounding L. amazonensis PVs but interpreted PVs as clusters of Lysotracker-positive vesicles. Each isospot has a statistic-based color corresponding to the Lysotracker RFI mean value (colored bar). By adjusting the thresholds of isospot detection based on Lysotracker RFI, the software can attribute isospots for the acidic vesicles (orange isospots in the second image, indicated by arrow), excluding the fluorescence signal of Leishmania PVs (asterisks). Blend and MIP filters, bar = 10 µm. Image acquisition started after 2 h of L. amazonensis infection, and the time of acquisition is shown (hh:mm). (B) Lysotracker RFIs from L. amazonensis PVs measured using isosurfaces (graph in the left) and Lysotracker RFIs from acidic vesicles measured using isospots (graph in the right) in the same multidimensional image (mean ± SEM, n = 7 [for PVs], n = 40−80 [for vesicles]). (C) L. amazonensis PVs increase in volume (graph in the left, mean ± SEM, n = 7), whereas macrophage acidic vesicles decrease in number (graph in the right) in the same macrophage. Gray dots in the graphs indicate the time points to which images presented in A are associated. (D) Quantification of the number of detected acidic vesicles (1 µm in diameter) in macrophages infected with L. amazonensis (green line) and non-infected macrophages (blue line). Images of these two conditions were separately acquired in the same experiment using multi-chamber dishes with the same acquisition parameters. Data are representative of 8 infected macrophages and 12 non-infected macrophages (mean and SEM) and reproduced in two experiments.

Figure 3. Fusion and remodeling of L. amazonensis PVs.

(A) Fusion between L. amazonensis PVs recorded by time-lapse microscopy of infected macrophage cultures loaded with Lysotracker (green). Asterisks indicate PVs involved in fusion; arrowheads indicate an intermediary PV morphology following membrane fusion. Time-lapse acquisition started after 48 h of infection, and the time of acquisition is shown as h:mm. Bar = 10 µm. (B–C) Multidimensional imaging of L. amazonensis PVs loaded with Lysotracker in live infected macrophages. In B, Lysotracker fluorescence of an infected macrophage containing four PVs. Asterisks indicate the PVs involved in fusion. In C, the isosurfaces representative of each PV in the multidimensional image. Isosurfaces have statistic-based color according to their measured volumes ranging from cyan (smaller volume) to magenta (larger volume). Fusion-prone PVs and fused vacuole are indicated. Image acquisition started after 48 h of infection, and the time of acquisition is shown as d:hh:mm. Bar = 10 µm. (D) Volume measurements of L. amazonensis PVs (a–d) in an infected macrophage (data acquisition started from 2 h of infection). Fusion between PVs is indicated by arrowheads, and the PVs involved in fusion are identified (i.e., when b fused with d, b–d). (E) Volume measurements of L. amazonensis PVs (a–d) in two different infected macrophages (data acquisition started from 48 h of infection). Fusion between PVs is indicated by arrowheads, and the PVs involved in fusion are identified. Another 5 multidimensional images of fusion-prone PVs were also analyzed and presented the same results.

Figure 4. Replication of L. major amastigotes in tight-fitting PVs.

(A) Multiplication of L. major-DsRed2 amastigotes recorded by fluorescent time-lapse microscopy of infected macrophage cultures. Arrowheads indicate dividing parasites (red fluorescence) that were located near each other for several hours. Time-lapse acquisition started after 2 h of infection, and the time of acquisition is shown as hh:mm. Phase contrast merged with DsRed2 fluorescence (red). Bar = 10 µm. Parasites were quantified per microscopic field (40× objective) by algorithm-based recognition of DsRed2 fluorescence, expressed by amastigotes (mean ± SEM, n = 10). The record comprises the period in which the parasite population doubles. Gray dots in the graph indicate the time points to which images are associated. (B) Immunostaining of LAMP1 and LAMP2 proteins for L. major PV identification in fixed samples of macrophages infected with L. major amastigotes for 48 h. Red represents LAMP1/LAMP2 immunostaining, and blue represents DAPI staining. Image shows amastigotes during division process sharing the same PV which presented polarized fission furrows (arrowheads). Asterisk show completely separated amastigotes in individualized PVs. Blend filter, bars = 1 µm. (C) Multidimensional imaging of L. major-DsRed2 (red) amastigotes hosted by live macrophages loaded with Lysotracker (green). First column: Lysotracker weakly stains individual tight-fitting PVs. During division, a Lysotracker cluster in the interface between two dividing L. major amastigotes is observed in some cases; MIP filter. Using the DsRed2 signal expressed by amastigotes (second column, Blend filter), isosurfaces were constructed representing the parasites before (a) and after division (a′ and a″), shown in the third column. The Lysotracker voxels can be detected by the isosurfaces (with transparency applied). Bar = 2 µm. (D) Volume measurements of L. major-DsRed2 amastigotes from isosurfaces in multidimensional images. A volumetric increase in isosurface a (before division) was detected, followed by division into isosurfaces a′ and a″, which present half of the initial value of isosurface a (line graph, in the left). The amastigotes volumes were also measured in other multidimensional images with z-stack intervals of 0.1 and 0.3 µm (bar graph, mean ± SEM, n = 3) with non-significant (ns) statistical difference (one-way ANOVA). (E) Mean Lysotracker RFI values detected in L. major isosurfaces before (a) and after parasite division (a′ and a″). Values diverge between divided isosurfaces a few hours hours after their identification. Results shown in D–E were reproduced in 5 other multidimensional images in which L. major amastigote divisions and lysotracker-positive interface were identified.

Figure 5. Fission of L. major PVs.

(A) Multidimensional image of dividing L. major-DsRed2 amastigotes (red) hosted by macrophages loaded with Lysotracker (green); a lysotracker-positive interface between the parasites is clearly observed at 1:30h. Image acquisition started after 48 h of infection, and the time of acquisition is shown as hh:mm. Blend filter, bar = 2 µm. Isosurfaces a′ and a″ were attributed to replicating amastigotes using the DsRed2 channel. (B) The Lysotracker RFIs surrounding parasites was measured during 10 h of image acquisition. An increase in the Lysotracker signal was detected for one of the dividing amastigotes, whereas the signal remained low in the other amastigote. After 8 h of acquisition, although both amastigotes remained next to each other, they were surrounded by Lysotracker at low intensities, and no Lysotracker-positive vacuolar interface was observed between them. Gray dots in the graphs indicate the time points to which images presented in E are associated. The data are representative of 5 cases with similar results. (C) Multidimensional image of RAW 264.7 macrophages expressing LAMP1-GFP and Rab7-GFP (green) infected with L. major-DsRed2 (red). Arrowheads indicate the complete division of double-occupancy PV into two individual PVs. This process was followed in 5 other multidimensional images and the time period in which amastigotes share a single vacuole before fission was about 100 minutes. Acquisition started after 4 h of intracellular infection and the time of acquisition is shown as hh:mm. MIP filter, bar = 5 µm. (D) The Lysotracker-positive cluster is observed between two dividing parasites (first row, MIP filter). The attribution of an isosurface to the Lysotracker-positive interface (second row, Blend filter) allowed the measurement of its volume at the image time points. The isosurface contains statistic-based color information ranging from cyan (smaller volumes) to magenta (larger volumes). Image acquisition started after 2 h of infection, and the time of acquisition is shown as hh:mm. Bar = 5 µm. (E) The graph presents the volume measured from the cluster between dividing parasites in the multidimensional imaging. Using a z-stack interval of 1 µm, a detection limit for volumetric measurement of this structure was determined to be 5 µm³. Gray dots in the graphs indicate the time points to which images presented in D are associated. These results were reproduced in 5 other cases.

Figure 6. Numerical decrease of host cells acidic vesicles during L. major division.

(A) Multidimensional imaging of a macrophage loaded with Lysotracker and hosting L. major-DsRed2 replicating amastigotes after 48 h of infection. On the left, Lysotracker (green) and DsRed2 (red) merged signals, MIP filter. On the right, isospots were attributed by software for macrophage acidic vesicles (green) considering Lysotracker fluorescence intensity; isosurfaces for parasites (constructed from DsRed2 fluorescence intensity) were also attributed (in the detail, gray). Bar = 10 µm. (B) Quantification of the number of amastigotes detected isosurfaces and the number of detected acidic vesicles (1 µm in diameter) in two macrophages (green and blue lines). (C) Quantification of the number of detected acidic vesicles (1 µm in diameter) in other four macrophages (lines colored accordingly) - arrowheads indicate the moments when at least one amastigote began to replicate. (D) Mean number of detected acidic vesicles in macrophages infected with L. major-DsRed2 (green line) and non-infected macrophages (blue line). Images of these two conditions were separately acquired in the same experiment using multi-chamber dishes, and the same acquisition parameters. Data are representative of 14 infected macrophages and 12 infected macrophages (mean and SEM) and reproduced in two experiments.