article: 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.
  Leishmania spp. are dimorphic trypanosomatid parasites that alternate between extracellular promastigote forms found in insect vectors and intracellular amastigote forms found in mammalian hosts. In infected cells, Leishmania amastigotes are sheltered within phagolysosome-like structures called parasitophorous vacuoles (PVs). The PV membranes and contents change as PVs fuse with the endoplasmic reticulum (ER), late endosomes, lysosomes, or other host cell vesicular elements, conferring to them distinctive properties and a hybrid nature [1]–[3]. In the majority of Leishmania species, including L. major, one or two amastigotes are enclosed within PVs, which display a modest vacuolar space. In contrast, the large PVs that shelter parasites of the L. mexicana complex, such as L. amazonensis, can contain numerous amastigotes, often bound by their posterior poles to the internal face of the PVs [4]. The biogenesis of these two types of PVs involves the acquisition of host cell late endosomes membrane markers, as shown in infected cells immunostained for lysosome-associated membrane proteins (LAMPs), Rab GTPases, cathepsin, proton ATPases, and MHC class II molecules [2], [5]–[8]. The acquisition of these markers is a coordinated event that results in a “mature” PV, which is presumably required for the survival and multiplication of the parasites. Because the relatively large dimensions of their PVs that allow them to be easily recognized at low magnification, L. amazonensis and L. mexicana PVs have often been used in studies of the fusogenic properties of Leishmania PVs. These PVs could be demonstrated to selectively fuse with each other or with phagosomes containing macromolecules, colloids, inert particles, and other live parasitic microorganisms [9]–[17]. The fusogenicity and easy access of certain particles and molecules to these large structures increase with the duration of infection [1]. The spacious PVs also incorporate acidic pH markers such as Lysotracker and neutral red [17], [18] and may be probed using pH-sensitive dyes [19], [20]. By taking advantage of differences in fluorescein emission under different pH conditions, it was reported that the pH of L. amazonensis PVs falls from approximately 5. 2 at 24 h to 4. 8 after 48 h of intracellular infection, whereas the pH of secondary lysosomes of around 5. 4 remained constant in non-infected control cells [19]. These studies led to the characterization of the biochemical and functional features of Leishmania PVs, which may not apply to the majority of Leishmania species studied that are lodged in tight PVs and assumed to undergo fission as parasites divide [21], [22]. The accessibility of particles, macromolecules, and probes to these tight-fitting PVs and the identification of their contents are hindered by the limited vacuolar space available between the parasites and their PV membranes. Chang and Dwyer [21] and Berman and colleagues [23] observed by electron microscopy that thorium dioxide (“Thorotrast”) particles, which were pre-loaded in lysosomes, were transferred to the small vacuolar space of L. donovani and L. major PVs, respectively. The granules were absent within PVs when parasites and PV membranes were only in close contact. These studies suggest that tight-fitting Leishmania PVs can fuse with lysosomes, although the retention of lysosomal markers differs accordingly to PV dimensions. Additionally, the pH in tight-fitting PVs may be different from that within loose vacuoles: the pH within L. donovani tight PVs was reported to reach 5. 5 after 2 h of infection [24]. Most of the available information on Leishmania PV biogenesis has been obtained by experiments on fixed cells, a drawback we sought to overcome in the present study. We examined the biogenesis of large or tight-fitting, membrane-bound Leishmania PVs recorded by the multidimensional imaging of live infected macrophages. The fission of Leishmania tight-fitting PVs was studied for the first time in live infected cells and characterized as a two-step process that involves the replication of amastigotes in a single PV prior to separation into two distinct PVs that accumulate transient amounts of lysosomotropic probe. The process is accompanied by the depletion of macrophage small acidic compartments, as previously described for Leishmania large vacuoles [25]. The biogenesis of these large structures was also studied and revealed to involve PV enlargement in volume and diameter to the detriment of other PVs in the same infected cells. The homotypic PV fusion between L. amazonensis PVs was recorded and involves PV volume restoration.
  All experiments involving animal work were conducted under Brazilian National Commitee on Ethics in Research (CONEP) and French National Committee on Ethics and Animal Experimentation (CNREEA) ethic guidelines, which are in accordance with international standards (CIOMS/OMS, 1985). The present study was approved by CEP/UNIFESP (Comitê de Ética em Pesquisa da Universidade Federal de São Paulo/Hospital São Paulo) under the protocol number 0856/07. BALB/c and BALB/c nude mice (8 weeks of age) were used as sources of bone marrow macrophage precursor cells and lesion-derived Leishmania amastigotes. Macrophages were obtained from bone marrow precursor cell suspensions cultivated in vitro for 7 days in RPMI 1640 medium with 10% fetal calf serum, 5% L929 cell conditioned medium, 100 U/ml penicillin, and 100 µg/ml streptomycin [7]. RAW 264. 7 macrophage-like cells were cotransfected with LAMP1-GFP and Rab7-GFP plasmids (using FuGene HD transfection reagent, ROCHE) kindly donated by Dr. Norma Andrews (Maryland University) and employed in infection experiments in order to observe PV membranes in live recordings. Macrophages were transferred to glass coverslips or round dishes (ibidi, GmbH or Mattek Corporation) suitable for maintaining living cells in incubators coupled to microscopes. Before their use in experiments, cultures were incubated overnight at 37°C in a humidified air atmosphere containing 5% CO2. BALB/c nude mice footpads were inoculated with wild-type L. (L.) amazonensis LV79 (MPRO/BR/72/M1841) or DsRed2-transfected L. (L.) major NIH173 (MHOM/IR/-/173). Isolation of amastigotes from footpad lesions was performed as previously described [26] after 2 months of inoculation. Leishmania amastigotes were added to macrophage cultures at a multiplicity of infection of 5 and incubated at 34°C and 5% CO2 in complete medium for different periods according to the experiment. Cultures were washed with Hanks' Buffered Salt Solution to remove free parasites and cultivated in complete medium at 34°C in a 5% CO2 atmosphere. Observation and image acquisition of live or fixed macrophage cultures under the employed microscopes started after periods ranging from 2 to 48 h depending on the experiment. Cultures were maintained at 34°C and 5% CO2 within the incubators coupled to the microscopes. Macrophages on coverslips were washed and fixed for 1 h with 3. 5% formaldehyde in phosphate-buffered saline (PBS). Leishmania PVs and other compartments were identified by immunolabeling of the membrane proteins LAMP1 and LAMP2 (monoclonal antibodies obtained from DSHB, Iowa University, USA). L. amazonensis amastigotes were immunostained with 2A3-26 antibody conjugated to FITC (kindly provided by Dr. Eric Prina, Institut Pasteur, France) or loaded with 5 µM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen, Life Technologies). Samples were stained for 15 min with 100 µg/ml 4′, 6-diamidino-2-phenylindole (DAPI, Invitrogen, Life Technologies) and mounted with 50% glycerol in PBS containing 0. 01% p-phenylenediamine. Confocal images were obtained using a Bio-Rad 1024 UV system coupled to a Zeiss Axiovert 100 microscope or a Leica TCS SP5 II system. Images acquired with a 100× (1. 44 NA) oil immersion objective were rendered with Imaris Software (Bitplane AG) by using Blend filters. Live imaging of cultures was performed using a Nikon Biostation IM-Q Live cell recorder system (Nikon Corporation), a Perkin-Elmer UltraView RS Nipkow-disk system (PerkinElmer Inc.) attached to a Zeiss Axiovert 200 M microscope with a Hamamatsu ORCA II ER CCD camera, or in a Leica TCS SP5 II system (Leica Microsystems). To identify Leishmania PVs, 50 nM Lysotracker green DND-26 (Invitrogen, Life Technologies), a lysosomotropic probe for acidic compartments, was added to complete medium 1 h before microscopic recordings and maintained throughout image acquisition. In contrast with L. amazonensis PVs that were rich in Lysotracker, vacuoles containing a single L. major-DsRed2 parasite displayed a feeble Lysotracker signal, possibly due to the lower acidity of their PVs. The effect of a tight vacuolar space between PV membranes and amastigotes on Lysotracker intensity is not discarded, although Lysotracker is a relatively small molecule (MW = 398. 69). Alternatively, 1 mg/ml FITC-dextran (average mol wt 42,000, Sigma-Aldrich) was used as lysosomotropic probe, with a 1 hour pulse, removal of the probe by 6 washings prior to image acquisition. The Nikon Biostation IM-Q was used to acquire, in 10 different microscopic fields, serial images of infected macrophage cultures in dishes. The Biostation acquired images in phase contrast and in two fluorescent channels (for Lysotracker- and DsRed2-labeled parasites) with a 40× (0. 8 NA) objective in 5-min intervals. Points in time of time-lapse image acquisitions are displayed as day, hours, and minutes (d: hh: mm). Images of the DsRed2 fluorescence signal displayed by L. major were processed by Acapella software (PerkinElmer Inc.) for algorithm-based quantification of these parasites during infection in macrophage cultures [17]. The Perkin-Elmer UltraView RS and the Leica TCS SP5 II system were used to acquire stacks of 20 to 30 optical sections from live infected cells in 5 to 12 microscopic fields. Stacks along the z- axis (z-stacks) were obtained with an optical section separation (z-interval) of 0. 2 to 1 µm. We acquired images of infected cell cultures after different post-infection times. Some image acquisitions began 2 h after parasite addition to macrophages; other started after 24 h or 48 h. Thus, we chose to present temporal data as “time of image acquisition” instead of “time of infection” due to different time ranges of intracellular infection. The time of multidimensional acquisition is displayed as d: hh: mm. All statistics were performed using SPSS software (SPSS Inc.). The statistical tests employed are indicated in the figure legends, and they were chosen on the basis of normal and non-normal distributions and equal and non-equal variances. Figures represent the same result reproduced in at least 2/3 out of all analyzed multidimensional images acquired in 3 different experiments, using 2 different equipments.
  PVs are the host intracellular compartments in which Leishmania parasites differentiate and multiply. They are lined by a dynamic membrane originated at the host cell plasma membrane and formed by successive and coordinated fusion/fission events with vesicles from early/late endocytic pathways, secondary lysosomes, ER, and possibly autophagic vesicles, resulting in an acidic compartment similar to phagolysosomes [3], [5], [7], [19], [27]. Most of this information has been obtained in experiments with Leishmania cell-cycling amastigotes of the L. mexicana group in fixed preparations. This study presents high-resolution morphological characterization of the main features displayed by Leishmania PVs in live infected macrophages, such as PV volumetric expansion/retraction and homotypic PV fusion/fission, using the fluorescent lysosomotropic probe Lysotracker. Additionally, a software-based methodology permitted the quantification of host cell acidic vesicles, which were distinguished from Leishmania PVs in the same fluorescence channel. As secondary lysosomes are considered the main source of membrane for Leishmania PV biogenesis, we investigated how macrophage acidic vesicles reservoirs (which include, but are not restricted to secondary lysosomes) were related to Leishmania PV biogenesis. The development of spacious PVs is an exception rather than a rule for most Leishmania parasites studied. Soon after the internalization of L. amazonensis, PV membranes acquire Rab5 and EEA1, two early endosomes markers; by membrane remodeling, PV membranes lose these early markers and rapidly acquire the late endosome and lysosome markers Rab7p and LAMP1 [7]. In the first hours of intracellular infection, fusion between PVs and lysosomes could be a determinant of PV enlargement, as the depletion of secondary lysosomes [25] and acidic vesicles, as shown in this report, is observed during parasite establishment. Additionally, the up-regulation of host cell lipid biosynthesis triggered by the parasite [28] could increase the repertoire of membrane donors to Leishmania PVs development. This up-regulation was not observed in the analysis of gene expression of macrophages infected with L. major promastigotes [29]. After lysosomal marker acquisition (by means of fusion with acidic vesicles, lysosomes and/or late endosomes), the membrane input into Leishmania PVs and their stability as large compartments could be related to continuous fusion with ER-derived or Golgi-derived vesicles [1], [3] instead of fusion with secondary lysosomes, late endosomes, or vicinal L. amazonensis PVs. The homotypic fusion between L. amazonensis PVs was described by Real and colleagues and occurs mainly after 24–48 h of intracellular infection [16]. Fusion between PVs before 12 h of infection was seldom observed. Indeed, Lippuner and colleagues found that a minority of Rab5-positive L. mexicana PVs fuse with each other in the first hours of intracellular infection [30]. The fusion between L. amazonensis PVs did not contribute to sustain PV enlargement because fused PVs regained their dimensions, a process probably related to compensatory membrane recycling. Although the results suggest a selective nature for PV membrane composition, the host cell/parasite components retained by large PVs to maintain their homeostasis remain to be elucidated. The output of membrane from large PVs could be incorporated by the plasma membrane (displaying parasite proteins on host cell surface) and other cytoplasmic organelles such as the ER. The spacious PV represents a strategy to subvert host cell defenses, providing an environment with lower activities and/or concentrations of hydrolytic enzymes [9], [31]–[33]. Considering that Leishmania from the L. mexicana complex are resistant to IFN-γ–mediated macrophage activation and high NO concentrations [34], the unique morphology of large vacuoles could be vital for the establishment of these parasites. Recent studies documented that L. major procyclic promastigotes survive and multiply within L. amazonensis PVs after interspecific PV fusion in doubly infected macrophages [17]. As expected, the procyclic promastigotes were destroyed within their own phagolysosomes but were spared from destruction once they entered the spacious L. amazonensis PVs. L. major, the first organism in the genus to have its genome sequenced [35], is one of several Leishmania species in which amastigotes develop tight PVs that undergo fission during parasite multiplication in host cells. These amastigotes are generally individualized in PVs that maintain their dimensions and possibly require different strategies to avoid host cell defenses. PV fission is displayed by several important pathogens that live in vacuoles throughout their intracellular life cycle, such as Leishmania and Mycobacterium [36]. The process is likely to require the mobilization of membrane sources because replicating amastigotes require increases in PV dimensions prior to division, which implies an increase in PV membrane surface area and volume. After parasite division, there is an intermediary state of double-occupancy PVs in which a vacuolar interface between recently-divided parasites and the PV membrane is visible using lysosomotropic probes. This detectable vacuolar space is polarized in agreement with the polarized event of amastigote division, which suggests a period of hours in which two dividing L. major amastigotes share the same PV with a small, acidic lumen. Indeed, in the late 1970s, a Thorotrast-rich vacuolar space located between two dividing L. major amastigotes was documented by electron microscopy [23]. Amastigote division within tight PVs could cause increase in PV fusogenicity with small vesicles by modifying the membrane curvature, which could physically assist SNARE machinery for membrane fusion [37]. An alternative interpretation of the Lysotracker clusters on dividing L. major amastigotes is that acidic vesicles could be mobilized to PV membrane sites in hotspots, where membrane input preferentially occurs. The numeric decrease of host acidic vesicles during L. major PV fission or L. amazonensis PV enlargement is likely related to a higher demand of host membrane sources for the biogenesis of Leishmania PVs, at least in the first 4 days of intracellular infection. The host cell reservoirs of acidic vesicles would partially account for membrane incorporation of fission-prone L. major PV and partition in two new PVs or massive incorporation of vesicles into enlarging L. amazonensis PVs. Considering the membrane surface area of Leishmania PVs, a large L. amazonensis PV with 20 µm of diameter has the approximate surface area of 16 L. major tight-fitting PVs, indicating that Leishmania PVs would require approximate amounts of host cell membrane for their biogenesis regardless of the different PV architectures (tight-fitting vs. loose vacuoles). The contribution of each acidic vesicle to PV biogenesis may be also hypothesized: approximately 50 detected acidic vesicles (1 µm in diameter) in macrophages infected with L. major or L. amazonensis are consumed in the course of intracellular infection. This represents a hypothetical volume contribution of 30 µm3 to Leishmania PVs, which only partially accounts for L. major or L. amazonensis PV dimensional doubling. Indeed, other mechanisms of Leishmania PV volume control were addressed in the literature and include fusion with ER vesicles, acquisition of water and ion transport channels, and parasite secretion of exosomes and macromolecules inserted in PV membranes, or displayed in parasite membrane surfaces [3], [33], [38]–[41]. Additionally, Leishmania can internalize PV membrane components: in a process resembling host membrane “clearance, ” L. amazonensis can actively internalize and digest MHC class II, possibly by its posterior pole that interacts with PV membranes [42]. This process could also be conserved in Leishmania with membrane-bound PVs participating in the control of membrane input into the PVs. The polarization of lysotracker clusters in dividing L. major parasites suggests that parasite poles could also participate in the PV biogenesis of tight-fitting phenotype. Although L. major PVs incorporate phagolysosomal markers on their membranes, they were unable to retain amounts of lysosomotropic content probes comparable to other tight-fitting phagosomes (i. e. latex beads or aldehyde-fixed amastigotes). A less acidic environment could account for Lysotracker-negative L. major PVs, which could present lower abundance of vesicular proton ATPases than L. amazonensis PVs. Osorio y Fortea and colleagues [28] showed that eight isoforms of vesicular proton ATPases subunits are up-regulated in macrophages infected with L. amazonensis amastigotes; in contrast, Gregory and colleagues [29] showed that only one isoform of these ATPases, V1 subunit H, is up-regulated in macrophages infected with L. major promastigotes. In these same experiments, a V0 subunit A2 is down-regulated and other lysosomal components (such as two isoforms of a lysosomal-associated transmembrane protein 5 and an isoform of acid phosphatase 2) are down-regulated. Non-acidic phagosomes displaying phagolysosomal markers on their membranes were associated with exclusion of these proton ATPases in Mycobacterium phagosomes [43]. It is possible that L. major PVs maintain the exclusion of vesicular proton ATPases for amastigote replication, in a process similar to what occurs in L. donovani promastigote PVs [44]. By presenting these fundamental pre-mechanistic data obtained by live imaging, we highlighted that Leishmania PVs are dynamic structures that remodel their shapes, allowing these structures to develop a privileged intracellular niche where Leishmania parasites survive and multiply. Several pathogens can utilize this process; describing and understanding the nature of these vacuoles in live infected cells are crucial steps towards the understanding of how parasites evade host immune responses [45].
summary: Leishmania parasites lodge in host cells within phagolysosome-like structures called parasitophorous vacuoles (PVs). Depending on the species, amastigote forms can be individually hosted within small, tight-fitting PVs or grouped within loose, spacious PVs. Using multidimensional live cell imaging, we examined the biogenesis of the two PV phenotypes in macrophages exposed to L. major (a representative of the tight PV phenotype) or L. amazonensis (an example of the loose PV phenotype) amastigotes. L. major PVs undergo fission as parasites divide; we demonstrate that in the course of fission there are transients of the lysosomotropic fluorescent probe Lysotracker. In contrast, during the course of amastigote population size expansion, L. amazonensis PVs do accumulate Lysotracker while increasing in diameter and volume. The large PVs fuse together, and the products of fusion undergo size and shape remodeling. The biogenesis/remodeling of the two types of Leishmania PVs is accompanied by a reduction in the number of macrophage acidic vesicles. The present imaging study adds new morphometric information to the cell biology of Leishmania amastigote intracellular parasitism.
keywords: parastic protozoans
  protozoology
  biology
  microbiology
  parasitology
title: The Diverse and Dynamic Nature of Leishmania Parasitophorous Vacuoles Studied by Multidimensional Imaging