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      The Diverse and Dynamic Nature of Leishmania Parasitophorous Vacuoles Studied by Multidimensional Imaging

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      PLoS Neglected Tropical Diseases
      Public Library of Science

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          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.

          Author 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.

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          Most cited references43

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          An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.

          Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.
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            Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus

            We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.
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              Evasion of innate immunity by parasitic protozoa.

              Parasitic protozoa are a major cause of global infectious disease. These eukaryotic pathogens have evolved with the vertebrate immune system and typically produce long-lasting chronic infections. A critical step in their host interaction is the evasion of innate immune defenses. The ability to avoid attack by humoral effector mechanisms, such as complement lysis, is of particular importance to extracellular parasites, whereas intracellular protozoa must resist killing by lysosomal enzymes and toxic metabolites. They do so by remodeling the phagosomal compartments in which they reside and by interfering with signaling pathways that lead to cellular activation. In addition, there is growing evidence that protozoan pathogens modify the antigen-presenting and immunoregulatory functions of dendritic cells, a process that facilitates their evasion of both innate and adaptive immunity.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                February 2012
                14 February 2012
                : 6
                : 2
                : e1518
                Affiliations
                [1]Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, UNIFESP, São Paulo, Brazil
                Institut Pasteur, France
                Author notes

                Conceived and designed the experiments: FR. Performed the experiments: FR. Analyzed the data: FR RAM. Contributed reagents/materials/analysis tools: RAM. Wrote the paper: FR RAM.

                Article
                PNTD-D-11-00620
                10.1371/journal.pntd.0001518
                3279510
                22348167
                7fe74725-bf5f-4dfd-94a3-f29f26765421
                Real, Mortara. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 29 June 2011
                : 22 December 2011
                Page count
                Pages: 13
                Categories
                Research Article
                Biology
                Microbiology
                Parasitology
                Protozoology
                Parastic Protozoans

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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