Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages (original) (raw)
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The Journal of Protozoology, 1975
SYNOPSIS. Secondary lysosomes of cultured mouse peritoneal macrophages were labeled with the electron-dense colloid saccharated iron oxide; the identity of the labeled structures was checked by the Gomori reaction for acid phosphatase. Amastigotes of Leishmania mexicana mexicana derived from mouse lesions were used to infect these macrophages in vitro. In electron micrographs of thin sections of infected macrophages the labeled secondary lysosomes were seen fused with the parasitophorous vacuoles without preventing subsequent multiplication of the parasites. A similar fusion probably occurs in vivo, and may provide a pathway through which not only nutrients but also drugs and host antibodies could reach the intracellular parasite. Index Key Words: Leishmania mexicana mexicana ; amastigote; macrophage ; intracellular parasitism; host lysosome, fusion with parasitophorous vacuoles; possible role of host lysosome in parasite nutrition, antibody transfer, and chemotherapy ; electron microscopy.
Trends in Microbiology, 1998
L eishmania spp. are the etiological agents of leishmaniases, a broad spectrum of diseases that affects 15 million people, mainly in tropical and subtropical areas of the world, and has a major impact on health in developing countries. The clinical manifestations of infection are dependent both on the parasite species involved and on the genetic and immunological status of the host 1. The various clinical forms observed in humans can be partially reproduced in mice of different inbred strains, which prove to be potent experimental models. Leishmania are protozoa belonging to the Trypanosomatidae family; numerous species have been described. These parasites cycle between insect vectors (the phlebotomine sandflies) and several mammals, including humans. In the sandfly, Leishmania multiply in the form of flagellated promastigotes, which are extracellular parasites of the digestive tract of their hosts. Here, they undergo metacyclogenesis, a differentiation process resulting in the appearance of infective (or metacyclic) promastigotes, which accumulate in the anterior part of the duct. Metacyclogenesis can be reproduced in vitro, at least partially, using semi-defined culture media 2. In mammals, Leishmania multiply almost exclusively in cells of the mononuclear phagocytic system as amastigotes. This is a nonmotile stage, although the parasites possess a short flagellum. Growth of amastigotes occurs within organelles of macrophages known as parasitophorous vacuoles (PVs), which exhibit properties similar to those of endocytic compartments 3,4. Several cell biology approaches have recently been used to gain insights into the biogenesis and characteristics of PVs. An important goal of these studies has been to understand the adaptive mechanisms used by the parasites to survive and multiply in this intracellular site. Another important aspect is understanding the role played by these organelles in the immune responses developed by infected hosts, as parasitized macrophages are thought to be an essen
Zeitschrift f�r Parasitenkunde Parasitology Research, 1981
Intracellular forms of Leishmania mexicana amazonensis divide inside the phagocytic vacuole of macrophages. Some parasites attach to the membrane of the phagocytic vacuole while others remain free in the vacuole. Examination of thin sections of the attachment region by electron microscopy revealed a space of 2 nm between the membrane of the phagocytic vacuole and the plasma membrane of the parasite. Freeze-fracture replicas showed an array of intramembranous particles in some areas of the parasite's plasma membrane resembling a gap junction which, in other cells, is involved in the process of intercellular communication.
Suppression of macrophage lysosomal enzymes after Leishmania donovani infection
Biochemical Medicine and Metabolic Biology, 1989
Leishmania donovani is an obligate intracellular parasite residing in the phagolysosome of the host's mononuclear phagocytes (1). Infection with this parasite begins with the introduction by the sandfly vector of the flagellated promastigote into the blood stream, leading to intracellular parasitism of macrophages. Macrophages constitute one of the primary defense mechanisms of the body against microbial invasion and are capable of fulfilling a variety of microbicidal functions such as the production of free oxygen radicals and lysosomal enzymes and the ability to activate host's immune system. The means used by Leishmania parasites to overcome the formidable array of macrophage's killing response is one of the still unsolved mysteries of Leishmania biology. Work on other parasites which live in a similar environment has revealed several possible mechanisms. Thus Toxoplasma (2,3) and Tuber&e bacilli (4) appear to be able to prevent fusion of the destructive macrophage lysosomes with the phagocytic vacuole, whereas Mycobacterium lepraemurium achieves immunity through a physical barrier in the form of a thick enveloping capsule (5,6). Trypanosoma crud is able to escape from the original phagosome and live in direct contact with the cytoplasm (7), thus avoiding the essentially vacuole-bound lysosomal enzymes. The most intriguing event is the finding that lysosome-phagosome fusion occurs after leishmanial entry into macrophages, and that Leishmania species not only survive, but continue to multiply in the phagolysosomes (8,9). Leishmania species are thus processed by macrophages as other digestive particles are, but do not suffer the consequences of lysosomal degradation. How they are able to resist the lysosomal degradation is yet to be elucidated. There are several suggestions: (i) leishmanial cell surface may be naturally resistant to enzyme degradation (9), (ii) carbohydrate moieties of surface glycoproteins may protect against proteolytic degradation (lo), and (iii) release of excretory factors as lysosomal enzyme inhibitors (11). Another possibility might be the suppression of host lysosomal enzyme machinery by Leishmania directly or through some inhibitors. The present 46
Journal of Cell Science, 1999
In their amastigote stage, Leishmania are obligatory intracellular parasites of mammalian macrophages, residing and multiplying within phagolysosomal compartments called parasitophorous vacuoles (PV). These organelles have properties similar to those described for the MHC class II compartments of antigen-presenting cells, sites where peptide-class II molecule complexes are formed before their expression at the cell surface. After infection with Leishmania amazonensis or L. mexicana, endocytosis and degradation of class II molecules by intracellular amastigotes have also been described, suggesting that these parasites have evolved mechanisms to escape the potentially hazardous antigen-presentation process. To determine whether these events extend to other molecules of the antigen-presentation machinery, we have now studied the fate of the MHC molecule H-2M in mouse macrophages infected with Leishmania amastigotes. At least for certain class II alleles, H-2M is an essential cofactor, ...
Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH
Infection and immunity, 1990
Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h...
European Journal of Immunology, 1999
We have previously demonstrated that murine macrophages (M ¤ ) infected with Leishmania promastigotes, in contrast to M ¤ infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I-A d -restricted T cell hybrids and to the T cell clone 9.1-2. These T cells react with the LACK (158-173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK-specific T cell hybridoma OD12 by promastigote-infected M ¤ is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log-phase or stationary-phase promastigotes of L. major or of L. amazonensis, M ¤ strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by M ¤ infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, M ¤ infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected M ¤ correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158-173)-I-A d complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158-173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by M ¤ infected with log-phase or stationary-phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4 + T lymphocytes. Abbreviations: BFA: Brefeldin A BMDM ¤ : Bone marrowderived M ¤ LACK: Leishmania homologue of receptors for activated C kinase Leu-oMe: L-Leucine methyl ester LPG: Lipophosphoglycan MOI: Multiplicity of infection PV: Parasitophorous vacuole 762 N. Courret et al.
Subverted transferrin trafficking in Leishmania- infected macrophages
Parasitology Research, 1998
The intracellular fate of human transferrin (HTf) in macrophages infected by Leishmania was investigated. Binding of HTf-gold complexes at 4°C was competitively inhibited by native holoHTf but not by apoHTf. Infected and uninfected macrophages displayed rather distinct HTf tracking. Pulse-chase experiments using uninfected macrophages loaded with 15-nm gold-conjugated bovine serum albumin (BSA) and then incubated with 5-nm gold-conjugated HTf revealed a remarkable segregation of these tracers in distinct compartments. Nevertheless, Leishmania-infected macrophages presented extensive particle colocalization at both 60 min and 18 h. Light and electron microscopy immunolabeling indicated that HTf was delivered to the parasitophorous vacuole, formed patches on the amastigote surface, and was endocytosed via thē agellar pocket. Double-staining assays showed the colocalization of biotinylated HTf and its receptor in association with the parasitophorous vacuole. To approach the Tf-binding sites of amastigotes we performed HTf-¯uorescein isothiocyanate (FITC) assays. Staining was diuse at 4°C and punctate at 35°C, and only the former was sensitive to ethidium bromide, indicating an eventual temperature-dependent endocytic process. Within parasites, HTf was found in cysteine-proteinaserich structures, suggesting that the protein can be endocytosed by intracellular amastigotes and sorted to the parasite endosomal-lysosomal compartments rather than being recycled. The treatment of infected macrophages with holoHTf, but not apoHTf, promoted the parasite's intracellular survival. These results suggest that Leishmania amastigotes can exploit and subvert the host-cell endocytic system and indicate the role of Tfcarried iron in the outcome of leishmanial infection.
Intracellular growth and pathogenesis of Leishmania parasites
Essays in biochemistry, 2011
Parasitic protozoa belonging to the genus Leishmania are the cause of a spectrum of diseases in humans, as well as chronic long-term infections. These parasites exhibit a remarkable capacity to survive and proliferate within the phagolysosome compartment of host macrophages. Studies with defined Leishmania mutants in mouse models of infection have highlighted processes that are required for parasite survival in macrophages. Parasite mutants have been identified that (i) are poorly virulent when the insect (promastigote) stage is used to initiate infection, but retain wild-type virulence following transformation to the obligate intracellular amastigote stage, (ii) are highly attenuated when either promastigotes or amastigotes are used, and (iii) are unable to induce characteristic lesion granulomas, but can persist within macrophages in other tissues. From these analyses it can be concluded that promastigote stages of some species require the surface expression of lipophosphoglycan, ...
Experimental Parasitology, 2008
Leishmania (L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected-or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24 h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.