Biogenesis ofLeishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites

Abstract

Protozoan parasites Leishmania alternate between a flagellated promastigote form and an amastigote form. In their mammalian hosts, Leishmania survive and multiply in macrophages. Both forms can be internalized by these host cells at different stages of the infectious process and eventually establish themselves within parasitophorous vacuoles exhibiting phagolysosomal properties. To determine whether the biogenesis of these organelles differs according to the parasitic stage used to initiate infection, we compared their formation kinetics after phagocytosis of either metacyclic promastigotes or amastigotes of L. amazonensis or of L. major by mouse bone-marrow-derived macrophages pre-exposed or not to IFN-γ. After 10 minutes of contact, an accumulation of F-actin was observed around the promastigotes and amatigotes undergoing phagocytosis or those that had already been internalized. This accumulation was transient and rapidly disappeared at later times. At 30 minutes, most of the promastigotes were located in long, narrow organelles that were exactly the same shape as the parasites. The latter were elongated with their cell bodies near to the macrophage nucleus and their flagella towards the periphery. This suggests that promastigote phagocytosis mainly occurs in a polarized manner, with the cell body entering the macrophages first. Most, if not all, of the phagocytosed promastigotes were located in organelles that rapidly acquired phagolysosomal properties. At 30 minutes, lamp-1, macrosialin, cathepsins B and D were detected in 70-98% of these compartments and about 70% of them were surrounded by rab7p. These late endosome/lysosome `markers' were recruited through fusion with late endocytic compartments. Indeed, when late endosomes/lysosomes were loaded with fluorescein dextran, 81-98% of the promastigote-harbouring compartments contained the endocytic tracer 30 minutes after infection. Electron microscopy of infected macrophages previously loaded with peroxidase confirmed that the phagosomes rapidly fused with late endocytic compartments. When the amastigote stage of L. amazonensiswas used to initiate infection, the kinetics of acquisition of the different late endosome/lysosome `markers' by the phagosomes were similar to those measured after infection with metacyclics. However, more rab7p+-phagosomes were observed at early time points (e.g. 90% were rab7p+ at 30 minutes). The early endosome `markers', EEA1 and the transferrin receptor, were hardly detected in parasite-containing compartments regardless of the parasitic stage used to infect macrophages and the time after infection. In conclusion, both metacyclic- and amastigote-containing phagosomes fuse with late endosomes/lysosomes within 30 minutes. However, with L. amazonensis, the time required for the formation of the huge parasitophorous vacuoles, which are characteristic of this species, was much shorter after infection with amastigotes than after infection with metacyclic promastigotes. This indicates that the initial fusions with late endosomes/lysosomes are followed by a stage-specific sequence of events.

The Paradox of a Phagosomal Lifestyle: How Innate Host Cell-Leishmania amazonensis Interactions Lead to a Progressive Chronic Disease

“…In fact, the formation of the L.a. phagolysosome is relatively rapid, occurring within 30 minutes post infection, when the phagolysosomes of most infected macrophages have already acquired lysosomal features in the lumen and membrane. This process occurs faster when the infection is initiated by the amastigote form of the parasite but is otherwise quite similar compared to promastigotes and it is not influenced by previous exposure to IFN-g activation (26). Altogether, Leishmania amastigotes are extremely well-adapted to survive and proliferate inside mature phagolysosomes and L.a., in particular, does this without significantly interfering with this host defense mechanism compared with other species.…”

“…growth (39). In vitro, the PV enlargement process is rapid and seen 8 h post-infection with promastigotes, reaching their maximum volume by 24 h (26,39). CD36 likely induces PV enlargement by enhancing the fusion with late endocytic vesicles, more specifically with lysosomes (39).…”

“…Curiously, L.a. parasites do not seem to interfere with phagolysosome maturation, an observation that is somewhat counterintuitive. Rather, several studies show that acidification, fusion between the phagosome and endosomes/lysosomes, lysosomal enzymes and protease activities and the expression of lysosome markers (Rab7, LAMP1, V-ATPase) are all present on L.a. containing phagolysosomes (26,(33)(34)(35)(36)(37). In fact, the formation of the L.a. phagolysosome is relatively rapid, occurring within 30 minutes post infection, when the phagolysosomes of most infected macrophages have already acquired lysosomal features in the lumen and membrane.…”

“…After binding to the phagocytic receptors, pseudopods surround Leishmania parasites and form the phagosome. During phagocytosis of L.a. metacyclic promastigotes, entrance is often mediated via the cell body rather than the flagellum, in a process that can take up to 10 minutes in vitro employing bone-marrow derived macrophages (BMDMs) (26). The engulfment of either promastigotes or amastigotes is associated with an arrangement of the cytoskeleton and a transient polymerization of F-actin around the parasites (26,27).…”

Host–Pathogen Interaction in Leishmaniasis: Immune Response and Vaccination Strategies

“…The pinocytic rate of macrophages increases following Leishmania infection, resulting in the expression of leishmanial antigens on their surfaces [70]. PV of mouse bone marrow derived macrophage infected with L. amazonensis WHOM/BR/75 Josefa strain consisted of host plasma membrane, phagosomal organelles, and parasite-derived [78] proteases, phosphatases, and cytosolic expression of Rab7, LAMP1, and LAMP2 under highly acidic pH [79,80]. Promastigote transforms into amastigote inside the PV and its intracellular metabolism requires a neutral pH.…”