Leishmania donovani-infected macrophages: characterization of the parasitophorous vacuole and potential role of this organelle in antigen presentation
Authors: Lang Thierry , Hellio Raymond , Kaye Paul M. , Antoine Jean-Claude 
Published: April 2021

ABSTRACT
Leishmania donovani amastigotes, the etiological agents of visceral leishmaniasis, are obligate intracellular parasites residing in membrane-bound compartments of macrophages called parasitophorous vacuoles (PV). The study of these organelles is of paramount importance to understanding how these parasites resist the microbicidal mechanisms of macrophages and how they escape the immune response of their hosts. Confocal microscopy of mouse bone marrow-derived macrophages infected with L. donovani amastigotes and stained for various prelysoso-mal/lysosomal markers and for major histocompatibility complex (MHC) molecules was used to define PV with respect to the endocytic compartments of the host cells and to address the issue of their potential role in antigen processing and presentation. Forty-eight hours after infection, many PV contained cathepsins B, D, H and L and they were all surrounded by a membrane enriched for the lysosomal glycoprotein lgp120/lamp1 but apparently devoid of the cation-independent mannose 6-phosphate receptor, a membrane protein generally absent from the lysosomes. These data suggested that PV acquire within 48 hours the characteristics of a lysosomal compartment. However, both macrosialin and the GTP-binding protein rab7p (specific markers of the prelysosomal compartment) were found to be highly expressed in/on PV membrane. Thus, at this stage, PV appear to exhibit both lysosomal and prelysoso-mal features. Infected macrophages activated with IFN-γ before or after infection showed PV strongly stained for MHC class II molecules but not for MHC class I molecules.
This suggests that, if infected macrophages can act as antigen-presenting cells for class I-restricted CD8+ T lymphocytes, Leishmania antigens must exit the PV. MHC class II molecules reached the PV progressively, indicating that they were not plasma membrane-bound molecules trapped during internalization of the parasites. The redistribution of class II observed in infected cells did not alter their quantitative expression on the plasma membrane at least during the first 48 hours following the phagocytosis of the parasites. The invariant chains, which are transiently associated with class II molecules during their intracellular transport and which mask their peptide-binding sites, did not reach PV or were rapidly degraded in these sites, suggesting that PV-associated class II are able to bind peptides. This last assumption is strengthened by the fact that class II located in PV could bind conformational antibodies that preferentially recognize class II with tightly associated peptides. Together, these data showed that L. donovani amastigotes live in hydrolytic compartments of the host cells where the processing of parasite antigens could eventually occur as well as the binding of parasite peptides to MHC class II molecules, both events being required before the presentation of antigens to specific CD4+ T lymphocytes.