Parasitophorous vacuoles of Leishmania mexicana acquire macromolecules from the host cell cytosol via two independent routes

The intracellular parasite Leishmania survives and proliferates in host mac rophages. In this study me show that parasitophorous vacuoles of L, mexican a gain access to cytosolic material via two different routes. (1) Small ani onic molecules such as Lucifer Yellow are rapidly transported into the vacu oles by an active transport mechanism that is sensitive to inhibitors of th e host cell's organic anion transporter. (2) Larger molecules such as fluor escent dextrans introduced into the host cell cytosol are also delivered to parasitophorous vacuoles. This transport is slower and sensitive to modula tors of autophagy, Infected macrophages were examined by two novel assays t o visualize and quantify this process. Immunoelectron microscopy of cells l oaded with digoxigenin-dextran revealed label in multivesicular endosomes, which appeared to fuse with parasitophorous The inner membranes of the muit ivesicular vesicles label strongly with antibodies against lysobisphosphati dic acid, suggesting that they represent a point of confluence between the endosomal and autophagosomal pathways, Although the rate of autophagous tra nsfer was comparable in infected and uninfected cells, infected cells retai ned hydrolyzed cysteine proteinase substrate to a greater degree. These dat a suggest that L. mexicana-containing vacuoles have access to potential nut rients in the host cell cytosol via at least two independent mechanisms. va cuoles.