In mammalian hosts, Leishmania amastigotes are obligatory intracellular parasites of macrophages and multiply within parasitophorous vacuoles of phagolysosomal origin. To understand how they escape the harmful strategies developed by macrophages to kill ingested microorganisms, it is important to obtain information on the functional state of parasitophorous vacuole. For this purpose, we studied the intracellular distribution and activity of host lysosomal
proteases in rat bone marrow-derived macrophages infected with Leishmania amazonensis amastigotes. Localization of
cathepsins B, H, L, and D was investigated by using specific
immunoglobulins. In uninfected macrophages, these
enzymes were located in perinuclear granules (most of them were probably secondary lysosomes) which, after
infection, disappeared progressively. In infected macrophages,
cathepsins were detected mainly in the parasitophorous vacuoles, suggesting that the missing secondary lysosomes had fused with these organelles. Biochemical assays of various
proteases (
cathepsins B, H, and D and
dipeptidyl peptidases I and II) showed that
infection was accompanied by a progressive increase of all activities tested, except that of
dipeptidyl peptidase II, which remained constant. No more than 1 to 10% of these activities could be attributed to amastigotes. These data indicate that (i)
Leishmania infection is followed by an increased synthesis and/or a reduced catabolism of host lysosomal
proteases, and (ii) amastigotes grow in a compartment rich in apparently fully active
proteases. Unexpectedly, it was found that infected and uninfected macrophages degraded endocytosed
proteins similarly. The lack of correlation in infected macrophages between increase of
protease activities and catabolism of exogenous
proteins could be linked to the huge increase in volume of the lysosomal compartment.