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Localization and activity of various lysosomal proteases in Leishmania amazonensis-infected macrophages
E Prina1, J C Antoine, B Wiederanders
1Département de Physiopathologie Expérimentale, Unité d'Immunophysiologie Cellulaire de l'Institut Pasteur et du Centre National de la Recherche Scientifique, Paris, France.
Abstract:
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.
Insights
Leishmania amazonensis infection leads to increased host lysosomal protease activity within macrophages, suggesting parasites thrive in a protease-rich environment. This occurs despite similar degradation of external proteins in infected and uninfected cells.
Area of Science:
- Cell Biology
- Parasitology
- Immunology
Background:
- Leishmania amastigotes are intracellular parasites residing in macrophages.
- Understanding parasite survival mechanisms requires knowledge of the parasitophorous vacuole's functional state.
Purpose of the Study:
- To investigate the distribution and activity of host lysosomal proteases in Leishmania-infected macrophages.
- To determine if Leishmania amastigotes manipulate host cell lysosomal function for survival.
Main Methods:
- Immunolocalization of cathepsins B, H, L, and D in infected and uninfected rat macrophages.
- Biochemical assays to measure activities of cathepsins B, H, D, and dipeptidyl peptidases I and II.
- Assessment of exogenous protein degradation in infected and uninfected macrophages.
Main Results:
- Lysosomal proteases (cathepsins) relocated from perinuclear granules to parasitophorous vacuoles upon Leishmania infection.
- Infection increased the activity of most tested host lysosomal proteases, with minimal contribution from amastigotes.
- Despite increased protease activity, infected macrophages showed no enhanced degradation of endocytosed proteins compared to uninfected cells.
Conclusions:
- Leishmania infection triggers increased synthesis and/or reduced degradation of host lysosomal proteases.
- Leishmania amastigotes survive within a compartment rich in active host proteases.
- The enlarged lysosomal compartment in infected macrophages may hinder exogenous protein degradation despite elevated protease levels.