SURFACE ZN-PROTEINASE AS A MOLECULE FOR DEFENSE OF LEISHMANIA-MEXICANA AMAZONENSIS PROMASTIGOTES AGAINST CYTOLYSIS INSIDE MACROPHAGE PHAGOLYSOSOMES

The role of the surface membrane Zn-proteinase in protecting the cellu lar integrity of the macrophage parasite Leishmania mexicana amazonens is from intraphagolysosomal cytolysis was studied. These cells lose th eir infectivity to host macrophages after prolonged cultivation in axe nic growth medium. The virulent and attenuated variants of the parasit e cells were cloned. Failure of these attenuated parasite cells to sur vive inside macrophage phagolysosomes is associated with 20- to 50-fol d reduction in the expression of surface gp63 protein. In situ inhibit ion of gp63 proteinase activity inside Leishmania-infected macrophage phagolysosomes with targeted delivery of an inhibitor of gp63 proteina se activity, 1,10-phenanthroline, selectively eliminated intracellular Leishmania amastigotes, further suggesting the importance of this pro teinase in phagolysosomal survival of the parasite. An upstream sequen ce (US) of the gp63 gene was cloned in front of the bacterial chloramp henicol acetyltransferase (CAT) gene in plasmid pCATbasic. Transfectio n oft. mexicana amazonensis cells with this recombinant plasmid showed that expression of the CAT gene from this US is 15- to 20-fold higher in virulent clones than in avirulent clones of the parasite. Band shi ft analysis with the cloned US also showed that binding of protein(s) was 15- to 20-fold higher in virulent cell extract than in avirulent c ell extract. Coating of attenuated cells or liposomes with proteolytic ally active gp63 protects them from degradation inside macrophage phag olysosomes. These results suggest a novel mechanism of survival of thi s phagolysosomal parasite with the help of its surface Zn-proteinase.