CATHEPSIN B-LIKE CYSTEINE PROTEINASE-DEFICIENT MUTANTS OF LEISHMANIA-MEXICANA

Mutants null for the cathepsin B-like cysteine proteinase gene (cpc) o f Leishmania mexicana have been generated by targeted gene disruption. The gene deletion was confirmed using a polymerase chain reaction (PC R) method with cpc-specific primers and genomic DNA isolated from the mutants. cpc was re-expressed in the null mutants from an episomal vec tor. Re-expression of the enzyme (CPC) was detected by Western blottin g with a specific anti-peptide antiserum. The cpc null mutants grew ap parently normally as promastigotes and amastigotes in axenic cultures, but they showed greatly reduced infectivity to macrophages in vitro w ith only a low percentage of the cells being infected. Re-expression o f cpc in the null mutant increased the parasite's infectivity in vitro . The null mutant parasites formed lesions in mice at a similar rate a s wild type parasites, although somewhat smaller lesions were produced . The results suggest that although the cysteine proteinase encoded by cpc plays a role in the parasite's interaction with macrophages it al one is not crucial for infectivity or virulence. (C) 1997 Elsevier Sci ence B.V.