The major surface protease (msp or gp63) of Leishmania plays a major role in the host–parasite interaction. We analysed here the structure of the msp gene locus in Leishmania (Viannia) braziliensis and compared it to results obtained in other species. Physical mapping of cosmid contigs revealed a minimum of 37 genes per haploid genome and at least 8 different msp gene families. Within the same organism, these genes showed a nucleotide sequence varying in certain stretches from 3 to 34%, and a mosaic structure. From an evolutionary point of view, major differences were observed between subgenera Viannia and Leishmania, both in terms of msp gene number and sequence. Within subgenus Viannia, phenetic analysis revealed three clusters in which sequence variants of L. (Viannia) braziliensis and L. (Viannia) guyanensis were interspersed. Functional implications of our results were explored from predicted L. (Viannia) braziliensis protein sequences: regions encoding the msp catalytic site showed a conserved sequence, while regions encoding surface domains possibly involved in the host–parasite interaction (macrophage adhesion sites and immunodominant B-cell and T-cell epitopes) were variable. We speculate that this would be an adaptive strategy of the parasite.

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.