title: Studies on the cysteine proteinases of Leishmania mexicana
Creators: name: Frame, Mhairi Jane
Publisher: name: University of Glasgow
Year: 1997

description: Three genes (Imcpa, Imcpb and Imcpc) that encode cysteme proteinases (CPs) in L. mexicana have previously been cloned and sequenced. The enzymes encoded differ significantly from each other and host CPs. In an attempt to elucidate the parts played by each CP in the parasite's life history, mutant lines of the parasite which lack Imcpa, Imcpb and Imcpc have been examined in order to determine their phenotypes. Null mutants which have been further modified by transfecting the cells with genes encoding L. mexicana CPs and CPs of other trypanosomatids have also been studied. These lines have been analysed to determine how they differ from the parent line in terms of proteinase and protein content, growth and differentiation axenically in vitro and in peritoneal macrophage cells, infectivity to mice and susceptibility to CP inhibitors. Null mutants for the Imcpa CP gene were found to have a cryptic phenotype, since differences between the Imcpa null mutants and wild type L. mexicana were not detectable. This suggested that either the enzyme has a role which was not detected with the phenotypic tests used or that its activity was being compensated for by one of the other CPs present in L. mexicana. In contrast to previously published conclusions, studies using null mutants for lmcpa revealed that LmCPa was not at highest levels in the amastigote stage. Triton X-114 phase separation revealed the protein encoded by lmcpa to be hydrophobic, suggesting it could be membrane-associated. Stationary phase promastigotes of mutants null for the multicopy lmcpb genes showed a marked phenotype with respect to virulence, as their ability to infect macrophages in vitro was severely impaired. The infectivity was restored by re-expression of an enzymatically active internal copy of the gene array (g2.8), suggesting that the enzymes play a role in intracellular survival. Time course experiments demonstrated that the null mutants invaded macrophages in large numbers but were unable to survive in the majority of the cells. Despite their reduced virulence to PECs, the mutants were capable of infecting mice although the time taken for lesions to form was significantly longer than with wild type and the lesions were 100-fold smaller. Interestingly, lesion amastigotes of the mutants were able to infect macrophages in vitro as successfully as wild type, but took three months longer to produce lesions in mice. These data showed that the CPs encoded by lmcpb are important for parasite virulence, but are not essential for survival in the host. Double lmcpa/lmcpb null mutants were found to have a similar phenotype to the lmcpb null mutants in terms of their ability to infect macrophages in vitro, suggesting that lmcpa was not compensating for the loss of lmcpb in this phenotypic test, although it was found that they did not cause lesions in mice. Studies utilising null mutants re-expressing different copies of the lmcpb array revealed that individual isoenzymes differ in their substrate preferences and ability to complement the loss of virulence associated with the null mutant, suggesting that the individual isoenzymes have distinct roles in the parasite's interaction with the host. Immunogold labelling of the megasome in transfectants re-expressing an lmcpb CP gene with a truncated C-terminal extension (gl) demonstrated that the C-terminal extension is not necessary for intracellular targeting or activity. All life-cycle stages of the mutants null for the cathepsin B-like CP gene (lmcpc) could be cultured in vitro, demonstrating that this CP is not essential for the growth or differentiation of the parasite. However, they showed greatly reduced infectivity to macrophages in vitro, resulting in only a low percentage of the cells being infected. Re-expression of lmcpc in the null mutant increased the parasite's infectivity to macrophages in vitro. However, the null mutants for lmcpc formed lesions in mice at a rate comparable with wild type parasites, suggesting that although the CP encoded by lmcpc may play a role in the parasite-macrophage interaction it alone is not crucial for infectivity or virulence. Peptidyl-diazomethane CP inhibitors, which entered the parasites and inhibited LmCPb in situ, had little or no effect on parasite growth or differentiation axenically in vitro. However, at the same concentration, N-benzoyloxycarbonyl-phe-ala-diazomethane reduced the infectivity of wild type parasites to macrophages by 80%, a similar percentage to that seen with the lmcpb null mutant. However, further experiments suggested that LmCPb may not be the prime target of this inhibitor. With respect to drug target validation it is essential to determine which CPs are necessary for parasite survival. The overall results of this study suggest that while none summary of the CPs of L. mexicana are vital, some appear to have important and distinct roles in differentiation and intracellular survival.