An investigation of the CPB cysteine proteinases of Leishmania mexicana and their role in intracellular survival

Abstract

The cysteine proteinases of Leishmania mexicana have long been suspected of being important in the virulence of the parasite. This was recently confirmed using analysis of the in vitro and in vivo infectivity of mutant lines lacking in different cysteine proteinase genes (Mottram et al., 1996; Alexander et al., 1998). One component of my study was an investigation of the effect of peptidyl cysteine proteinase inhibitors on L. mexicana. None of the inhibitors tested had any effect on the growth of promastigotes in vitro. However, analysis of the effects on axenic amastigotes demonstrated that cysteine proteinase inhibition reduced growth. The inhibitors were also effective at reducing the cysteine proteinase activity and prevented the complete processing of the pro-enzymes of the cysteine proteinases encoded by CPA and different genes within the CPB array. These effects were consistent with the autocatalytic nature of the processing, although the results also suggested a partial role for CPA in CPB maturation. The loss of mature enzyme and the resultant increase in the number and abundance of precursor enzyme forms manifested itself as the appearance of large matrix-filled vacuoles, indicating a perturbation of intracellular protein trafficking. Analysis of the effect of cysteine proteinase inhibition upon intracellular infection demonstrated that it was possible to reduce the survival of the amastigotes. Interestingly, removal of the CPB array did not reduce the parasite's susceptibility, indicating that other cysteine proteinases are important targets of these inhibitors. My findings, in conjunction with the work of others, confirm the potential of cysteine proteinases as targets for the design of chemotherapeutic agents. An understanding of the mechanisms by which cysteine proteinases are important for the intracellular survival of L. mexicana is essential if these enzymes are to be effectively exploited through chemotherapy. Other components of my study addressed this issue; an investigation of the role of the CPB enzymes in parasite virulence being undertaken. This part of my investigation involved an analysis of the effect of loss of the CPB enzymes using a CPB null mutant (DeltaCPB). An analysis of the response of macrophages to infection with wild-type parasites or DeltaCPB indicated that comparable levels of reactive nitrogen and oxygen intermediates were produced. This suggested that other factors within the host cell were responsible for the reduced virulence of DeltaCPB. The CPB enzymes were shown to have a role in the turn-over of cellular proteins; loss of these enzymes was demonstrated to cause a reduction in the rate of protein turn-over from the cell surface. Furthermore, analysis of the kinetics of attachment and internalisation of wild-type and DeltaCPB promastigotes demonstrated that loss of the CPB array resulted in a prolonged period of interaction of the parasite with the surface of the macrophage. The mechanisms of attachment were investigated and it was demonstrated that promastigotes of both wild-type parasites and DeltaCPB/g2.8 (DeltaCPB re-expressing a single gene copy from the CPB array; this mutant line had been previously demonstrated to have wild-type virulence) rely heavily on attachment to complement receptors, CR3 and CR4, during interaction with the macrophage surface. DeltaCPB, however, was shown to attach via different receptor-ligand interactions. Therefore, loss of the CPB array was shown to alter the surface architecture of promastigotes such that they could no longer enter macrophages via the route utilised by wild-type promastigotes. This alteration in the mechanism of uptake is hypothesised to be important in the poor intracellular survival of the mutants, in that it results in the parasites encountering a different and more hostile intracellular environment