Characterisation of Leishmania major metacaspase

Metacaspases are distant orthologues of mammalian caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. In this study, I found that the single MCA gene of Leishmania major (LmajMCA or MCA) is expressed in actively replicating amastigotes and promastigotes, whereas expression level is significantly lower in non-dividing stationary phase promastigotes. In order to determine the role of the metacaspase during promastigote division, an analysis of the events of the L. major cell cycle was performed. Based on these findings, immunofluorescence experiments revealed the presence of LmajMCA in punctate structures throughout the cytoplasm of interphase cells and its accumulation in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation, LmajMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. LmajMCA null mutants could not be generated using standard gene deletion techniques. When LmajMCA was expressed from an episome, the only mutants that were viable were those expressing LmajMCA at physiological levels. Over-expression of LmajMCA in promastigotes leads to changes in ploidy and a severe growth retardation, associated with defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. Several LmajMCA variants lacking their N-terminal and/or C-terminal domains or with active site mutation(s) (C201G, C201-C202G, C202G, H147A) were expressed, with or without tags, in L. major promastigotes, in Escherichia coli, in the yeast Pichia pastoris, and using an in vitro translation kit. In all the systems used, the expression of most LmajMCA variants was barely detectable and was apparently toxic for the host cells. Some encouraging results were obtained using P. pastoris, with the expression and secretion in the culture medium of N- and C-terminally truncated LmajMCA. Thus, this system may facilitate production of sufficient enzyme for the characterisation of the biochemical properties of LmajMCA. Insights into the putative function of the different domains of LmajMCA were gained when it was observed, that the first 101 N-terminal amino acids of LmajMCA worked, in L.major, as a mitochondrion targeting signal. A potential role of the LmajMCA proline-, glutamine- and tyrosine-rich C-terminal domain in protein- protein interactions was also hypothesized. Together these data suggest that LmajMCA is essential for the correct segregation of the nucleus and kinetoplast and cytokinesis and that the tight regulation of the amount of LmajMCA within the cell is crucial for the development of Leishmania promastigotes. Trypanosoma brucei possesses five metacaspase genes. Two of the encoded proteins, TbMCA1 and TbMCA4, possess substitutions at their putative active site residues and thus might lack peptidase activity. Therefore functional studies were performed only on the three other metacaspases. Triple RNAi analysis showed TbMCA2, TbMCA3 and TbMCA5 to be essential in the bloodstream form, with induced parasites showing a delay in kinetoplast segregation and being blocked in pre-cytokinesis. Triple null mutants (DeltaTbmca2/3DeltaTbmca5) were found to be initially affected in their development, but recovered after several weeks of in vitro adaptation. The initial slow growth rate of the DeltaTbmca2/3DeltaTbmca5 mutant was associated with a delayed entry in cytokinesis. This phenotype was similar, although less extreme, to that produced upon triple RNAi of TbMCA2, TbMCA3 and TbMCA5. These data suggest that these metacaspases are essential for T. brucei bloodstream forms, but they have overlapping functions and their progressive loss can be compensated for by activation of alternative biochemical pathways. Analysis of DeltaTbmca2/3DeltaTbmca5 revealed no greater or lesser susceptibility to stresses reported to initiate programmed cell death, such as treatment with prostaglandin D2. These metacaspases co-localise with RAB11, a marker for recycling endosomes but variant surface glycoprotein (VSG) recycling processes and the degradation of internalised anti-VSG antibody were found to occur similarly in wild type, DeltaTbmca2/3DeltaTbmca5 and triple RNAi-induced parasites. Thus the data provide no support for the direct involvement of T. brucei metacaspases in programmed cell death and suggest that the proteins have a function associated with RAB11 vesicles that is independent of known recycling processes of RAB11-positive endosomes. As both the L major and the T. brucei metacaspases appear to play crucial roles in cell cycle progression and because metacaspases are absent from the mammalian genome, these metacaspases have potential as novel drug targets.