Bloodstream formTrypanosoma bruceidepend upon multiple metacaspases associated with RAB11-positive endosomes

Abstract

Trypanosoma brucei possesses five metacaspase genes. Of these, MCA2 and MCA3 are expressed only in the mammalian bloodstream form of the parasite, whereas MCA5 is expressed also in the insect procyclic form. Triple RNAi analysis showed MCA2, MCA3 and MCA5 to be essential in the bloodstream form, with parasites accumulating pre-cytokinesis. Nevertheless, triple null mutants (Δmca2/3Δmca5) could be isolated after sequential gene deletion. Thereafter, Δmca2/3Δmca5 mutants were found to grow well both in vitro in culture and in vivo in mice. We hypothesise that metacaspases are essential for bloodstream form parasites, but they have overlapping functions and their progressive loss can be compensated for by activation of alternative biochemical pathways. Analysis of Δmca2/3Δmca5 revealed no greater or lesser susceptibility to stresses reported to initiate programmed cell death, such as treatment with prostaglandin D2. The metacaspases were found to colocalise with RAB11, a marker for recycling endosomes. However, variant surface glycoprotein (VSG) recycling processes and the degradation of internalised anti-VSG antibody were found to occur similarly in wild type, Δmca2/3Δmca5 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.

Inhibition of active nuclear transport is an intrinsic trigger of programmed cell death in trypanosomatids

“…Furthermore, the L. major metacaspase seems to have an essential function in cell-cycle progression rather than in PCD. 28 And, in T. brucei, metacaspases appear to be associated with RAB11-positive endosomes, 15 and not to intervene in apoptosis. Still, intriguingly, some reports have described 'caspase-like' activities in these protozoa.…”

“…Although metacaspase genes have been identified in the genome of all trypanosomatids, their involvement in apoptosis is still debated. [14][15][16][17] Moreover, no homologue for members of the Bcl-2 protein family has been identified in T. brucei. Finally, two genes whose transcription is upregulated late during the identification of concanavalinA-induced PCD in the procyclic form of T. brucei rhodesiense, a homologue of prohibitin and a protein called TRACK, homologous to the RACK family (receptor for activated kinase C).…”

Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation

“…However, in T. brucei bloodstream forms, triple metacaspase null mutants (Dmca2/3, Dmca5) did not prevent cell death induced by prostaglandin D 2 when compared with wild-type parasites. 12 Nevertheless, RNA interference of the three MCAs together resulted in a rapid arrest of growth with a delay in kinetoplast segregation and a cytokinesis block. In addition, Ambit et al 13 showed that limited levels of LmjMCA are essential for normal cell proliferation.…”

“…5 Their substrate specificity is for basic residues at P1 position, making them unable to cleave caspase substrates. 6 Evidences suggest that metacaspases modulate cell death, [7][8][9][10][11] cell cycle progression [12][13][14] and protein aggregation, 15 but there is still controversy about their functions and their relation with caspases. [16][17][18] The fact that metacaspases are not present in humans and fulfil important roles in trypanosomatids make them attractive drug targets, and a first series of inhibitors with trypanocidal activity has been developed recently.…”

Crystal structure of a Trypanosoma brucei metacaspase

“…MCA2, MCA3, and MCA5 have the conserved catalytic residues (histidine/cysteine dyad) (27) and are collectively required for the bloodstream form of the parasite (28). MCA2, which is an active peptidase, is located in RAB11-positive endosomes and has roles in precytokinesis cell cycle control (28).…”