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 (deltamca2/3deltamca5) could be isolated after sequential gene deletion. Thereafter, deltamca2/3deltamca5 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 deltamca2/3deltamca5 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, deltamca2/3deltamca5 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.
Subject(s)
Caspases/metabolism , Cysteine Endopeptidases/metabolism , Endosomes/metabolism , Genes, Protozoan , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Animals , Apoptosis/genetics , Blood-Borne Pathogens , Caspases/genetics , Cattle , Cysteine Endopeptidases/genetics , Endosomes/genetics , In Vitro Techniques , Mice , Protozoan Proteins/genetics , RNA Interference , Trypanosoma brucei brucei/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Parasitic protozoa contain an abundance of cysteine peptidases that are crucial for a range of important biological processes. The most studied cysteine peptidases of parasitic protozoa belong to the group of papain-like enzymes known as clan CA. However, several more recently identified cysteine peptidases differ fundamentally from the clan CA enzymes and have been included together in clan CD. Enzymes of this clan have now been identified in parasitic protozoa. Many have important roles and also differ significantly from known mammalian counterparts. The main characteristics of clan CD enzymes are outlined here, in particular glycosylphosphatidylinositol (GPI):protein transamidase, metacaspase and separase, and their differences from the clan CA enzymes are described.
