Metacaspases of T. cruzi: possible candidates for programmed cell death mediators of protozoan parasite.

KOSEC, G.; ALVAREZ, V.; AGUERO, F.; SÁNCHEZ, D.; TURK, B.; TURK, V.; CAZZULO, J.J.

Chania, Creta, Grecia.

Congreso; 12th Euroconference on Apoptosis.; 2004

European Cell Death Organization (ECDO).

Metacaspase genes are present in genomes of non-metazoan eukaryotic lineages (plants, fungi and protozoa). Based on catalytic domain amino acid sequence and predicted secondary structure metacaspases are putative clan CD cysteine peptidases. Yeast's metacaspase was suggested to be a functional homologue of caspases, which makes metacaspases an attractive research field also in trypanosomatids, kinetoplastid protozoan parasites that cause endemic diseases in humans and animals. So far 5 metacaspase genes have been identified in Trypanosoma brucei. In unassembled shotgun reads of T. cruzi genome, however, we found only sequences with homology to TbMCA3 and TbMCA5. The TcMCA3 is present in approx. 16 copies per haploid genome, which encode a 358 aa protein with His – Cys catalytic dyad intact. Slight variations appear among the copies, which share 92-95 % identity. Apart from the catalytic domain the single copy TcMCA5 gene predicts a Cterminal extension rich in P, Q and Y. Performing a Southern blot of digested T. cruzi genomic DNA and PFGE of its chromosomes we determined TcMCA3 copies to be arranged in 2 head-to-tail tandems with large intergenic regions separating the ORFs. In the vicinity of TcMCA3 ORFs we detected several retrotransposons, which probably caused a great amplification of TcMCA3 copy number in T. cruzi and disruption in gene syntheny in comparison to related T. brucei. We detected transcripts of TcMCA3 and TcMCA5 in T. cruzi mRNA and found metacaspase-3 to be present in cell free protein extracts of epimastigotes and bloodstream trypomastigotes. Moreover, metacaspase-3 is highly immunogenic in human infections with T. cruzi (chronic Chagasic patients). During the PCD of T. cruzi epimastigotes metacaspase-3 changes its subcellular localization and colocalizes with fluorescent in vivo caspase probe (SR-VAD-FMK) signal. Our results suggest that metacaspases could be involved in the PCD of trypanosomatids although further studies need to be carried out to confirm the mechanism of this process.