REPOSITORY > RESULTS

Abstract

Cysteine peptidases have been demonstrated to be important virulence factors of protozoan parasites, including Trypanosoma cruzi. With genome sequence determination the existence of several novel families of cysteine peptidases can be envisioned in this parasite. We focused on two families, metacaspases from the clan CD and autophagins (or Atg4 proteases) from the clan CA. During our previous work on the metacaspase family in T. cruzi we identified two genes, metacaspase-3 and metacaspase-5. Metacaspase-3 gene was shown to be present in about 16 copies per haploid genome and now we prove that there are two tandem arrays of these copies located on two different chromosomes. We also show that metacaspase-3 is expressed in all mayor developmental stages of T. cruzi, a result supported by the fact, that almost all patients with chronic Chagas disease possess antibodies against the metacaspase-3. We were able to express N-terminally truncated form of the metacaspase-3 in soluble form. We used this protein to screen fluorescence quenched combinatorial libraries of synthetic substrates, however we were unable to detect proteolytic activity. Using polyclonal antibodies we detected that metacaspases are translocated form the cytosol to the nucleus during fresh human serum induced programmed cell death of epimastigotes and could mediate this process in T. cruzi.
We identified two genes belonging to the autophagin family in the genome of T. cruzi, TcATG4.1 (autophagin-1) and TcATG4.2 (autophagin-2). We also identified two genes homologous to ubiquitin-like ATG8 genes, predicted substrates of autophagins, TcATG8.1 and TcATG8.2. We expressed all four genes in E. coli and purified the proteins. Both TcAtg4.1 and TcAtg4.2 are active cysteine peptidases, although only TcAtg4.1 efficiently processes TcAtg8.1 and TcAtg8.2. Reconstitution experiments of knock out yeast strains showed that TcATG4.1 and TcATG4.2 could replace S. cerevisiae ATG4 gene reconstituting autophagy whereas only TcATG8.1 and not TcATG8.2 could do so in ScATG8 knockout strain. Further on, using polyclonal antibodies against TcAtg8.1 in immunofluorescence studies in T. cruzi we could observe the translocation of this protein to the autophagosomal membranes during prolonged starvation of epimastigotes. This suggests that TcATG8.1 is functionally homologous to yeast ATG8 gene and mammalian MAP1 LC3 mediating autophagosome growth and closure. Finally, autophagin activity is expressed in all developmental stages of T. cruzi suggesting a constitutive basal level of autophagy in this organism.