Overexpression of Cruzipain, the Major Cysteine Proteinase of Trypanosoma cruzi, is Associated with Enhanced Metacyclogenesis

Abstract

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected 7: cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandenily repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteiiie proteinase inhibitor Cbz-Phe-Phe-CHN, (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

“…The process, therefore, requires fast and extensive protein degradation and recycling of building blocks for the synthesis of new macromolecules. Initial studies suggested that cruzipain, the most abundant lysosomal/reservosomal cysteine peptidase of T. cruzi belonging to the CA clan (21) of proteases, is critical for the differentiation as the cell-permeable cruzipain inhibitors effectively inhibited the process (19,51), whereas overexpression of this enzyme is associated with enhanced metacyclogenesis (52). Our results clearly demonstrate that autophagy is also involved in cell remodeling most probably by delivering large parts of the cytoplasm and organelles to lysosomes/reservosomes.…”

Golgi UDP-GlcNAc:Polypeptide O -α- N -Acetyl- d -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

“…1C). These values are comparable to those from other studies using pTEX or pTEX-derived vectors to overexpress proteins in the cytosol (78,79), mitochondria (80), glycosomes (81), endosomal/lysosomal system and reservosomes (75,82), ER (68), flagellar pocket (83), or plasma membrane (69,(84)(85)(86) of T. cruzi. The protein was stable, with no indication of a C-terminal proteolytic cleavage event observed when expressed in L. tarentolae (21), and it appeared to be inserted into the rER and N-glycosylated normally (Fig.…”

A Lipid-modified Phosphoinositide-specific Phospholipase C (TcPI-PLC) Is Involved in Differentiation of Trypomastigotes to Amastigotes of Trypanosoma cruzi

“…Overexpression of superoxide dismutase resulted in increased sensitivity to the trypanocidal agents crystal violet and benzidazole (39), although overexpression of cruzipain, the major cysteine proteinase of T. cruzi, was associated with enhanced metacyclogenesis (40). We found that overexpression of TcPI-PLC resulted in an increased rate of differentiation of trypomastrigotes into amastigotes (Figs.…”