High expression of a functional cruzipain by a non-infective and non-pathogenic Trypanosoma cruzi clone

Abstract

We compared a Trypanosoma cruzi clone unable to infect or induce pathology in mice (CL-14), with virulent T. cruzi (Y and CL strains) in terms of cruzipain expression, subcellular distribution and functional activity. Our results showed that (1) intracellular Y amastigotes expressed R1 (carboxy-terminal) and R2 (catalytic) domains concentrated in cytoplasmic vesicles, while CL-14 presented R1 labelling on membrane clusters and R2 in intracellular compartments, (2) CL-14-trypomastigotes presented R1 and R2 staining preferentially on flagellar and cellular membranes, similar to CL, but different from Y strain intracellular labelling pattern, (3) flow-cytometry revealed higher expression of R1 by CL-14-trypomastigotes than virulent strains, but R2 staining similar to CL-trypomastigotes, (4) CL-14-trypomastigotes presented normal cruzipain activity in gelatin gels, but different banding patterns were found in CL-14 versus CL and Y strains. Our data rule out failure in cruzipain expression, activity or subcellular distribution as an explanation for CL-14 biological behaviour, but suggest the expression of a different isoform. These results also cast doubt on the putative role of cruzipain as a target of immunopathological responses, since high levels of functional cruzipain are expressed by a non-pathogenic T. cruzi.

Cell signaling during Trypanosoma cruzi invasion

“…Cruzipain, the major T. cruzi cysteine proteinase expressed in all developmental forms of different strains (Murta et al, 1990; Paiva et al, 1998), participates in TCT internalization and in intracellular parasite development (Meirelles et al, 1992). From experiments using human umbilical vein endothelial cells or CHO cells overexpressing B 2 type of bradykinin receptor (B 2 R), it was postulated that cruzipain acts on cell-bound kininogen and generates bradykinin that, upon recognition by B 2 R triggers IP 3 -mediated Ca 2+ influx (Scharfstein et al, 2000; Figure 1B ), thus promoting parasite invasion, a mechanism that is not ubiquitous, its activation depending on the cell type and the parasite isolate used.…”

“…From experiments using human umbilical vein endothelial cells or CHO cells overexpressing B 2 type of bradykinin receptor (B 2 R), it was postulated that cruzipain acts on cell-bound kininogen and generates bradykinin that, upon recognition by B 2 R triggers IP 3 -mediated Ca 2+ influx (Scharfstein et al, 2000; Figure 1B ), thus promoting parasite invasion, a mechanism that is not ubiquitous, its activation depending on the cell type and the parasite isolate used. Higher expression of functional cruzipain does not correlate with parasite infectivity (Paiva et al, 1998). …”

Biological Roles of Peptidases in Trypanosomatids~!2009-11-26~!2010-02-15~!2010-03-18~!

“…The enzyme is found in all developmental forms of different T. cruzi isolates, with levels 10-fold higher in epimastigotes and is active in the pH range of 5.0 to 7.5 [58][59][60]. Cruzipain has an N-terminal catalytic domain linked to an antigenic C-terminal extension, the gp25 [61,62].…”

Molecular analysis of early host cell infection by Trypanosoma cruzi

“…It is a gp57/51 cysteine proteinase, which is active in pH ranges of 5 to 7.5. The enzyme is expressed in all developmental forms of different T. cruzi isolates (87)(88). The involvement of cruzipain in host cell invasion and intracellular development was suggested by using peptidyl diazomethane derivatives, a class of irreversible inhibitors of cysteine proteinase (89).…”