Nucleoside triphosphate diphosphohydrolase1 (TcNTPDase-1) gene expression is increased due to heat shock and in infective forms of Trypanosoma cruzi

Abstract

Background: Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hydrolyze tri- and/or di-phosphate nucleotides. Evidences point to their participation in Trypanosoma cruzi virulence and infectivity. In this work, we evaluate TcNTPDase-1 gene expression in comparison with ecto-NTPDase activity, in order to study the role of TcNTPDase-1 in parasite virulence, infectivity and adaptation to heat shock.

Findings: Comparison between distinct T. cruzi isolates (Y, 3663 and 4167 strains, and Dm28c, LL014 and CL-14 clones) showed that TcNTPDase-1 expression was 7.2 ± 1.5 times higher in the Dm28c than the CL-14 avirulent clone. A remarkable expression increase was also observed in the trypomastigote and amastigote forms (22.5 ± 5.6 and 16.3 ± 3.8 times higher than epimastigotes, respectively), indicating that TcNTPDase-1 is overexpressed in T. cruzi infective forms. Moreover, heat shock and long-term cultivation also induced a significant increment on TcNTPDase-1 expression.

Conclusions: Our results suggest that TcNTPDase-1 plays an important role on T. cruzi infectivity and adaptation to stress conditions, such as long-term cultivation and heat shock.

Figure 1

TcNTPDase-1 expression in epimastigote forms from distinct T. cruzi isolates. A. TcNTPDase-1 mRNA levels estimated by RT-qPCR. The relative quantification by ∆∆Ct method was performed using the avirulent Cl-14 clone as calibrator. B. Ecto-ATPase and ecto-ADPase activities between distinct T. cruzi strain/clones. The ecto-nucleotidase activities were estimated in T. cruzi epimastigotes, using 5 mM ATP or ADP as substrate. The inset shows the ecto-nucleotidase activities represented as Fold Change versus Cl-14 clone. C. Alignment of DNA sequence from PCR products for the TcNTPDase-1. The asterisks indicate identity between the nucleotides. *, # p < 0.05 (versus Cl-14 clone, Mann-Whitney Rank-Sum test).

Figure 2

TcNTPDase-1 expression in infective and non-infective forms of T. cruzi . A. TcNTPDase-1 mRNA levels in epimastigotes, amastigotes and trypomastigotes, estimated by RT-qPCR. The relative quantification by ∆∆Ct method was performed using the epimastigote form as calibrator. B. Ecto-ATPase and ecto-ADPase activities in distinct developmental forms. The ecto-nucleotidase activities were estimated in epimastigotes, amastigotes and trypomastigotes, using 5 mM ATP or ADP as substrate.*, #p < 0.05 (versus epimastigotes, Mann-Whitney Rank-Sum test).

Figure 3

TcNTPDase-1 gene expression during epimastigote in vitro cultivation and induced by heat-shock. A. Growth curve of T. cruzi epimastigotes, in BHI medium, at different temperatures. (●) 28°C, (●) 37°C. B. TcNTPDase-1 mRNA levels during T. cruzi growth curve. The relative quantification by ∆∆Ct method was performed using epimastigotes (Y strain) from the first day of cultivation as calibrator. (●) 28°C, (●) 37°C. *p < 0.05 (versus day 1, Student’s t test).