Influence of Ecto-nucleoside triphosphate diphosphohydrolase activity on Trypanosoma cruzi infectivity and virulence

Abstract

Background: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models.

Methodology/principal findings: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites.

Conclusions/significance: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Figure 1. Ecto-nucleotidase activities of trypomastigotes from different strains/clone of T. cruzi.

Trypomastigotes were obtained from the first VERO cells passage (P1). The Ecto-ATPDase activities were measured at 37°C during 1 hour. Data are mean±SE of two independent experiments in triplicate.

Figure 2. T. cruzi infectivity and ecto-ATPase/ADPase ratio decrease during in vitro cultivation.

A) Ecto-ATPDase (solid bars) and Ecto-ADPDase (open bars) activities from live trypomastigotes from different cellular passages (P1, P3 and P4). P1-1 and P1-2 are the first and second massive exits of parasites from the 1^st passage; in the same way P3-1 and P3-2 are the first and second massive exits of parasites for the 3^rd passage. Data are mean±SE of triplicate assays from one experiment. The inset shows the ATPase/ADPase hydrolytic activities ratio. B) Microphotograph of infected VERO cells culture after 24 h of parasite-cell interaction at the 3^rd to 4^th passage. Spherical bodies are non-internalized amastigote-like parasites. C) Zoom from box section shown in B. Black arrow exemplifies a non-internalized amastigote-like and white arrow a non-internalized trypomastigote parasite.

Figure 3. Effect of inhibitors in the ecto-ATPDase activity of Y strain P1 trypomastigotes.

Parasites were pre-incubated for 10 min with different concentrations of Suramin (Panel A), ARL 67156 (Panel B) or Gadolinium (Panel C) and ecto-ATPDase activities were measured. Ecto-ATPase (Δ) and ecto-ADPase (□) activities are expressed as percentage of control activity (without inhibitors). Data are mean±standard error of two independent experiments in triplicate.

Figure 4. Effects of ecto-ATPDase inhibitors on in vitro T. cruzi infectivity.

Microphotographs of VERO cell monolayers infected with trypomastigotes not treated (panels A,C,E) or treated with Ecto-ATPDase inhibitors (panels B,D,F). P1 trypomastigotes (Y strain) were pre-treated with ARL 67156, Gadolinium or Suramin at the indicated concentrations, resuspended in medium without inhibitor and used in VERO cells invasion assays. After 24 hours post infection each assay slide was washed and stained with Giemsa. The arrows indicate many internalized amastigotes.

Figure 5. Heterologous expression of T. cruzi Ecto-NTPDase-1 and the effect of ecto-ATPase inhibitors on purified protein.

A) SDS-PAGE stained with coomassie blue. A 15 µL sample of each step of production and purification of Ecto-NTPDase-1 was applied in each lane, showing the purified protein with approx. 66 kDa. MW, molecular weight markers. B) ATPDase activity of purified Ecto-NTPDase-1 in the presence of ecto-ATPase inhibitors. The results represent the percent activity with inhibitor related to the activities in the absence of inhibitors. Data are mean±SE of two independent experiments, each assayed in triplicate.

Figure 6. Inhibition of T. cruzi Ecto-ATPDases decreased virulence to mouse.

A) parasitemia curves and B) mortality in Swiss mice infected with 5,000 parasites/0.1 mL blood (Y strain P1 trypomastigotes). Parasites were pre-treated with ARL67156, Gadolinium or Suramin as indicated; a negative control assay is included, omitting the parasites' drug pre-treatment. Data are from one experiment using the mean value of a group of 10 mice in each treatment.