Knocking Down TcNTPDase-1 Gene Reduces in vitro Infectivity of Trypanosoma cruzi

Abstract

Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.

Keywords: TcNTPDase-1; Trypanosoma cruzi; infectivity; knockout; virulence.

FIGURE 1

Growth curve of hemi-knockout epimastigote clones in LIT medium at 28°C, up to 10 days of cultivation. (A) Hygromycin-resistant hemi-knockout clones. (●) Dm28c WT; (■) Hygro (#1); (▲) Hygro (#2); (▼) Hygro (#3); (■) Hygro (#4). (B) Neomicyn-resistant hemi-knockout clones. (●) Dm28c Wt; (■) Neo (#1); (▲) Neo (#2); (▼) Neo (#3); (■) Neo (#4).

FIGURE 2

TcNTPDase-1 gene expression in distinct evolutive forms of T. cruzi hemi-knockouts. (A,B) TcNTPDase-1 mRNA levels in epimastigote forms of HYGRO and NEO hemi-knockout clones, respectively. The relative quantification by ΔΔCt method was performed using the wild type (WT) clone as the calibrator. (C) TcNTPDase-1 mRNA levels in trypomastigote forms of WT, HYGRO, and NEO hemi-knockouts. Epimastigote forms of the respective clones were used as calibrators. (D) TcNTPDase-1 mRNA levels in trypomastigote forms of the hemi-knockouts (HYGRO and NEO) in comparison to the WT clone. Trypomastigote forms of the WT clone were used as the calibrator. *p < 0.05 (Student’s t-test).

FIGURE 3

Estimation of TcNTPDase-1 protein levels in hemi-knockout epimastigote clones by Western blots. (A) HYGRO hemi-knockout epimastigote clones. (B) NEO hemi-knockout epimastigote clones. 1: WT; 2: (#1); 3: (#2); 4: (#3); 5: (#4). Insets: (A) Anti-potato apyrase serum (1:5000). (B) Anti-tubulin antibodies (1:1000). ^∗,^&p < 0.05 (Student’s t-test).

FIGURE 4

Adhesion assay of hemi-knockout parasites to VERO cells. The adhesion of WT, HYGRO, and NEO hemi-knockout trypomastigotes to VERO cells were estimated after 2 h of interaction. (A) Percentage of VERO cells with adhered trypomastigotes. (B) Number of trypomastigotes adhered per VERO cells. (C) Adhesion index. The asterisks show significant differences (*p < 0.05, Student’s t-test) between the control and hemi-knockout parasites. At the top, representative light microscopy images show VERO cells with adhered trypomastigotes. The arrows indicate adhered trypomastigotes and the scale bars correspond to 40 μm.

FIGURE 5

Infection assay of hemi-knockout parasites to VERO cells. The internalization of WT, HYGRO, and NEO hemi-knockout trypomastigotes to VERO cells were estimated after 48 h of interaction. (A) Percentage of VERO cells infected. (B) Number of intracellular amastigotes per VERO cells. (C) Endocytic index. The asterisks show significant differences (*p < 0.05, Student’s t-test) between the control and hemi-knockout parasites. At the top, representative light microscopy images show infected VERO cells. The arrows indicate amastigotes and the scale bars correspond to 40 μm.

FIGURE 6

Parasitic load quantification in VERO cells infected with TcNTPDase-1 hemi-knockouts. VERO cells were infected for 48 h with metacyclic trypomastigotes of WT, HYGRO, and NEO hemi-knockout clones. DNA was extracted and parasitic load was estimated by real-time PCR. *p < 0.05 (Student’s t-test).

FIGURE 7

Overexpression of TcNTPDase-1 gene from WT or hemi-knockout parasites. (A) TcNTPDase-1 mRNA levels were estimated by RT-qPCR in WT and parasites overexpressing the gene. The relative quantification by ΔΔCt method was performed using the WT clone as the calibrator. *p < 0.05. (B) Adhesion index to the in vitro infection of VERO cells using the parasites overexpressing TcNTPDase-1. ^∗p < 0.05. (C) Endocytic index to the in vitro infection of VERO cells with the parasites overexpressing TcNTPDase-1. (D,E) Parasitic load quantification in VERO cells infected with parasites overexpressing TcNTPDase-1 gene. VERO cells were infected for 48 h with metacyclic trypomastigotes of WT, OE WT, and OE Neo clones. DNA was extracted and parasitic load was estimated by real-time PCR. (D) Standard curve to the absolute quantification experiments. (E) Parasitic load in VERO Cells infected by WT and parasites overexpressing TcNTPDase-1. *p < 0.05 (Student’s t-test).