Nucleoside triphosphate diphosphohydrolase1 (TcNTPDase-1) gene expression is increased due to heat shock and in infective forms of Trypanosoma cruzi
© Silva-Gomes et al.; licensee BioMed Central Ltd. 2014
Received: 14 March 2014
Accepted: 27 September 2014
Published: 5 October 2014
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.
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.
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.
Chagas disease is a neglected illness caused by the protozoan parasite Trypanosoma cruzi, which affects 8 million people in endemic areas of Latin America ,. The different disease spectrums and the course of chronic infection may be consequences of complex interactions between genetic variability of T. cruzi subpopulations (classified into TcI to TcVI) , host immunogenetics and eco-epidemiological characteristics -. Current chemotherapy is based on the drugs nifurtimox and benznidazole, which present a lack of effectiveness on the chronic phase of the disease . In this scenario, the search for new drugs and targets to chemotherapy is pivotal.
Ecto-nucleoside triphosphate diphosphohydrolases (Ecto-NTPDases, EC 188.8.131.52) are enzymes that hydrolyze tri- and/or di-phosphate nucleotides ,. Since extracellular ATP is an immune-modulatory molecule that stimulates the secretion of IFN-γ and IL-2 , it is hypothesized that ecto-ATPase activity in parasites can be important to the evasion mechanism from the host immune defense, although the mechanism is not clearly elucidated . In 2004, a 2,282 base pair mRNA encoding a full-length NTPDase was cloned, sequenced  and named T. cruzi NTPDase-1 (TcNTPDase-1; Genbank: AY540630.1), which presents a single copy gene in the genome. In this work, we have developed a quantitative Real-Time RT-PCR assay to quantify TcNTPDase-1 mRNA levels, using TcGAPDH and TcCalmoduline as housekeeping genes (Additional file 1: Figure S1), aiming to contribute to the knowledge about the role of this NTPDase to T. cruzi infectivity and virulence. In this sense, we evaluated the TcNTPDase-1 expression in distinct developmental forms (epimastigotes and cell culture-derived amastigotes and trypomastigotes) and parasite isolates, such as the Cl-14, which is described as an avirulent T. cruzi clone, since it is unable to promote infection and to induce immune response in a murine model ,.
During its life cycle, T. cruzi undergoes profound adaptations triggered by a wide range of environmental conditions between the vertebrate or invertebrate hosts, such as variations in pH and temperature. The epimastigote and metacyclic trypomastigote forms interact with the triatomine insect vector at 28°C and the amastigote and trypomastigote forms interact with the mammalian host at 37°C. This heat shock may induce a response from the parasite, promoting the modulation of ecto-ATPase activity . Thus, considering the importance of the ecto-NTPDase activity on the parasite’s purine salvage pathway  and heat shock adaptation, the expression of TcNTPDase-1 was also analyzed during T. cruzi epimastigote cultivation, at different temperatures, to investigate the expression regulation in response to long-term cultivation or induced by heat shock.
The T. cruzi Y, 3663, 4167 strains, and Dm28c, LL014 and CL-14 clones were obtained from the Coleção de Protozoários da Fundação Oswaldo Cruz (COLPROT-FIOCRUZ). T. cruzi laboratory-adapted epimastigotes were cultivated in BHI medium, supplemented with 10% heat-inactivated fetal bovine serum, at 28°C for 5 days, to reach late-log growth phase.
Production of culture derived trypomastigotes and amastigotes
The isolation of trypomastigote and amastigote forms was carried out using Vero cells, as detailed elsewhere . Briefly, cell cultures were infected with mice-derived bloodstream trypomastigotes, in a 10:1 parasite/host cell ratio. Infected cells were maintained at 37°C in a 5% CO2 atmosphere. After 5–6 days, the supernatant was collected, centrifuged at 500 × g for 5 min, and allowed to stand at 37°C for 30 min for the migration of trypomastigotes into the supernatant. The amastigotes remained in the pellet.
Ecto-ATPase and ecto-ADPase activity measurements
The extracellular hydrolysis of ATP or ADP by intact parasites was carried out through the measurement of inorganic phosphate (Pi) released in the supernatant, as previously described by De Souza et al. . Briefly, ecto-ATPase and ecto-ADPase activities were estimated by the incubation of intact cells (0.5 × 108 parasites) for 1 h at 28°C, in a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose, 5 mM MgCl2, and 50 mM Hepes–Tris buffer, in the presence of 5 mM ATP or ADP (Sigma-Aldrich), in a final volume of 0.5 mL. The reaction was started by the addition of living parasites and terminated by the addition of 1 mL of ice cold HCl 0.2 M. The cell suspensions were pelleted and supernatant aliquots were used for inorganic phosphate (Pi) quantification .
RNA isolation and cDNA synthesis
Total RNA from T. cruzi (1x108 cells) was extracted using TRIzol Reagent (Invitrogen, USA) and treated with DNAse I (Sigma-Aldrich, USA), following manufacturer’s instructions. RNA quantity and purity was estimated by spectrophotometry at 260/280/230 nm. RNA integrity was verified through electrophoresis on a 1.5% (w/v) agarose gel. All reverse transcriptase reactions were performed from 3 μg of RNA using a Superscript III First-strand System (Invitrogen, USA), according to the manufacturer’s instructions.
-NTPDase-gene expression quantification by Real-Time RT-PCR
Real-time quantitative PCR assays were performed in ABI Prism 7500 fast sequence detection system using Power SYBR Green PCR mastermix (Applied Biosystems, USA). The following primers and concentrations were used: TcNTPDase-I Fw (600 nmol/L), 5’-GCGGAACCGCAACACCCTCA-3’; TcNTPDase-I Rv (600 nmol/L), 5’-CGGTCGAGCTGAAGCGCCAA-3’; TcCalmoduline Fw (600 nmol/L), 5’-CCCGACGGAGGCGGAGCTGC-3’; TcCalmoduline Rv (600 nmol/L), 5’-GTCCACGTCGGCCTCGCGGA-3’; TcGAPDH Fw (300 nmol/L), 5’-GTGCGGCTGCTGTCAACAT-3’; and TcGAPDH Rv (300 nmol/L), 5’-AAAGACATGCCCGTCAGCTT- 3’. The conditions for the RT-qPCR were as follows: 95°C for 10 minutes, followed by 40 cycles at 95°C for 15’seconds and 62°C for 1 minute. To monitor the primers specificity, melting curves were performed after each experiment, resulting in a single peak. Reactions were performed in duplicates using 2 μL of cDNA template, in a total volume of 20 μL. The relative quantitative measurement of target gene levels was performed using the ΔΔCt method . As endogenous housekeeping control genes, T. cruzi Calmoduline and GAPDH genes were used. PCR assays were in triplicate and data were pooled.
All experiments were performed at least in biological triplicates and experimental duplicates. Data are expressed as arithmetic mean × Standard Deviation. Student’s t test or Mann-Whitney Rank-Sum test were adopted to analyze the statistical significance of the apparent differences. All statistical tests were performed with SigmaPlot for Windows Version 12 (Systat Software). Differences were considered statistically significant when p < 0.05.
In order to perform the experimental infections with Trypanosoma cruzi, swiss mice obtained from the animal facilities of the Oswaldo Cruz Foundation (CECAL/Fiocruz, Rio de Janeiro, Brazil) were housed under specific pathogen free conditions in a 12-hour light-dark cycle with access to food and water ad libitum. Our protocols were approved by the Institutional Committee for Animal Ethics of Fiocruz (CEUA/Fiocruz, License LW-16/14).
Results and discussion
There are lines of evidence that NTPDases are related to virulence and infectivity in protozoan parasites -. However, most of the studies reported ecto-NTPDase enzymatic activities in intact parasites or plasma membrane fractions. Taking into account that plasma membranes share distinct ecto-nucleotidase activities, such as the Mg2+-dependent and Mg2+-independent ecto-ATPase activities , there is a lack of information regarding the specific contribution of each enzyme to these processes. It is possible that distinct ecto-enzymes could contribute to the parasite adaptation to stress conditions, as nutrients starvation or heat shock. Recently, Mariotini-Moura et al.  performed heterologous expression, purification and molecular characterization of TcNTPDase-1. By using specific polyclonal antibodies, they confirmed the presence of TcNTPDase-1 not only on the surface of T. cruzi, but also in the kinetoplast, nucleus, intracellular vesicles, flagellum and flagellum insertion region. The two latter localizations suggest that the enzyme may have a role in nutrient acquisition. It was also shown that the treatment of the parasite with anti-TcNTPDase-1 antibody decreases adhesion of T. cruzi to Vero cells, corroborating the importance of this enzyme to parasite-vertebrate host interaction.
Identification and classification of T. cruzi strains/clones used in this work
T. cruzi I
T. cruzi II
São Paulo, Brazil
T. cruzi III
T. cruzi IV
T. cruzi V
T. cruzi VI
Rio Grande do Sul, Brazil
The authors thank Dr. Luciana P. Rangel for critical reading of the manuscript and English revision, as well as the Program for Technological Development in Tools for Health (PDTIS-Fiocruz) for the facilities on the Real Time PCR and DNA sequencing platforms and Coleção de Protozoários da Fundação Oswaldo Cruz (Colprot), for having provided the T. cruzi isolates used in this study. This work was supported in by a grant from FIOCRUZ and CNPq (PAPES VI – Process 407688/2012-9).
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