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.
Highlights
Chagas disease is an endemic zoonosis in some countries of Central and South America that has Trypanosoma cruzi as its etiological agent, which affects 6–7 million individuals and still remains a major public health problem (World Health Organization [WHO], 2017)
These enzymes have multiple roles associated with T. cruzi virulence such as ability of the parasites to regulate the cell signaling triggered by extracellular ATP and other nucleotides (Silverman et al, 1998; Sansom et al, 2007), that are generated during the lysis of the T. cruzi-infected cells (Schnurr et al, 2000; Sansom et al, 2007)
Once confirmed the insertion of the resistance gene into the TcNTPDase-1 genomic locus and established the resistance of these parasites to neomycin and hygromycin, parasites were separated through cell sorting, in four clones for each hemiknockout: Hygro (#1), (#2), (#3), and (#4) and Neo (#1), (#2), (#3), and (#4)
Summary
Chagas disease is an endemic zoonosis in some countries of Central and South America that has Trypanosoma cruzi as its etiological agent, which affects 6–7 million individuals and still remains a major public health problem (World Health Organization [WHO], 2017). The chemotherapy currently used is based on antiparasitics such as, nifurtimox and benznidazole, which cause many side effects and present a lack of effectiveness on the chronic phase of the disease (Bern, 2011; Morillo et al, 2015) In this scenario, the search for new drugs and targets to chemotherapy is pivotal. Since extracellular ATP is an immunemodulatory molecule that in normal conditions stimulates the secretion of proinflammatory cytokines (IFN-7 and IL-2) to control the infection (Langston et al, 2003), it is hypothesized that ATP hydrolysis by ecto-ATPases activity in parasites can be important to subvert and avoid host defense mechanism, the mechanism is not clearly elucidated (Sansom et al, 2008) Another important role of these enzymes is in parasite nutrition, facilitating acquisition of extracellular purines since these parasites are not capable for performing de novo synthesis
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