Molecular Characterization of Tc964, A Novel Antigenic Protein from Trypanosoma cruzi.

Elizabeth Ruiz-Márvez,César Augusto Ramírez,Concepción J Puerta,Fanny Guzmán,Magda Mellisa Flórez,Gabriela Delgado,Paulino Gómez-Puertas,Eliana Rocío Rodríguez,José María Requena

doi:10.3390/ijms21072432

Abstract

The Tc964 protein was initially identified by its presence in the interactome associated with the LYT1 mRNAs, which code for a virulence factor of Trypanosoma cruzi. Tc964 is annotated in the T. cruzi genome as a hypothetical protein. According to phylogenetic analysis, the protein is conserved in the different genera of the Trypanosomatidae family; however, recognizable orthologues were not identified in other groups of organisms. Therefore, as a first step, an in-depth molecular characterization of the Tc946 protein was carried out. Based on structural predictions and molecular dynamics studies, the Tc964 protein would belong to a particular class of GTPases. Subcellular fractionation analysis indicated that Tc964 is a nucleocytoplasmic protein. Additionally, the protein was expressed as a recombinant protein in order to analyze its antigenicity with sera from Chagas disease (CD) patients. Tc964 was found to be antigenic, and B-cell epitopes were mapped by the use of synthetic peptides. In parallel, the Leishmania major homologue (Lm964) was also expressed as recombinant protein and used for a preliminary evaluation of antigen cross-reactivity in CD patients. Interestingly, Tc964 was recognized by sera from Chronic CD (CCD) patients at different stages of disease severity, but no reactivity against this protein was observed when sera from Colombian patients with cutaneous leishmaniasis were analyzed. Therefore, Tc964 would be adequate for CD diagnosis in areas where both infections (CD and leishmaniasis) coexist, even though additional assays using larger collections of sera are needed in order to confirm its usefulness for differential serodiagnosis.

Highlights

Chagas disease (CD) or American Trypanosomiasis and its etiological agent (Trypanosoma cruzi) were first described by Dr Carlos Chagas in 1909 [1,2]
The parasitaemia in chronic CD (CCD) patients is very low or undetectable, the presence of high levels of antibodies against T. cruzi antigens [12] serves for diagnosing CCD [13,14]
We report the characterization of a novel protein from T. cruzi, named Tc964, which was initially identified by mass spectrometry due to its binding to the 5 untranslated region of the LYT1 mRNAs [19] within a project aimed to study the regulatory mechanisms operating on LYT1 gene expression

Summary

Introduction

Chagas disease (CD) or American Trypanosomiasis and its etiological agent (Trypanosoma cruzi) were first described by Dr Carlos Chagas in 1909 [1,2]. Chagas disease evolves through two clinical stages, recognized as the acute and chronic phases The former corresponds to the initial stage of the infection, and its duration lasts between one to two months [10,11]. 10 to 30 years after the initial infection, about 30 to 40% of the patients progress to the symptomatic or determinate chronic phase, developing cardiac arrhythmias, progressive heart failure or digestive alterations as megaesophagus and megacolon. These alterations are linked to the tissue tropism of the parasite; during the chronic stages of the disease, T. cruzi mainly persists in the cardiac and mesenteric muscles. The parasitaemia in chronic CD (CCD) patients is very low or undetectable, the presence of high levels of antibodies against T. cruzi antigens [12] serves for diagnosing CCD [13,14]

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