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

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.

Keywords: Leishmania major; Trypanosoma cruzi; chagas disease; leishmaniasis; molecular modelling; serodiagnosis.

Figure 1

Localization of the gene coding for Tc964 protein in the T. cruzi genome and evolutionary relationship of this protein in different species of trypanosomatids. (A) Location of TcCLB.511467.70 gene (red label) on the 38-S chromosome of T. cruzi (TcChr_38S), from curated reference strain CL Brener Esmeraldo like, with the coordinates 58,780 to 59,715k. (B) Maximum Likelihood phylogenetic tree proposed to the evolutionary history of the Tc964 protein among taxa of the Trypanosomatidae family; the tree was obtained with the Jones-Taylor-Thornton model using 1000 replicates of Bootstrap and a site coverage cutoff of 75% with the MEGA 7 program [34]. The selected outgroup was the B. saltans species. Numbers at the branches indicate maximum likelihood bootstrap support.

Figure 2

Molecular dynamics and structures of Tc964 and Lm964 proteins. (A) Root Mean Square Deviation values obtained during the 50 nanoseconds (2500 frames) of unrestricted molecular dynamics trajectories of the obtained models. Black line: Tc964_2FYM (template PDB code:2FYM) [38]. Gray line: Tc964_5ZH3 (template PDB code:5ZH3) [39]. Blue and red lines: Tc964_5L3S, Lm964_5L3S (template PDB code:5L3S) [37]. (B) Structural models of the 964 proteins from T. cruzi and L. major. Left: 3D Structure of the template SRP54 (PDB code:5L3S). Center: 3D structure model for Tc964 protein. Right: 3D structure model for Lm964 protein. N, G and M domains are located on the structures. 3D figures were generated using PyMOL Molecular Graphics System, (version 2.0, Schrödinger LLC, Portland, OR, USA) [43]. (C) Alignment between the sequence of SRP54 template protein and the 964 proteins of T. cruzi and L. major using the Omega Clustal program [44]. The domains N, G, and M are shown with the representative colors of the 3D model. Identical amino acids are highlighted in gray and the motif LGMGD is highlighted in alcian blue.

Figure 3

Subcellular localization of the Tc964 protein. (A) Detection of the Tc964 protein (35-kDa) in epimastigotes from the Y strain (DTU II) using an anti-Tc964 as primary antibody. (B) Tc964 detection on trypomastigotes strain Y (DTU II), with the anti-Tc964 antibody. Antibodies against Histone 3 (nuclei) and Heat-Shock Protein 70 (cytoplasm) were used as controls of subcellular fractionation.

Figure 4

Antigenicity of the Tc964 and Lm964 recombinant proteins. (A)ELISA test, using a protein lysate of T. cruzi Y (DTU II) epimastigotes and sera from patients at different stages of CCD severity: Asymptomatic (A, n = 25; B, n = 11), symptomatic (C, n = 18; D, n = 9). (B) ELISA test, using a protein lysate of L. major promastigotes and sera from ACL (n = 12) and CCL (n = 11) patients. (C) ELISA test, using the rTc964 protein as antigen and sera from CCD patients at different stages of disease. (D) ELISA test, using rLm964 protein and sera from ACL (n = 7) and CCL (n = 4) patients. Mean values are indicated by horizontal lines. The RI corresponds to the average derived from two (2) replicas. The p values were calculated using the Mann-Whitney U test (ns p > 0.05) for (B) and (D) and one-way ANOVA nonparametric Kruskal–Wallis test with Dunn’s posttest (* p < 0.05, *** p < 0.001, **** p < 0.0001) for (A)and (C).

Figure 5

Cross-reactivity analysis of the Tc964 and Lm964 recombinant proteins. (A) ELISA test, using the rLm964 as antigen and sera of patients at different stages of CCD (B) ELISA test, using the rTc964 as antigen and sera of ACL and CCL patients. The recombinant protein Trigger Factor was used as negative control. Horizontal lines represent the mean ± SD of the values for each sera group. (C) Analysis of the antigenicity of the recombinant proteins rTc964 (37 kDa) and rLm964 (38 kDa) by Western blot. Left: 11% SDS-PAGE gel with the rTc964, rLm964 and recombinant protein Trigger Factor. Center: Western blot using the serum of a CCD patient with severity stage C (sample QX90). Right. Western blot using the serum of an ACL patient (sample 31A). In each assay, recombinant protein Trigger Factor was used as a negative control.

Figure 6

Antigenicity of the TcNV and TcKP peptides. (A) ELISA test, using the TcNV peptide and sera from patients at different stages of CCD and sera of ACL and CCL patients. The rTc964 protein was used as positive control. (B) ELISA test, using TcKP as antigen and as primary antibody sera from patients with CCD with different stages and sera of ACL and CCL patients. The RI values shown in the figures correspond to the average of three (3) replicas. The p values were calculated using one-way ANOVA nonparametric Kruskal–Wallis (ns p > 0.05, * p < 0.05, ** p < 0.01) for the rTc964, TcKP peptide vs CCD sera; one-way ANOVA Fisher’s LSD test (ns p > 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001) for the TcNV peptide vs CCD sera and the significance between two groups was calculated using the Mann-Whitney U test for both peptides vs ACL, CCL sera.