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. 2022 Dec;13(1):1827-1848.
doi: 10.1080/21505594.2022.2132776.

Trypanosoma cruzi pathogenicity involves virulence factor expression and upregulation of bioenergetic and biosynthetic pathways

Juan San Francisco  1 , Constanza Astudillo  1 , José Luis Vega  1   2   3 , Alejandro Catalán  1 , Bessy Gutiérrez  1 , Jorge E Araya  1 , Anibal Zailberger  4 , Anabel Marina  5 , Carlos García  5 , Nuria Sanchez  5 , Antonio Osuna  6 , Susana Vilchez  6 , Marcel I Ramírez  7 , Janaina Macedo  8 , Verónica Santiago Feijoli  8 , Giuseppe Palmisano  8 , Jorge González  1   3   9
Affiliations

Trypanosoma cruzi pathogenicity involves virulence factor expression and upregulation of bioenergetic and biosynthetic pathways

Juan San Francisco et al. Virulence. 2022 Dec.

Abstract

The molecular repertoire of Trypanosoma cruzi effects its virulence and impacts the clinical course of the resulting Chagas disease. This study aimed to determine the mechanism underlying the pathogenicity of T. cruzi. Two T. cruzi cell lines (C8C3hvir and C8C3lvir), obtained from the clone H510 C8C3 and exhibiting different virulence phenotypes, were used to evaluate the parasite's infectivity in mice. The organ parasite load was analysed by qPCR. The proteomes of both T. cruzi cell lines were compared using nLC-MS/MS. Cruzipain (Czp), complement regulatory protein (CRP), trans-sialidase (TS), Tc-85, and sialylated epitope expression levels were evaluated by immunoblotting. High-virulence C8C3hvir was highly infectious in mice and demonstrated three to five times higher infectivity in mouse myocardial cells than low-virulence C8C3lvir. qPCR revealed higher parasite loads in organs of acute as well as chronically C8C3hvir-infected mice than in those of C8C3lvir-infected mice. Comparative quantitative proteomics revealed that 390 of 1547 identified proteins were differentially regulated in C8C3hvir with respect to C8C3lvir. Amongst these, 174 proteins were upregulated in C8C3hvir and 216 were downregulated in C8C3lvir. The upregulated proteins in C8C3hvir were associated with the tricarboxylic acid cycle, ribosomal proteins, and redoxins. Higher levels of Czp, CRP, TS, Tc-85, and sialylated epitopes were expressed in C8C3hvir than in C8C3lvir. Thus, T. cruzi virulence may be related to virulence factor expression as well as upregulation of bioenergetic and biosynthetic pathways proteins.

Keywords: Trypanosoma cruzi; genetically related cell lines; proteomics; virulence.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Experimental design to study C8C3hvir and C8C3lvir Trypanosoma cruzi cell lines and compare biological behaviour, proteomics profile, and virulence factor expression. T. cruzi tissue culture-derived trypomastigotes from both cell lines were used to infect BALB/c mice and evaluate parasitaemia curves and parasite loads in organs and tissues of acute and chronically infected mice. Metacyclogenesis was also studied. Trypomastigotes were lysed and supernatants were submitted to SDS-PAGE. Total proteins were in-gel digested by trypsin. Tryptic peptides were analysed by label-free quantitative mass spectrometry-based proteomics. The entire workflow was performed in triplicate for each T. cruzi cell line. Virulence factor protein expression was evaluated by immunoblotting. Cruzipain and transialidase enzymatic activity were measured. Created with Biorender.Com.
Figure 2.
Figure 2.
In vitro and in vivo infectivity of T. cruzi C8C3hvir and C8C3lvir cell lines. (a) Parasitemia curves of mice infected with trypomastigotes from either C8C3hvir or C8C3lvir. Results are expressed as the mean ± SD and represent at least 3 experiments performed in triplicate. ** P < 0.01, *** P < 0.001; Two-way ANOVA. (b) Cardiomyocytes were infected with trypomastigotes from either C8C3hvir or C8C3lvir cell lines, using a parasite/cell ratio 5:1. After 3 h, cultures were washed three times with PBS. and stained with propidium iodide. Results are expressed as the mean ± SD of parasites/500 cells from three experiments performed in triplicate. * P < 0.01; Student’s t-test.
Figure 3.
Figure 3.
Organ and tissue parasitic load of mice infected with either T. cruzi C8C3hvir or C8C3lvir cell lines. Acutely or chronically infected mice were sacrificed and parasite load (parasites/50 ng DNA) was determined by qPCR in (a) heart, (b) liver, (c) quadriceps muscle, and (d) lung. The limit of detection was 0.1 parasite equivalent/50 ng DNA. Results are expressed as the mean ± SD and represent three experiments performed in triplicate. * P <0,01, ** P < 0,01 *** P < 0,001; Student’s t-test.
Figure 4.
Figure 4.
Epimastigote proliferation curve and metacyclogenesis of T. cruzi C8C3hvir and C8C3lvir cell lines. (a) Epimastigote growth. Results are expressed as the mean ± SD and represent at least three experiments performed in triplicate. ***P < 0.001; Two-way ANOVA. (b) Epimastigote differentiation into metacyclic trypomastigotes. Results are expressed as the mean ± SD and represent at least three experiments performed in triplicate. *P < 0.05; Student’s t-test.
Figure 5.
Figure 5.
Workflow of proteomics analysis. Low-virulence (C8C3lvir, n = 3) and high-virulence (C8C3hvir, n = 3) cell lines were analysed by nLC-MS/MS and the data were mapped using concatenated databases for the Dm28c, TCC, and CL Brener T. cruzi strains. (a) Chromatographic elution profile of the peptides. (b) Identification of differentially regulated proteins. (c) Principal component analysis of differentially regulated proteins. (d) MAplot of differentially regulated proteins. (e) Volcano plot of differentially regulated proteins (F). Heatmap of differentially regulated proteins. The red and blue colours correspond with upregulated and downregulated proteins, respectively. The grey colour indicates that the protein was not differentially expressed.
Figure 6.
Figure 6.
Gene ontology analysis combined with quantitative data for differentially expressed proteins in T. cruzi C8C3hvir cell line. Upregulated proteins in C8C3hvir associated with (a) biological processes, (b) cellular components, and (c) molecular functions. Only ontologies with q-value ≤0.05 are presented (Benjamini-Hochberg corrected). The word cloud diagram indicates terms in size and colour proportional to -log(p-value). The histogram indicates the fold enrichment of each ontology term, presenting overrepresented terms in comparison to a gene background. The bubble chart shows the geometric position of each identified ontology term, illustrating the proximity between them.
Figure 7.
Figure 7.
Gene ontology analysis combined with quantitative data for differentially expressed proteins in T. cruzi C8C3lvir cell line. Downregulated proteins in C8C3lvir associated with (a) biological processes, (b) cellular components, and (c) molecular functions. Only ontologies with q-value ≤0.05 are presented (Benjamini-Hochberg corrected). The word cloud diagram indicates terms in size and colour proportional to -log(p-value). The histogram indicates the fold enrichment of each ontology term, presenting overrepresented terms in comparison to a gene background. The bubble chart shows the geometric position of each identified ontology, illustrating the proximity between them.
Figure 8.
Figure 8.
KEGG pathway enrichment analysis of upregulated and downregulated proteins in T. cruzi C8C3hvir cell line. Pfam and Interpro domains enriched in (a) upregulated and (b) downregulated proteins. Only pathways with a q-value ≤0.05 (Benjamini-Hochberg corrected) are presented. (c) Results of the sparse PLS-DA analysis. (d) Proteins are classified into known groups. (e) Key variables that drive the discrimination. The top 20 variables that contribute to components 1 and 2 are inside the circle (dim = 1). The red and blue colours correspond to upregulated and downregulated proteins, respectively.
Figure 9.
Figure 9.
Cruzipain expression and metacyclogenesis in T. cruzi C8C3hvir and C8C3lvir cell lines. Two-dimensional electrophoresis results for cruzipain expression in (a) trypomastigotes and (b) epimastigotes from T. cruzi C8C3hvir and C8C3lvir cell lines. Densitometric analysis of immunoblots were performed using β actin as loading control. Bars are represented as the mean ± SEM of the least three independent experiments. ****P < 0.0001 vs corresponding control; Student’s t-test. (c) Cysteine proteinase activity of both cell lines. * P <0.5, **P < 0.05; Two-way ANOVA. (d) Metacyclogenesis in the presence or absence of cysteine proteinase inhibitor E64d, noted as percentage of metacyclic trypomastigotes.
Figure 10.
Figure 10.
Trans-sialidase expression in T. cruzi C8C3hvir and C8C3lvir cell lines. (A) Trans-sialidase expressionin C8C3hvir and C8C3lvircell lines. Densitometric analysis of immunoblots were performed using b actin as loading control. The results are represented as the mean + SEM of five independent experiments. ** P = 0.029 vs corresponding control; Student’s t-test. (B) Trans-sialidase activity of trypomastigotes from C8C3hvir and C8C3lvir T. cruzi cell lines. All assays were performed in triplicate. * P < 0.01;Student’s t-test.
Figure 11.
Figure 11.
Complement regulatory protein (CRP) expression and complement-mediated lysis resistance in T. cruzi C8C3hvir and C8C3lvir cell lines. (a) CRP expression in trypomastigotes from C8C3hvir and C8C3lvir cell lines. Densitometric analysis of immunoblots were performed using β actin as loading control. =the results are represented by the mean ± SEM of five independent experiments. ** P = 0.0014 vs corresponding control; Student’s t-test. (b) Susceptibility of trypomastigotes from C8C3hvir and C8C3lvir cell lines to complement-mediated lysis. The results are expressed as the mean ± SD of experiments performed in triplicate. * P < 0.01; Student’s t-test.
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Grants and funding

JG acknowledges the financial support extended by Seedlings Grants SEM-17-02 and the Bridge Fund for Research of Excellence, University of Antofagasta. JSF was supported by Antofagasta University Fellowship. We are grateful to Agencia Nacional de Investigación y Desarrollo (ANID)-Millennium Science Initiative Program-ICN09_016: Millennium Institute on Immunology and Immunotherapy (ICN09_016; former P09/016-F). Finally, GP and JMdS are supported by FAPESP (2018/18257-1, 2018/15549-1, 2020/04923-0 to GP and 2021/00140-3 to JMdS).
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