Abstract
Trypanosoma cruzi has a complex life cycle consisting of four morphological and distinct biological stages. Although some authors suggest that T. cruzi primarily follows clonal reproduction, recent genomic and transcriptomic studies indicate an unorthodox capacity for recombination. We aimed to estimate the differential gene expression of 10 meiosis/homologous recombination-related genes during the T. cruzi life cycle, including epimastigotes, under two different types of stress (oxidative stress and pH changes). We performed RT-qPCR tests using novel-designed primers to estimate the differential gene expression (∆Ct and ∆∆Ct) of nine genes (SPO11, HAP2, RAD50, MRN complex, BRCA2, DMC1, MND1, and RPA1) and RAD51, which was previously reported. Our results show basal expression of all genes during the life cycle, indicating their hypothetical role in several cellular processes but with specific signatures of differential gene expression during the life cycle (HAP2, RPA, RAD50, BRCA2, MND1, and DMC1) and oxidative stress (RPA, MRE11, NBS1, BRCA2, MND1, and RAD51). Additionally, we found that the MRN complex has an independent level of expression in T. cruzi, with profiles of MRE11 and NBS1 upregulated in some stages. Recent studies on other trypanosomatids have highlighted the influence of HAP2 and RPA in recombination and hybridization. If T. cruzi uses the same repertoire of genes, our findings could suggest that metacyclogenesis may be the putative step that the parasite uses to undergo recombination. Likewise, our study reveals the differential profiles of genes expressed in response to oxidative and pH stress. Further studies are necessary to confirm our findings and understand the recombination mechanism in T. cruzi.
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Data availability
All the sequences for meiosis and HR-related genes were deposited in NCBI GenBank under the accession numbers ON746177-ON746187.
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Acknowledgements
We thank Duvan Morales for his support during the RT-qPCR analysis. We would like to acknowledge the assistance of ChatGPT, a language model trained by OpenAI, for providing helpful insights and corrections during the process of improving the drafting and readability.
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This work was funded by the Dirección de Investigación e Innovación at the Universidad del Rosario.
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LCS designed the primers, standardized the RT-qPCR, made the RNA extraction, performed the RT-qPCR, analyzed the data, and wrote the manuscript. TC made the survival curves and RNA extraction. NB supported the RT-qPCR standardization. BD participated during the primers design. JDR conceived the manuscript, supervised the project, and wrote, reviewed, and edited the manuscript. All authors approved the final version of the manuscript.
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ESM 1
Supplementary file1 Primers and PCR conditions for nine pairs of primers including the previously designed primers that amplify RAD51 (Table. S1) (Alves et al. 2018). (XLSX 11 kb)
ESM 2
Supplementary file2 Anova one-way test comparing the expression of meiosis and HR-related genes during the life cycle of Trypanosoma cruzi. (XLSX 26 kb)
ESM 3
Electrophoresis agarose gel for the ten meiosis and HR related genes. (PNG 1813 kb)
ESM 4
Interaction plot of Meiosis and homologous recombination-related genes during the whole T. cruzi life cycle. The y axes correspond to the values of ΔCt calculated, the x axes list the genes evaluated. (PNG 1032 kb)
ESM 5
Interaction plot of Meiosis and homologous recombination-related genes during oxidative stress in epimastigotes, metacyclic trypomastigotes and cell-derived trypomastigotes. The y axes correspond to the values of ΔΔCt calculated, the x axes list the genes evaluated. A interaction plot per stage was included. (PNG 2181 kb)
ESM 6
Interaction plot of Meiosis and homologous recombination-related genes during pH changes in epimastigotes, metacyclic trypomastigotes and cell-derived trypomastigotes. The y axes correspond to the values of ΔΔCt calculated, the x axes list the genes evaluated. A interaction plot per stage was included. (PNG 2942 kb)
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Cruz-Saavedra, L., Caceres, T., Ballesteros, N. et al. Differential expression of meiosis and homologous recombination-related genes in the life cycle of Trypanosoma cruzi. Parasitol Res 122, 1747–1757 (2023). https://doi.org/10.1007/s00436-023-07850-2
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DOI: https://doi.org/10.1007/s00436-023-07850-2