Dividir para conquistar: como fragmentar o genoma do Trypanosoma cruzi pode aprimorar o seu entendimento e facilitar a busca de alvos conservados para aplicações diagnósticas

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease (CD), displays significant genetic diversity, being divided into seven Discrete Typing Units (DTUs). This high variability poses challenges for molecular and serological diagnosis of the infection, depending on the parasite population evaluated. In this context, it is crucial to further understand the parasite genome and enhance existing diagnostics by identifying conserved targets across parasite populations from distinct endemic areas. A substantial number of unassembled genomic sequences (reads) of T. cruzi are available in public databases. This dataset is ideal for identifying high-copy sequences that are difficult to assemble but may improve diagnostic sensitivity due to their repetitive nature. Thus, using bioinformatics tools without requiring genome assembly, we sought conserved motifs in six T. cruzi genotypes (TcI to TcVI). Read libraries of six DTUs were mapped onto assembled genomes, and each mapped read was divided into 80-nucleotide portions (k-mers) that were subsequently grouped. We identified conserved clusters whose consensus sequences were used to design primers capable of amplifying DNA from all T. cruzi genotypes, without cross-amplification with other trypanosomatids or hosts. Additionally, from sequences deposited in the NCBI, a conservation study of the T. cruzi satellite sequence, which has been used as a molecular diagnostic target, was conducted. It was observed that although most of the 195 nucleotides constituting the sequence are conserved, there were positions with less than 80% conservation, with sequences grouped by DTU. The primers designed from the satellite sequences were able to amplify as little as 50 fg of parasite gDNA without amplification in hosts or other trypanosomatids. Finally, the conserved genome-wide consensus were translated into peptide sequences, and after reactivity screening with pooled sera from CD patients, four peptides were selected for testing with sera from patients in different regions and experimentally infected animals. In mouse infections, the peptides were able to detect anti-T. cruzi IgM and IgG from the sixth week of infection. Furthermore, high sensitivity and specificity values were obtained with sera from patients from Bolivia and Brazil, but not from Peru. We believe that further investigation of the immune response profile of individuals from Peru, associated with an increase in the repertoire of genomic data that help to elucidate this particularity, may assist in the identification of better diagnostic targets for this region. Thus, these results suggest that bioinformatics approaches have the potential to identify conserved regions in T. cruzi isolates from distinct endemic areas, which may serve as potential targets for infection diagnosis.