Deciphering Trypanosoma lainsoni kDNA minicircles: insights into genetic diversity, mRNA editing, and molecular diagnosis.

The mitochondrial genome of kinetoplastids, organized in a unique DNA net called the kinetoplast, presents significant complexity compared to other eukaryotic mitochondrial genomes. Minicircles, which constitute over 90% of the kinetoplast mass, are essential for the post-transcriptional editing of maxicircle transcripts. This study focuses on Trypanosoma lainsoni, a trypanosomatid first discovered in northern Brazil and later reported in Argentina. Utilizing a combined second and third generation sequencing approach, we conducted an in-depth analysis of the kDNA minicircles of three Argentinian isolates of T. lainsoni. Through de novo assembly, minicircle molecules with two conserved regions interspaced by two hypervariable regions 180° apart, were identified. Guide RNAs encoded within hypervariable regions were inferred and the mitochondrial mRNA editing cascades mediated by these guide RNAs were fully reconstructed. We also developed a PCR-based approach for the detection and confirmation of T. lainsoni kDNA using minicircle-specific primers. A deeper analysis on the diversity of minicircle hypervariable regions revealed that the three isolates correspond to different genotypes circulating in the region. This research significantly advances our understanding of the genomic architecture of T. lainsoni and offers valuable tools for its molecular identification, differentiation from other trypanosomes, and the study of kinetoplastid biology.