Assembly of a Large Collection of Maxicircle Sequences and Their Usefulness for Leishmania Taxonomy and Strain Typing

Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named "non-JPC like", may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability.