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

Abstract

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.

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand

“…This was also clearly supported by the close phylogenetic relationship of the maxicircle of L. martiniquensis strain PCM3 and L. enriettii strain LEM3045 from the same subgenus Mundinia (Fig. 3 ), consistent with the work of Solana et al, which also used the coding regions of maxicircle DNA [ 48 ]; however, the maxicircle sequences of subgenus Mundinia in the database remained limited and required further data gathering. Although the divergent region of L. martiniquensis maxicircle might not be suitable for the phylogenetic inference, the distinctiveness of this region was helpful in identification of Leishmania species and exploring intra-species variation.…”

“…Phylogenetic trees were built from the coding region of the maxicircle DNAs of available Leishmania and Trypanosoma species in the NCBI database compared with that of L. martiniquensis , similar to the method described by Solana et al [ 48 ]. For minicircles, only universal minicircle invariant (CSB-3, CSB-1 and CSB-2) regions from L. martiniquensis strain PCM3 were compared with those available from the NCBI database.…”

Identification of Leishmania infantum Epidemiology, Drug Resistance and Pathogenicity Biomarkers with Nanopore Sequencing

“…Differentiation between L. infantum and L. donovani can be difficult due to their high nucleotide identity (i.e., chromosome 36 is 99.16% similar between references L. donovani BPK282A1 and L. infantum JPCM5); thus, it is not striking that a small percentage of reads (5%) is assigned as L. donovani. The remaining 1% consists of a mixture of assignments to other Trypanosomatidae, to high error reads or sequences belonging to the maxicircle and minicircle kinetoplasts, for which there is no complete reference for L. infantum yet [12,21].…”

“…In addition to species identification by random sequence fragments, maxicircle kinetoplast sequences from Trypanosomatidae have been previously described as a suitable molecular marker for species [11,21] and strain [12] phylogenies, akin to mitochondrial sequences from the kingdoms Plantae, Fungi, or Animalia. As shown in Figure S1, reconstruction of the L. infantum conserved region (CR) of the maxicircle is possible through guided consensus assembly with nanopore sequencing reads, with as little coverage as with a mean of 3X.…”

“…Some genetic variations have been associated with resistance to drugs: an increase in copy number of genes in the H locus (chromosome 23) has been associated with trivalent antimonial resistance, and a reduction in METK (LinJ.30.3560) gene copies has been associated with increased allopurinol resistance [10]. Other markers, such as the coding regions of the conserved region (CR) of the maxicircle, have recently been reported as suitable for Leishmania taxonomy for phylogenetic analysis, including subspecies divisions [11,12].…”