The Complete Mitochondrial DNA of Trypanosoma cruzi: Maxicircles and Minicircles

Abstract

The mitochondrial DNA of Trypanosomatids, known as the kinetoplast DNA or kDNA or mtDNA, consists of a few maxicircles and thousands of minicircles concatenated together into a huge complex network. These structures present species-specific sizes, from 20 to 40 Kb in maxicircles and from 0.5 to 10 Kb in minicircles. Maxicircles are equivalent to other eukaryotic mitochondrial DNAs, while minicircles contain coding guide RNAs involved in U-insertion/deletion editing processes exclusive of Trypanosomatids that produce the maturation of the maxicircle-encoded transcripts. The knowledge about this mitochondrial genome is especially relevant since the expression of nuclear and mitochondrial genes involved in oxidative phosphorylation must be coordinated. In Trypanosoma cruzi (T. cruzi), the mtDNA has a dual relevance; the production of energy, and its use as a phylogenetic marker due to its high conservation among strains. Therefore, this study aimed to assemble, annotate, and analyze the complete repertoire of maxicircle and minicircle sequences of different T. cruzi strains by using DNA sequencing. We assembled and annotated the complete maxicircle sequence of the Y and Bug2148 strains. For Bug2148, our results confirm that the maxicircle sequence is the longest assembled to date, and is composed of 21 genes, most of them conserved among Trypanosomatid species. In agreement with previous results, T. cruzi minicircles show a conserved structure around 1.4 Kb, with four highly conserved regions and other four hypervariable regions interspersed between them. However, our results suggest that the parasite minicircles display several sizes and numbers of conserved and hypervariable regions, contrary to those previous studies. Besides, this heterogeneity is also reflected in the three conserved sequence blocks of the conserved regions that play a key role in the minicircle replication. Our results using sequencing technologies of second and third-generation indicate that the different consensus sequences of the maxicircles and minicircles seem to be more complex than previously described indicating at least four different groups in T. cruzi minicircles.

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

“…A significant portion of the maxicircle (around 12 kb), named “divergent region” or DR, consists of low-complexity, tandemly repeats and its complete sequence remains undetermined for most species, but it is considered to be non-coding [ 48 ]. In contrast, the “coding region” (CR), completely sequenced years ago [ 49 ], has a remarkable conservation, in terms of both sequence and gene order among the trypanosomatids [ 19 , 22 , 25 ]. Figure 1 illustrates the absolute conservation in gene order (and strain polarity) in the maxicircles of evolutionary distant species such as L. tarentolae , L. infantum , and T. cruzi.…”

“…The kDNA has been proposed as a discrete target for phylogenetic analyses [ 18 , 19 ] because this DNA is uniparentally inherited [ 20 ], and is not affected by polymorphisms and recombination events occurring in the Leishmania inter-species/-strain hybrids [ 21 ]. By comparison, whereas minicircles are heterogeneous in terms of both copy number and sequence, maxicircles represent reliable markers for phylogenetic analyses [ 19 , 22 ]. Maxicircles contain two different regions: the coding region (CR) and the divergent region (DR).…”

“…Maxicircles contain two different regions: the coding region (CR) and the divergent region (DR). The low sequence complexity and repeated nature of the DR have been insurmountable hurdles to determining its complete sequence and length until the use of long-range PCR [ 23 ], or a combination of short and long reads from massive sequence technologies [ 22 , 24 ]. However, the gene order (synteny) in the maxicircle CR is highly conserved among the trypanosomatids [ 19 , 23 ], a feature that enables maxicircles to be reliable markers to determine evolutionary relationships among members of this order of organisms [ 25 ].…”

“…In previous work, we described a pipeline designed to assemble the complete maxicircle sequence using next genome sequencing (NGS) reads, and the maxicircles for a few Leishmania species were determined [ 19 ]. Subsequently, the methodology has been used to determine the maxicircle sequences of several L. infantum strains [ 35 ] and the T. cruzi maxicircle sequence [ 22 ]. Here, we assembled a collection of maxicircle sequences using publicly available WGS data, and generated a dataset consisting of 26 sequences that may be useful to conduct phylogenetic studies in trypanosomatids.…”

Genomics of Trypanosomatidae: Where We Stand and What Needs to Be Done?

“…Their analyses revealed novel non-canonical mechanisms, as well as species-specific differences in RNA editing [142,143]. Such studies can delineate not only the structure of maxicircles and minicircles [144][145][146][147], but also predict the guide RNA repertoire in a given species [143,148,149]. As judged from pre-genomic studies carried out on single genes, different lineages of trypanosomatids possess distinct kDNA editing patterns [150].…”