Leishmania Mitochondrial Genomes: Maxicircle Structure and Heterogeneity of Minicircles

Abstract

The mitochondrial DNA (mtDNA), which is present in almost all eukaryotic organisms, is a useful marker for phylogenetic studies due to its relative high conservation and its inheritance manner. In Leishmania and other trypanosomatids, the mtDNA (also referred to as kinetoplast DNA or kDNA) is composed of thousands of minicircles and a few maxicircles, catenated together into a complex network. Maxicircles are functionally similar to other eukaryotic mtDNAs, whereas minicircles are involved in RNA editing of some maxicircle-encoded transcripts. Next-generation sequencing (NGS) is increasingly used for assembling nuclear genomes and, currently, a large number of genomic sequences are available. However, most of the time, the mitochondrial genome is ignored in the genome assembly processes. The aim of this study was to develop a pipeline to assemble Leishmania minicircles and maxicircle DNA molecules, exploiting the raw data generated in the NGS projects. As a result, the maxicircle molecules and the plethora of minicircle classes for Leishmania major, Leishmania infantum and Leishmania braziliensis have been characterized. We have observed that whereas the heterogeneity of minicircle sequences existing in a single cell hampers their use for Leishmania typing and classification, maxicircles emerge as an extremely robust genetic marker for taxonomic studies within the clade of kinetoplastids.

Common Structural Patterns in the Maxicircle Divergent Region of Trypanosomatidae

“…It has been also proposed that there can be different structural classes of maxicircles within the same pool of kDNA [10,18,34], but our findings can neither confirm, nor disprove this. In most cases we might have insufficient coverage to detect alternative maxicircle variants that can be present, taking into account that the proportion of alternative variant can be as low as 2%.…”

“…The shortest sequence in this study of 23,201 bp belongs to Trypanosoma brucei (Lister 427), while the longest one of 47,384 bp is from T. cruzi (Dm28c). Table 1 demonstrates that size variation of maxicircles primarily reflects the differences in length of the DR, which is consistent with previous observations [18,19,21]. The CRs of all assembled maxicircles are similar in length and exhibit a high level of synteny.…”

“…Maxicircles are functional equivalents of mitochondrial genome of other eukaryotes. Mitochondrial genes are compactly located in the so-called coding region (CR) of a maxicircle, which is typically about 16 kbps, and demonstrate high level of synteny between species [18][19][20][21]. The rest of maxicircle's molecule is termed the divergent region (DR), sometimes also called the variable region [22][23][24][25].…”

“…With the development of sequencing technologies some maxicircles were sequenced completely [12,[30][31][32]. However, even paired-end Illumina sequencing was often not sufficient to assemble the complete maxicircle, and, because of that, most studies were focused only on coding region with short flanks [11,13,18], and even the most recent assembly from Illumina data had recovered only 3.5 kb of DR (as in [32]). Analysis of 3.5-kb and 5-kb regions of DR of Trypanosoma lewisi, flanking 12S and ND5 genes, respectively, revealed that these loci have different patterns of repeats [31].…”

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations

“…Similar findings were found by Rosenzweig and collaborators in the L. donovani promastigote proteome [20]. Several proteins of the electron transport chain are encoded by the kinetoplast DNA maxicircles [49] such as cytochrome oxidase subunits and NADH dehydrogenase, but they were not searched in this study.…”