Analysis of the mitochondrial maxicircle of Trypanosoma lewisi, a neglected human pathogen

Abstract

Background: The haemoflagellate Trypanosoma lewisi is a kinetoplastid parasite which, as it has been recently reported to cause human disease, deserves increased attention. Characteristic features of all kinetoplastid flagellates are a uniquely structured mitochondrial DNA or kinetoplast, comprised of a network of catenated DNA circles, and RNA editing of mitochondrial transcripts. The aim of this study was to describe the kinetoplast DNA of T. lewisi.

Methods/results: In this study, purified kinetoplast DNA from T. lewisi was sequenced using high-throughput sequencing in combination with sequencing of PCR amplicons. This allowed the assembly of the T. lewisi kinetoplast maxicircle DNA, which is a homologue of the mitochondrial genome in other eukaryotes. The assembly of 23,745 bp comprises the non-coding and coding regions. Comparative analysis of the maxicircle sequence of T. lewisi with Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma brucei and Leishmania tarentolae revealed that it shares 78%, 77%, 74% and 66% sequence identity with these parasites, respectively. The high GC content in at least 9 maxicircle genes of T. lewisi (ATPase6; NADH dehydrogenase subunits ND3, ND7, ND8 and ND9; G-rich regions GR3 and GR4; cytochrome oxidase subunit COIII and ribosomal protein RPS12) implies that their products may be extensively edited. A detailed analysis of the non-coding region revealed that it contains numerous repeat motifs and palindromes.

Conclusions: We have sequenced and comprehensively annotated the kinetoplast maxicircle of T. lewisi. Our analysis reveals that T. lewisi is closely related to T. cruzi and T. brucei, and may share similar RNA editing patterns with them rather than with L. tarentolae. These findings provide novel insight into the biological features of this emerging human pathogen.

Fig. 1

Morphology and ultrastructure of T. lewisi. a Diff Quick staining of bloodstream forms of T. lewisi from rat. Flagellum (F), Nucleus (N) and Kinetoplast (K) are indicated. b Electron micrograph of T. lewisi trypomastigote form. Attachment Zone (AZ), Basic Body (BB), Mitochondrial Cristae (MC), Flagellum (F), Flagellar Pocket (FP) and Kinetoplast (K) are indicated

Fig. 2

Diagram of the T. lewisi maxicircle. The diagram is composed of fourloops, from inner to outer are assembly coverage, assembled contigs, PCR sequencing and geneorganization, respectively

Fig. 3

Sequence analyses of the T. lewisi maxicircle divergent region. Self Dottup graph of the T. lewisi entire maxicircle (a), DR section I (b), DR section II (d). Each dot represents an exact match over of 10 nt. Two distinct sections of DR region (I and II) are indicated in (a) with red boxes. The repeated elements (α, β, γ, α’ and β’) identified from Dottup graph in (d) are illustrated, three palindromes within elements are indicated with white triangles. c The repetitive sequences from the section I were aligned and the position of three motifs is indicated with black line. LOGO diagrams show nucleotides at a given position of each motif and their relative frequency indicated by height. e The palindrome sequences from the DR of T. lewisi, T. rangeli and T. cruzi are shown with the inverted repeats underlined

Fig. 4

Phylogenetic tree of maxicircle coding sequences from trypanosomatid species. Phylogenetic tree is performed based on Neighbor joining or Maximum likelihood methods with 1,000 bootstrap replicates. The scale bar represents the number of nucleotide substitutions per site. Partial T. congolense (9 kb) was retrieved from Tritrypdb by BLAST using other trypanosome maxicircle sequences