Novel insertions in the mitochondrial maxicircle of Trypanosoma musculi, a mouse trypanosome

Abstract

Trypanosoma musculi is a, globally distributed, mouse-specific haemoflagellate, of the family Trypanosomatidae, which shares similar characteristics in morphology with Trypanosoma lewisi. The kinetoplast (mitochondrial) DNA of Trypanosomatidae flagellates is comprised of catenated maxicircles and minicircles. However, genetic information on the T. musculi kinetoplast remains largely unknown. In this study, the T. musculi maxicircle genome was completely assembled, with PacBio and Illumina sequencing, and the size was confirmed at 34 606 bp. It consisted of 2 distinct parts: the coding region and the divergent regions (DRs, DRI and II). In comparison with other trypanosome maxicircles (Trypanosoma brucei, Trypanosoma cruzi and T. lewisi), the T. musculi maxicircle has a syntenic distribution of genes and shares 73.9, 78.0 and 92.7% sequence identity, respectively, over the whole coding region. Moreover, novel insertions in MURF2 (630 bp) and in ND5 (1278 bp) were found, respectively, which are homologous to minicircles. These findings support an evolutionary scenario similar to the one proposed for insertions in Trypanosoma cruzi, the pathogen of American trypanosomiasis. These novel insertions, together with a deletion (281 bp) in ND4, question the role of Complex I in T. musculi. A detailed analysis of DRII indicated that it contains numerous repeat motifs and palindromes, the latter of which are highly conservative and contain A₅C elements. The comprehensively annotated kinetoplast maxicircle of T. musculi reveals a high degree of similarity between this parasite and the maxicircle of T. lewisi and suggests that the DRII could be a valuable marker for distinguishing these evolutionarily related species.

Keywords: Divergent regions; Trypanosoma lewisi; Trypanosoma musculi; indels; maxicircle; minicircle; palindrome.

Graphical abstract

Fig. 1.

Morphology and ultrastructure of T. musculi. (A) Giemsa staining of the epimastigote form of T. musculi from in vitro culture (scale bar 5 μm). Flagellum (F), nucleus (N) and kinetoplast (K) are indicated. (B) Electron micrograph of the T. musculi trypomastigote form (scale bar 200 nm). Basal body (BB), flagellum (F), flagellar pocket (FP) and kinetoplast (K) are indicated. (C) Immunofluorescence analysis (IFA) shows epimastigote-like forms of T. musculi from in vitro cultivation with antibody L8C4 detecting paraflagellar rods (green) and DAPI detecting kinetoplast DNA and nuclear DNA (blue) (scale bars 5 μm).

Fig. 2.

Circos plot of the T. musculi maxicircle. The outer track indicates the gene arrangements and gene distribution; the middle track represents the GC content (orange for above-average and green for below-average) and the inner track is a histogram of assembly coverage.

Fig. 3.

Analysis of insertions or deletions in the T. musculi maxicircle genes MURF2, ND5 and ND4. (A) A diagram of insertions or deletions in the T. musculi maxicircle genes MURF2 (630 bp insertion), ND5 (1278 bp insertion) and ND4 (281 bp deletion). Insertion regions are labelled as grey boxes. (B) PCR amplification of the T. musculi MURF2, ND5 and ND4 genes from T. musculi Partinico II (1), T. musculi Particino 2 (2), T. musculi Lincicome (3), T. musculi CDC (4), and fragments are analysed on a 1.0% agarose gel. M, DL2000 marker (Takara, China). N, negative control. The positions of primers are shown in (A) and Table S1. (C) Alignment of the conserved regions from the T. lewisi minicircle (MN447335.1) and insertion sequences of MURF2 and ND5. Conserved sequence blocks (CSB-I, II, III) are highlighted in grey.

Fig. 4.

Phylogenetic analysis of the Trypanosomatidae species using the maxicircle coding regions. Phylogenetic tree is performed based on Maximum likelihood /Neighbour joining/Minimum evolution methods with 1000 bootstrap replicates with the respective bootstrap confidences indicated at branch points. Branch lengths are indicated by the black line and the scale bar represents the number of nucleotide substitutions per site.

Fig. 5.

Sequence analyses of the DR of the T. musculi maxicircle. (A) Dot-plot analysis of the T. musculi maxicircle, the main diagonal line represents the sequence's alignment with itself and the lines about the main diagonal represent repetitive patterns within the maxicircle sequence. (B) Circos plot of the DR of T. musculi maxicircle, the outer track indicates gene arrangement and gene distribution. Ribbons inside the circle connect homologous regions, colour represents per cent of sequence identity in the range (70%; 100%) in the order yellow, green and blue. Four palindromes are shown with red arrows. (C) The repetitive sequences from DR I are aligned and 2 motifs predicted by MEME are indicated with a black line. LOGO diagrams show nucleotides at a given position of each motif and relative frequency represented by height. (D) The palindromes from the DR of the Trypanosomatidae species are shown with the palindromic bases highlighted.