Bi-parental inheritance of kinetoplast DNA following sexual reproduction maintains mitochondrial genome complexity in Trypanosoma and Leishmania parasites
Item statusRestricted Access
Embargo end date31/07/2022
Kinetoplastids are a group of unicellular, protozoan flagellates. Within this group are the Trypanosomatidae family, obligate parasites infecting a wide variety of invertebrates, vertebrates and plants. Of note are the genera Trypanosoma and Leishmania which include the causative agents of human diseases African sleeping sickness, Chagas disease and Leishmaniasis. These are classified as Neglected Tropical Diseases (NTDs) by the World Health Organisation (WHO) and predominantly affect people from low- and middle-income countries. Members of Trypanosoma are also significant parasites of wide range of economically relevant domestic animals, including cattle, sheep, pigs, horses, camels and water buffalo. As this parasitism involves a transition between mammalian and insect hosts, adaptations of these parasites to different environments can also answer key questions in the field of evolution. Curiously, trypanosomatids have a characteristic single mitochondrion within which the mitochondrial genome is condensed into a disk-like structure called the kinetoplast. This kinetoplast DNA (kDNA) consists of an interconnected network of large ~ 20-50 kb ‘maxicircles’ and small 400 bp - 2 kb ‘minicircles’. Maxicircles are the equivalent of mtDNA in other eukaryotes, and minicircles code for small RNAs which specify extensive and essential post-transcriptional editing of maxicircle-encoded mRNAs. Minicircles are heterogeneous and vary in size and gRNA number by species and strain. Minicircle sequences can be clustered into ‘classes’ based on sequence identity. In this work, we characterise two minicircle sequences as belonging to the same class if they are at least 95 % identical by alignment. Genetic exchange through putative sexual reproduction has been documented in Trypanosoma brucei and Leishmania spp. as early as 1980. This exchange has been demonstrated to be a non-obligatory part of the life cycle, occur in the insect vectors and consistent with meiosis with the observation of putative haploid gametes. Early studies using southern blotting and restriction digests suggested bi-parental inheritance of both minicircles and maxicircles, with subsequent loss of one parental maxicircle from the population over time. To probe this, we have isolated, sequenced and assembled kDNA of parents and hybrid populations from previously characterised mating events of Trypanosoma brucei. In general, the hybrid populations we analysed contained maxicircle sequences from only one parental strain and the majority of minicircle classes from both parents. This corresponds to previous models suggesting that kinetoplast DNA is interchanged during mating and that one parent maxicircle is subsequently lost from the population while minicircle classes from both parents are retained. Despite very little overlap in minicircle classes between the parental strains, we find that gRNAs are highly redundant, and the same or similar gRNAs are commonly found in each parent. We show that interchange of minicircles during sexual reproduction increases gRNA redundancy in the hybrids. By utilising publicly available whole-genome sequencing for kDNA assembly, we have further corroborated our findings in Leishmania. In all but 2 of 51 experimental intra- and inter-species hybrids of L. major, L. infantum and L. tropica, we show bi-parental inheritance of kDNA minicircles. Again, most hybrids had maxicircle sequences corresponding to only one parent. We also analysed naturally derived hybrids of L. donovani strains. Our findings further show hybrids had one parental maxicircle and minicircle classes from both parent lineages. This demonstrates that genetic interchange of kDNA during sexual reproduction also occurs within natural populations. We also investigated the kDNA of two T. brucei subspecies, T. b. equiperdum and T. b. evansi, which have previously been determined to have either partial or total loss of their kDNA. These subspecies are restricted to the mammalian bloodstream life cycle stage, where kDNA loss can be compensated for by a number of known mutations. This was done with both publicly available whole-genome sequencing and sequencing of isolated kDNA. Consistent with previous studies, we found maxicircle DNA to be present only in T. b. equiperdum samples and either one minicircle class or a complete absence of minicircle DNA in one strain of T. b. equiperdum and four strains of T. b. evansi. Surprisingly, we found over 40 minicircle classes in each of three T. b. equiperdum strains. These minicircle classes were enriched for A6 and RPS12 gRNAs, the only two edited kDNA genes which are thought to be essential for bloodstream survival. This suggests that kDNA maintenance and RNA editing may still be essential in these strains of T. b. equiperdum. As these subspecies have lost the ability to colonise the tsetse salivary gland they are hence unable to undergo genetic exchange. The loss of non-essential minicircle classes in these asexual subspecies further complements the hypothesis that sexual reproduction is important for maintenance of minicircle complexity. Overall, our findings suggest that kDNA is almost always bi-parentally inherited following sexual reproduction in Trypanosoma brucei and Leishmania. This leads to an increase in gRNA redundancy and may compensate for previously observed loss of minicircle classes during asexual blood-stage growth. This may be a key evolutionary adaptation in order to prevent the loss of genes which are essential in only part of the life cycle.