Discussion
Trypanosomatids are unicellular, flagellated eukaryotes that cause a number of diseases in humans and livestock. These parasites are all transmitted by different insect vectors and also renowned for their extraordinarily massive and complex mitochondrial DNA, the kinetoplast. The kinetoplast DNA (kDNA) in trypanosomatids forms a chainmail-like network that contains two types of interlinked DNA molecules: 20 to 50 copies of identical maxicircles and thousands of highly heterogeneous minicircles. Maxicircle genes encode subunits of the mito-ribosome, the electron transport chain and the F
OF
1-ATP synthase. The pre-mRNAs of several maxicircle genes (twelve in
Trypanosoma brucei) require post-transcriptional editing directed by short “guide RNAs” (gRNAs) encoded on minicircles. The plentitude of editing sites entails that a diverse population of minicircles is necessary for editing all maxicircle-encoded mRNAs.
The lifecycle of several trypanosomatid parasites involves developments in insect vectors and mammalian hosts. As typically not all maxicircle genes are needed in the mammalian stage, the selective pressure on kDNA relaxes temporarily and allows minicircle populations to change. During cell division, imperfect replication and segregation of kDNA result in fluctuation in the minicircle populations, and copy number of insect-stage specific gRNA genes may randomly drift towards a dangerous low level approaching elimination. Loss of essential gRNAs may render the parasites incapable of establishing themselves in the insect vector and deprived of the opportunity of transmission.
We propose that sexual reproduction is key in countering random genetic drift in kDNA. For trypanosomatids such as
T. brucei,
T. cruzi and
Leishmania spp, it has been shown that sexual reproduction happens exclusively in the insect vector and results in completing mixing of the mitochondrial genome in the progeny. Hence, sexual reproduction reshuffles minicircles among insect-transmissible isolates. The circulation potentially rescues underrepresented gRNA genes by replenishing it with copies from another parental cell line in which the gRNA gene is more abundant. Hence, sexual reproduction lowers the risk of losing the ability to express insect stage-specific genes after generations of clonal reproduction in mammalian host.
In support of this concept, we demonstrate that absence of sexual reproduction has profound impacts on kDNA of tsetse-transmissible and tsetse-independent subspecies (or ecotypes) of
T. brucei. We sequenced, assembled and compared kDNA genomes from 262
T. brucei isolates of diverse geographical origin. Compared to the highly complex kDNA in
T. brucei subspecies capable of sexual recombination (i.e.
T. b. brucei,
T. b. rhodesiense and
T. b. gambiense type 2), we observed different degrees of reduction in kDNA complexity in the asexual subspecies. We confirmed that in three groups of kDNA independent
T. b. evansi and
T. b .equiperdum, the minicircle genomes consist of thousands of a single minicircle class specific and therefore diagnostic of each group. Unexpected for a putatively kDNA independent subspecies,
T. b. equiperdum group OVI retains a minicircle population with moderate complexity and is potentially capable of generating fully edited mRNAs of A6 and RPS12, the only edited maxicircle genes required in the mammalian host. Further, we report a highly streamlined and conserved minicircle population characteristic of
T. b. gambiense type 1 isolates. The significantly lower gRNA coverage in
T. b. gambiense type I suggests that only a minor fraction of cells within each population still retain the ability to survive in the tsetse vector, which may help
