Abstract
While Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of Eukaryotic organisms, for many protozoan species its status remains elusive. In this work, we investigated the presence of C5 DNA methylation in Leishmania donovani, a Trypanosomatid parasite and the causative agent of the visceral leishmaniasis in humans. We show that the L. donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, of which the function is still unclear, and verified its expression at RNA level. We created a viable overexpressor and knock-out lines of this enzyme and characterised their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites and 0.0126% at CHH sites at genome scale. As none of these methylated sites were retained after manual verification, we concluded that there is no evidence for DNA methylation in this species. Similar results were obtained for the blood form of T. brucei, another Trypanosomatid species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation), can be used as the base of an enrichment strategy for parasite DNA. As such, we depleted methylated DNA from 1) mixes of Leishmania promastigote and amastigote DNA with human DNA and 2) THP-1 cells infected with Leishmania amastigotes. This resulted in 62x to 263x Leishmania:human enrichment, depending on the dilution and type of sample studied. These results open a promising avenue for including an unmethylated DNA enrichment step as a pre-enrichment before sequencing clinical samples.