The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, 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 the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples

A Screen against Leishmania Intracellular Amastigotes: Comparison to a Promastigote Screen and Identification of a Host Cell-Specific Hit

The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect–infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy

Treatment of infected THP-1 with DMSO and amphotericin B.

<p>A. Number of infected THP-1 counted per well treated or not with 1% DMSO or 2 µM amphotericin B. B. Number of parasites counted per well divided by the number of host nuclei per field. C. Dose response curve for amphotericin B plotting the percentage of parasite growth inhibition. Values are mean from at least 3 independent experiments.</p

Number of hits identified with the intracellular amastigote and the promastigote primary screens.

<p>White bar: number of compounds identified in both screens. Light grey and black bars: number of compounds specifically active against promastigotes and intracellular amastigotes respectively. Hatched bar: number of compounds active against the promastigote stage but determined as toxic against THP-1 host cell in the intracellular amastigote screen.</p

Structure and activity of naloxonazine and naloxone.

<p>Lower panels: Dose response curve for naloxonazine (left) and naloxone (right) against intracellular amastigotes (black diamonds), promastigotes (black squares), axenic amastigotes (white diamonds) and THP-1 (white triangles) plotting the percentage of parasite growth inhibition.</p

Infection of THP-1 with <i>L. donovani</i>: detection, segmentation and growth of host cell and parasite.

<p>A–D. Detection and segmentation of THP-1 host cell and <i>L. donovani</i> intracellular amastigotes. Images obtained with the INCell Analyzer 1000 (20X) of THP-1 cells infected with <i>L. donovani</i> and treated with 1% DMSO (A, B) or 2 µM amphotericin B (C, D). Insert shows the relative fluorescence of DAPI-stained parasite kinetoplast (k) and nucleic DNA (n) and host cell nucleus (N). Segmentation of host cell nuclei and parasite kinetoplast using INCell developer toolbox software (B, D). Red outline: parasite kinetoplast, blue outlines: host cell nucleus and border representing the boundary of the host cell. E. Evolution of the number of parasites and THP-1 host cells in a 72 h time course. THP-1 and <i>L. donovani</i> were counted at several time points after infection using the INCell 1000. White squares: average number of host nuclei per well (n = 8); black circles: average number of parasites counted per well divided by the total number of host nuclei per well (n = 8).</p

Available therapeutics for HAT.

<p>Available therapeutics for HAT.</p

Live trypanosome fluorescence imaging.

<p>Diffuse cytoplasmic fluorescent signal of fluorophore conjugate 30, (a) indicating the uptake and accumulation of the compound into the cells. No fluorescence was detected in parasites treated with unlabeled compound <b>1</b> (b) or with free coumarin dye <b>31</b> (c).</p