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Single cell genomics of Leishmania: tracking genetic diversity between and within hosts directly in clinical samples (SINGLE)

In a previous project (GeMInI), our research group performed pioneering work on the genome diversity of Leishmania donovani. Focusing on a cohort of clinical isolates from the Indian subcontinent, we described a large epidemic population, highly homogeneous from a sequence point of view (in average 173 SNPs/strain in 94 % of the strains), but highly heterogeneous in its genome structure, essentially in the form of massive aneuploidy, extra-chromosomal gene amplification (episomes) and expansion or contraction of tandem arrays. Experimental evolution studies (induction of drug resistance, long-term in vitro maintenance) showed that the consequent changes in gene dosage are rapid, probably adaptive and can even occur between different life stages. Noteworthy, previous studies were made with cultivated, extra-cellular life stages (promastigotes), with associated quantitative and qualitative biases. Quantitatively, knowing that isolation success from patients is usually less than 50% and more than 90% of L. donovani infections are asymptomatic and not accessible to isolation, only the ‘tip of the iceberg’ is currently accessed by population genomics. Qualitatively, the perception of gene dosage will likely be different if a parasite’s DNA is obtained after in vitro isolation and cultivation or directly from intracellular stages (amastigotes), in vivo. The latter is obviously more adequate when aiming to understand natural evolution, unravel traits of parasitism like virulence or drug resistance or assess the impact of gene dosage at transcription level. Considering (i) the potential functional importance and the flexibility of gene dosage at the genome level (aneuploidy and local CNV) in Leishmania and (ii) the current biases in the representation of the parasite population among sequenced isolates, it is essential to pursue further genome diversity studies on intracellular parasites, directly in the host’s tissues. In SINGLE, we aim to (i) develop and validate protocols for enrichment and sequencing of the whole L. donovani genome and transcriptome from pooled and single cells, isolated directly from human samples and (ii) apply these methods for characterising the inter- and intra-host parasite diversity, with a special emphasis on genome dynamics of gene dosage. In order to succeed in this endeavour, we will collaborate with experts in genome sequencing and single-cell genomics (KU-Leuven and Sanger Institute) and with a clinical team in Nepal (BPKIHS).
Datum:1 jul 2013  →  30 dec 2018