Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance

Abstract

Visceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-quality reference genome sequence for a strain of L. donovani from Nepal, and use this sequence to study variation in a set of 16 related clinical lines, isolated from visceral leishmaniasis patients from the same region, which also differ in their response to in vitro drug susceptibility. We show that wholegenome sequence data reveals genetic structure within these lines not shown by multilocus typing, and suggests that drug resistance has emerged multiple times in this closely related set of lines. Sequence comparisons with other Leishmania species and analysis of single-nucleotide diversity within our sample showed evidence of selection acting in a range of surface-and transport-related genes, including genes associated with drug resistance. Against a background of relative genetic homogeneity, we found extensive variation in chromosome copy number between our lines. Other forms of structural variation were significantly associated with drug resistance, notably including gene dosage and the copy number of an experimentally verified circular episome present in all lines and described here for the first time. This study provides a basis for more powerful molecular profiling of visceral leishmaniasis, providing additional power to track the drug resistance and epidemiology of an important human pathogen.[Supplemental material is available for this article.]Leishmaniases are a complex of diseases that range from self-curing lesions to gross disfigurations and potentially deadly visceral disease. The diseases are caused by protozoan parasites that are transmitted by sandflies in 88 countries and infect an estimated 12 million people (www.who.int/leishmaniasis/en/). Parasites of the Leishmania genus are remarkably biologically, clinically, and epidemiologically diverse and present enormous differences in disease tropism. The mildest form is cutaneous leishmaniasis, which is caused by Leishmania major and other species, and is largely limited to lesions around the area of a sandfly bite-though a diffuse form can also occur. Disfiguring mucocutaneous leishmaniasis is due to the destruction of nasopharyngeal tissue by parasites such as L. braziliensis. More significantly, visceral leishmaniasis is caused by parasites of the L. donovani species complex that can spread to internal organs and cause death.In 2005, sequencing the genome of L. major identified 8311 protein-coding genes and provided a framework for future comparative genomic studies (Ivens et al. 2005). The genome elucidated the full structural architecture of Leishmania chromosomes, which includes an unusual pattern of genes distributed in large directional clusters. Subsequently, the genomes of L. braziliensis and L. infantum were described-the latter is a member of the L. donovani complex (Peacock et al. 2007). A detailed comparison of these first three Leishmania genomes re...

Adaptive mechanisms in pathogens: universal aneuploidy in Leishmania

“…Recent advances in next-generation sequencing technologies boosted this research and provided a reference genome for other Leishmania species: a Nepalese Leishmania donovani [16], a Leishmania mexicana strain from Guatemala [17], as well as an African lizard-infecting sample, Leishmania tarentolae [18]. Data for Leishmania panamensis and Leishmania tropica and close relatives of Leishmania, Endotrypanum and Crithidia, are also available (www.ncbi.nlm.nih.gov/sra; www.tritrypdb.org).…”

Antimony-resistant but not antimony-sensitiveLeishmania donovaniup-regulates host IL-10 to overexpress multidrug-resistant protein 1

“…Nevertheless, it is still used as a first-line treatment in Africa and Latin America (6). Interestingly, 78% of the recent clinical isolates from the hyperendemic zone of Bihar State still showed in vitro resistance to antimonials (7), which might be explained by an increased fitness of the corresponding parasites (8).Recently, we have demonstrated three unique characteristics of antimony-resistant LD (Sb R LD). First, it shows a higher concentration of terminal glycoconjugates (N-acetylgalactosamine residues) on its surface than antimony-sensitive LD (Sb S LD) (7).…”

“…Nevertheless, it is still used as a first-line treatment in Africa and Latin America (6). Interestingly, 78% of the recent clinical isolates from the hyperendemic zone of Bihar State still showed in vitro resistance to antimonials (7), which might be explained by an increased fitness of the corresponding parasites (8).…”

Constitutive aneuploidy and genomic instability in the single‐celled eukaryote Giardia intestinalis

“…2014), as has been documented in Leishmania populations, another parasitic protist with a constitutive aneuploidy (Downing et al. 2011; Rogers et al. 2011).…”