Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent

Abstract

Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG, both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1 versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the ISC, especially in a post-VL elimination context.

Keywords: Genome diversity; Genomics; Leishmania donovani; Metabolomics; Multi-omics integration; Virulence; Visceral leishmaniasis.

Fig. 1

Neighbour joining tree based on whole genome SNP loci. Bootstrap support was calculated on 1000 bootstrap replicates. LdonLV9 = East African L. donovani LV9, SRI_CLB = Sri Lankan L. donovani SRI_CLB.

Fig. 2

Somy levels in ISC1 and Core Group strains (CG). ISC1 strains are depicted in shades of red and yellow, while CG strains in shades of blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Principal component analysis of the metabolic profiles of the 5 Leishmania donovani ISC1 isolates and 4 CG strains in this study. This analysis was based on the quantitative measurements of all 290 putatively identified metabolites. In this PCA plot, average values are shown for each strain based on four biological replicates. For each principal component, the proportion of variation of the total dataset it explains is mentioned.

Fig. 4

The 40 differentially expressed glycerophospholipids (|Log₂FC| > 1 & p < 0.05) between ISC1 and CG parasites.

Fig. 5

The 3 key differences between ISC1 and CG that are shared between genome and metabolome. 1. Argininosuccinate (ASS) has a lower genomic copy number in ISC1 vs CG. More citrulline and less argininosuccinate were detected in ISC1, suggesting a corresponding downregulation of the ASS protein. Although the urea cycle might be incomplete in Leishmania, links have been established with Trypanothione biosynthesis and virulence. 2. Several glycerophospholipid (or precursor) biosynthetic enzymes had higher gene copy numbers in ISC1 which could be linked to increased GPL amounts (See also Fig. 4). 3. More copies of a p1/s1 nuclease gene were found in ISC1, associated with higher concentrations of several salvage pathway metabolites. Genomic copy number information is underlined and in bold. Pathway names are in italics. Arrows in grey represent potentially missing enzymes in the urea cycle of Leishmania. Blue = down in ISC1, Red = up in ISC1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)