Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent

Abstract

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (Sb^III) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline Sb^III susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to Sb^III resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to Sb^III resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of Sb^III The main driver of this preadaptation was shown to be MRPA, a gene involved in Sb^III sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE The "antibiotic resistance crisis" is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

Keywords: Leishmania; antimonials; drug resistance mechanisms; genomics; metabolomics.

FIG 1

Sb^III susceptibility of promastigotes from ISC1 and core group (CG) strains. The three ISC1 strains (red) and the seven strains of CG (blue) are representative of the overall genetic diversity previously described in reference . Sb^III susceptibility for each strain was determined by three independent experiments using potassium antimony tartrate as an Sb^III source. *, P < 0.05 by ANOVA.

FIG 2

Dynamics of Sb^III resistance selection. (A and B) Flowchart of Sb^III resistance selection for CG strains BPK282 and BPK275 (A) and ISC1 strain BPK026 (B). Boxes indicate the increasing Sb^III concentrations (6 to 382 µM), and black lines are for the stepwise selection (SW) and blue lines for the flash selection (FS) of the different replicates (populations A to D). “5P” represents 5 in vitro passages. Red crosses show replicates that did not survive the selection pressure at a given drug concentration. Solid-line boxes represent samples analyzed at the genomic and metabolomics levels, and dashed-line boxes represent samples analyzed at the genomic level only. If no box is present, the samples were not analyzed. (C) Time to resistance in weeks for the 3 Sb^III stepwise-selected (SW [black lines]) and flash-selected (FS [blue lines]) strains.

FIG 3

Genomic analysis of the selected Sb^III-resistant strains. (A) Evolution of allele frequency over the Sb^III resistance selection process of BPK026 for a fusaric acid resistance protein-like protein (LdBPK_120011000-H1104T) and the intraflagellar transport protein (LdBPK_210017400-Δ1699). (B and C) Heat map representing the karyotype dynamics across the Sb^III resistance selection of BPK282 and BPK275. (D) High hierarchical representation of BPK026 karyotype evolution. Heat maps show median normalized read depths of chromosomes found within each cell population for each of the 36 chromosomes (y axis) and each sample (x axis), replicate populations called A, B, C, and D are shown for each selecting Sb^III concentration (from 6 to 382 µM), and the samples “WT” framed in a black box represent the parental Sb^III-sensitive population for each strain. The color key for panels B and C shows the normalized chromosome read depth (S). S ranges are as follows: monosomy, S < 1.5 (dark blue); disomy, 1.5 ≤ S < 2.5 (light blue); trisomy, 2.5 ≤ S < 3.5 (green); tetrasomy, 3.5 ≤ S < 4.5 (orange); pentasomy, 4.5 ≤ S < 5.5 (red). In panel D, the ranges are as follows: monosomy, S < 1.5 (dark blue); disomy, 1.5 ≤ S < 2.5 (blue); trisomy, 2.5 ≤ S < 3.5 (light blue); tetrasomy, 3.5 ≤ S < 4.5 (green); pentasomy, 4.5 ≤ S < 5.5 (yellow); hexasomic, 5.5 ≤ S < 6.5 (orange); heptasomic, 6.5 ≤ S < 7.5 (red). A black triangle in an upper right corner indicates a significant change in S value (>0.5, with a shift from one S range to another and a P value of ≤10⁻⁵) in comparison to the S value of the starting population (sample “0” framed in a black box). (E) The left panel shows the normalized read depth of chromosome 23 (trisomic) compared, as a control, to the normalized read depth of chromosome 27 (disomic) in the parental strain BPK026 (BPK026 WT), and the right panel shows the normalized read depth for these two chromosomes in BPK026 Sb^III-R. (F) Normalized read depth of the fragment of chromosome 23 harboring the H locus. The left panel displays the parental strains (BPK026 WT, BPK275 WT, and BPK282 WT), while the right panel shows Sb^III-R line B for the three isolates.

FIG 4

Importance of H and M loci for Sb^III resistance. (A) Expression levels of genes of the M locus (above) and H locus (below) in parental lines (BPK026 WT, BPK282 WT, and BPK275 WT) and one respective Sb^III-resistant population (BPK026 Sb^III-R pop B, BPK282 Sb^III-R pop B, and BPK275 Sb^III-R pop B) were measured by qRT-PCR. Data were first normalized to the stable SAT gene and LdBPK_240021200 genes and then rescaled to the levels of BPK026 WT. Data are means ± standard deviation (SD) from two independent experiments. (B) Sb^III susceptibility of the BPK026 WT and BPK026 WT transfected with the H or M locus or with the individual genes of these loci. Data are means from three independent experiments. *, P < 0.05 by ANOVA.

FIG 5

Metabolomics analysis of the selected Sb^III-resistant strains. (A) Principal-component analysis based on 300 metabolites and including the initial parental strains (WT) BPK026 (green), BPK275 (red), and BPK282 (blue), the final populations resistant to 382 µM Sb^III (BPK026 382B, BPK275 382B, -C, and -D, and BPK282 382B, -C, and -D) as well as BPK026 intermediates (12B and 24B, respectively, resistant to 12 and 24 µM Sb^III). (B) Venn diagrams showing metabolites significantly different between strains. (C) High hierarchical representation of amino acid abundance in the different strains, including the parental strains (BPK026 WT, BPK282 WT, and BPK275 WT) and the selected populations resistant to 382 µM Sb^III (BPK275 382B, -C, and -D, BPK026 382B, and BPK282 382B, -C, and -D).

FIG 6

Flash selection with 382 µM Sb^III on ISC1 and CG strains. Shown are growth curves over 5 weeks of three ISC1 strains (red) and seven CG strains (blue) in the presence of 382 µM Sb^III. Each point represents the average value of promastigote counts in three independent flasks.