Fig 1.
Leishmania sterol biosynthetic pathway.
Fig 2.
In vitro evolution of AmB resistance in L. donovani.
(A) Chemical structure of amphotericin B. (B) Schematic representation of the generation of AmB-resistant cell lines in L. donovani. Each passage of cells in culture (circles, lines 1–4) is indicated with cell lines 1–4 indicated in black, grey, blue, and red, respectively. (C) Dose-response EC50 values for AmB were determined for WT (white) and cloned resistant cell lines 1–4 (black, grey, blue, and red, respectively). These representative curves are the nonlinear fits of data using a two-parameter EC50 equation provided by GraFit. An EC50 value of 19 ± 2 nM was determined for AmB against WT promastigotes. EC50 values for resistant clones AmB R1–4 were 915 ± 118, 100 ± 8, 126 ± 0.8 and 55 ± 8 nM, respectively. These EC50 values represent one biological replicate, composed of two technical replicates. Collated datasets reporting the weighted mean ± SD of multiple biological replicates are summarised in Table 1.
Table 1.
Collated AmB EC50 values for WT, resistant and transgenic promastigote cell lines.
Fig 3.
Investigating the impact of SMT1 and SMT2 deletions on AmB susceptibility.
(A) Schematic representation of SMT1-related deletions identified in AmB R2–4 cell lines. The site of the single amino acid change between SMT1 (V indicating valine) and SMT2 (I indicating isoleucine) are shown. The site of the new stop codon in AmB R3 is denoted by an asterisk. (B) Dose-response curves for WT (white), AmB R3 (blue), AmB R3 plus SMT1WT add-back (green) and AmB R3 plus SMT2WT add-back (red) clones treated with AmB. EC50 values of 30 ± 1, 234 ± 36, 33 ± 2 and 31 ± 1 nM were determined for WT, AmB R3, AmB R3 plus SMT1WT add-back and AmB R3 plus SMT2WT add-back promastigotes, respectively. (C) Dose-response curves for WT (white), SMT1 SKO (black), SMT1 DKO (grey), SMT2 DKO (blue), and SMT1/2 DKO (red). EC50 values of 28 ± 1, 35 ± 1, 128 ± 6, 30 ± 0.6 and 149 ± 4 nM were determined for WT, SMT1 SKO, SMT1 DKO, SMT2 DKO, and SMT1/2 DKO promastigotes, respectively. These EC50 curves and values represent one biological replicate, composed of two technical replicates. Collated datasets reporting the weighted mean ± SD of multiple biological replicates are summarised in Table 1. (D) Plot of total SMT RNA versus level of AmB resistance, relative to WT.
Fig 4.
Investigating the impact of P450R1 functional loss on AmB susceptibility and sterol composition.
(A) Schematic representation of the homozygous 24-bp deletion within P450R1 in the AmB R1 cell line. (B) Dose–response curves for AmB R1 (grey), AmB R1 plus P450R1WT add-back (red) and AmB R1 plus P450R1Δ605–612 add-back (black) promastigote clones treated with AmB. EC50 values of 1530 ± 116, 34 ± 3.9, and 1450 ± 211 nM were determined for AmB R1, AmB R1 plus P450R1WT add-back, and AmB-R1 plus P450R1Δ605–612 addback, respectively. (C) Dose-response curves for WT (white), P450R1 DKO (blue), P450R1 DKO plus P450R1WT add-back (green), P450R1 DKO plus P450R1 Δ605–612 add-back (red) and P450R1Δ605–612 (grey) promastigotes treated with AmB. EC50 values of 22 ± 0.1, 2170 ± 204, 20 ± 0.7, 1990 ± 193 and 1550 ± 99 nM were determined for WT, P450R1 DKO, P450R1 DKO plus P450R1WT add-back, P450R1 DKO plus P450R1Δ605–612 add-back and P450R1Δ605–612 promastigotes, respectively. These EC50 curves and values represent one biological replicate, composed of two technical replicates. Collated datasets reporting the weighted mean ± SD of multiple biological replicates are summarised in Table 1. (D) Sterol profiling of WT and P450R1 mutant promastigotes. Values are the mean ± SD from biological replicates.
Fig 5.
Investigating the impact of P450R1 and CYP51 functional loss on ketoconazole susceptibility.
Dose–response curves for WT (white), CYP51 DKO (blue) and P450R1 DKO (red) promastigote clones treated with ketoconazole. EC50 values of 0.03 ± 0.01 (lower) and 10 ± 8 μM (upper) were determined for WT promastigotes while values of 11 ± 3 and 7 ± 1 μM were determined for P450R1 DKO and CYP51 DKO parasites, respectively. These EC50 curves and values represent one biological replicate, composed of two technical replicates. Collated datasets reporting the weighted mean ± SD of multiple biological replicates are summarised in S8 Table.
Fig 6.
AlphaFold model of LdP450R1 in closed conformation.
The FMN, FAD and NADPH binding domains are highlighted in blue, magenta and green, respectively. The N-terminal membrane attachment domain is highlighted in grey. FMN (orange), FAD (yellow) and NADPH (cyan) are shown in stick representation with binding modes modelled from the rat P450R structure (1J9Z.pdb). Helix 21 (H21), known to directly interact with partner CYPs is highlighted in yellow. Amino acids 605–612, deleted in our AmB R1, form part of helix 20 (H20). Deleted amino acids are highlighted in red.