Article Figures & Data
Figures
- FIG 1
Validation of LinARM58. (A) Putative domain structure of LinARM58 and LinARM58rel. The boxes represent 4 putative domains of unknown function (DUF1935) for each protein. TMD, transmembrane domain; insertion, 31-amino-acid sequence absent from ARM58rel. The numbers below the structures show the positions within the amino acid sequence. (B) Dose-inhibition experiment for growth of L. infantum carrying the indicated transgenes at the indicated antimonyl tartrate (SbIII) concentrations. Growth over 72 h was normalized to that of the 0 μM samples (100%). The dotted lines indicate the respective IC50s. **, P < 0.01; ***, P < 0.001 (n = 8). (C) As for panel B, but with L. donovani 1SR as the acceptor strain. (D) In vitro infection of bone marrow-derived macrophages and treatment with sodium stibogluconate. L. infantum transfected with vector or with the ARM58 transgene was used to infect BMMs at a 10:1 ratio. After 4 h, extracellular parasites were removed. Sodium stibogluconate was added to the cultures at 160 μg/ml, and incubation was continued for 72 h. After DAPI staining, infection rates relative to the untreated control infections (data not shown) were determined by fluorescence microscopy (10 experiments with 100 macrophages each). The horizontal bars depict the median values. **, P < 0.01. (E) As for panel D, except that L. donovani 1SR served as the acceptor strain. *, P < 0.05 (n = 6). (F) As for panel B, except that parasites were treated with the indicated concentrations of miltefosine. (G) As for panel B, except that parasites were treated with the indicated concentrations of pentamidine.
- FIG 2
(A) Schematic depiction of ARM58 deletion constructs used for panels B and C. The shaded boxes represent the four DUF1935 putative domains. The black box signifies the putative transmembrane domain (TMD). The numbers indicate the positions relative to the wild-type amino acid sequence; insertion, 31-amino-acid sequence absent from ARM58rel. (B) SbIII IC50s (μM) for L. infantum carrying the indicated ARM58 variants. *, P < 0.05 (n = 4) relative to full-length ARM58. (C) As for panel B, but with further shortened ARM58 variants (n = 4).
- FIG 3
(A) Schematic depiction of mutated ARM58 constructs used for panel B. The shaded boxes represent the 4 DUF1935 putative domains. The black box signifies the putative transmembrane domain (TMD). The numbers indicate the positions relative to the wild-type amino acid sequence. (B) SbIII IC50s (μM) for L. infantum carrying the indicated ARM58 mutants. *, P < 0.05 (n = 4) relative to full-length ARM58. (C) Schematic depiction of ARM58rel, ARM58, and two DUF1935-III domain swapping mutants, ARM58_3Drel and ARM58rel_D3. (D) As for panel B, but testing domain swapping mutants of ARM58 and AMR58rel (n = 4).
- FIG 4
Subcellular localization of mCH::ARM58 fusion proteins. (A) Schematic depiction of the episomal expression plasmid pCL2N-mCH::ARM58. (B) Functional test of mCherry::ARM58 fusion protein. L. infantum overexpressing either ARM58 (solid squares) or mCH::ARM58 (diamonds) was subjected to a 72-h dose effect growth experiment with various SbIII concentrations and compared to a control strain transfected with pCLN (vector). Dotted lines show IC50s; error bars show standard deviations (n = 4). (C to F) Fluorescence microscopy imaging of L. infantum(pCL2N-mCH::ARM58) showing differential inference contrast (DIC) (C), DAPI (D), and mCherry (E) channels plus overlay of DAPI and mCherry (F). n, nucleus; k, kinetoplast. (G to K) Confocal laser microscopy imaging of L. infantum(pCL2N-mCH::ARM58) showing DIC (G), DAPI (H), and mCherry (I) channels plus overlay of DAPI and mCherry (K). n, nucleus; k, kinetoplast. Size bar, 5 μm.
- FIG 5
Intracellular Sb concentration following SbIII exposure. L. infantum(pCLN) and L. infantum(pCLN-ARM58) were incubated for 48 h with 0 μM or at 400 μM SbIII. Cells were then counted (A). From each culture, 5 × 107 promastigotes were precipitated, washed, and lysed in nitric acid. Sb concentration in each lysate was measured by inductively coupled plasma mass spectrometry (ICPMS) (n = 3) (B). (C and D) Induction of SbIII resistance in strains receiving 10 μM verapamil (C) or 50 μM sodium vanadate (D). Vector controls and ARM58 overexpressing L. infantum were grown for 72 h. Cell densities were measured and plotted against the indicated SbIII concentrations. Values are the medians from 4 independent experiments. V, verapamil; SV, sodium vanadate.
