The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in <i>Leishmania amazonensis - Table 3 </i>

<p>The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in <i>Leishmania amazonensis - Table 3 </i></p

Tyr-18, Tyr-80 and Tyr-129 differentially regulate LHR1-mediated uptake of a toxic heme analog.

<p><i>S</i>. <i>cerevisiae</i> W303 expressing vector alone or yeast codon-optimized LHR1-HA WT, Y18A, H36A, Y80A or Y129A were serially diluted and spotted onto agar plates with a range of GaPPIX concentrations, incubated at 30°C for three days and imaged. Images are representative of two experiments using independent transformants.</p

Tyrosine mutations resulting in decreased heme uptake cause equivalent defects in <i>L</i>. <i>amazonensis</i> intracellular replication.

<p><b>A</b>) <i>L</i>. <i>amazonensis</i> Δ<i>lhr1</i>/<i>LHR1</i> (SKO) amastigotes expressing WT, Y18A, H36A, Y80A, or Y129A <i>LHR1</i>-HA constructs were used to infect mouse BMMs, and intracellular growth was determined microscopically at different time points after infection. The assay was performed in triplicate, and results are representative of three independent experiments. Student’s <i>t</i>-test values comparing 72 h time points: WT vs. SKO = 0.005, WT vs. Y18A = 0.004, WT vs. H36A = 0.06, WT vs. Y80A = 0.01, WT vs. Y129A = 0.04; Student’s <i>t</i>-test comparing 36 h time point: WT vs. Y18A = 0.03. *** p ≤ 0.005, ** p ≤ 0.01, * p ≤ 0.05. <b>B</b>) Deconvolution fluorescence images of infected macrophages showing the localization of wild type and mutant LHR1-HA proteins and the ER marker BiP at 24h post-infection. The arrows point to HA-tagged proteins localized on the plasma membrane of intracellular amastigotes. Images were acquired under similar conditions. Blue = DAPI, red = anti-BiP, green = anti-HA. Scale bar = 6 μm.</p

Amino acid residues targeted for mutagenesis in <i>L</i>. <i>amazonensis</i> LHR1.

<p><b>A</b>) Alignment of <i>C</i>. <i>elegans</i> CeHRG-4 with <i>L</i>. <i>amazonensis</i> (LmxA) LHR1 and LHR1 homologues from <i>L</i>. <i>major</i> (LmjF) and <i>L</i>. <i>infantum</i> (LinJ) highlighting the residues selected for mutation analysis. Light blue indicates residues found to be involved in heme uptake by <i>C</i>. <i>elegans</i> HRG-4; orange indicates residues selected for mutagenesis in <i>L</i>. <i>amazonensis</i> LHR1; light gray boxes indicate predicted transmembrane domains. <b>B</b>) Predicted structure of LHR1 with amino acids selected for mutation highlighted. Red = tyrosine; purple = histidine; yellow = arginine; light purple = cysteine.</p

Mutation of Tyr-18, Tyr-80 and Tyr-129 causes varying growth inhibition levels in yeast respiration assays.

<p><b>A</b>) Western blot of <i>S</i>. <i>cerevisiae</i> Δ<i>hem1</i> (6D) expressing vector alone or yeast codon-optimized LHR1-HA WT, Y18A, H36A, Y80A or Y129A. <b>B</b>) Deconvolution fluorescence images of <i>S</i>. <i>cerevisiae</i> Δ<i>hem1</i> (6D) expressing vector alone or yeast codon-optimized WT or mutant LHR1-HA proteins. The arrows point to the yeast cell plasma membrane. Bar = 2 μm. <b>C</b>) <i>S</i>. <i>cerevisiae</i> Δ<i>hem1</i> (6D) expressing vector alone or yeast codon-optimized WT or mutant LHR1-HA proteins were cultivated for 18 h in glycerol/lactate medium, serially diluted and spotted onto agar plates containing glycerol/lactate and supplemented with either 250 μM ALA (positive control) or a range of heme concentrations, incubated at 30°C for four days and imaged. Images are representative of two experiments using independent transformants.</p

LHR1-mediated growth rescue of a yeast strain defective in heme biosynthesis depends on tyrosine residues.

<p><b>A</b>) <i>S</i>. <i>cerevisiae</i> Δ<i>hem1</i> (6D) expressing vector alone or LHR1, WT or mutant proteins, were serially diluted and spotted onto agar plates containing a range of heme concentrations or 250 μM ALA (positive control), incubated at 30°C for four days and imaged. Images are representative of two to four experiments using independent transformants. <b>B</b>) Heme-dependent β-galactosidase production in <i>S</i>. <i>cerevisiae</i> Δ<i>hem1</i> (6D) expressing vector alone or LHR1, WT or mutant proteins, grown in 1 or 10 μM heme for 16 h. The results are representative of five-eight experiments performed with independent transformants. Student’s <i>t</i>-test: *** p ≤ 0.005, ** p ≤ 0.01, * p ≤ 0.05 compared to WT. <b>C</b>) Western blot of <i>S</i>. <i>cerevisiae</i> Δ<i>hem1</i> (6D) expressing vector alone or LHR1, WT or mutant proteins, grown in 10 μM heme.</p

LIR1 expressed in <i>Arabidopsis thaliana</i> is targeted to the plasma membrane and decreases the iron content of leaves.

<p>(a) Confocal images of root sections of <i>Arabidopsis thaliana</i> expressing LIR1 fused to GFP at the C-terminus. Red, cell wall/plasma membrane staining with propidium iodide (PI). Green, LIR1-GFP. Scale bars: 50 μm (upper panel) and 10 μm (lower panel). (b) ICP-MS quantification of the iron total content from leaves of wild type (WT), WT expressing LIR1, and <i>irt1-/- Arabidopsis thaliana</i>. A-C, independent <i>Arabidopsis</i> transgenic lines containing the 35S promoter-driven <i>LIR1</i> cDNA (35S::LIR1). D-F, independent transgenic lines of 35S::LIR1+GFP (35S promoter-driven LIR1 cDNA fused to GFP). The data represent the mean ± SD of 3 samples for each strain, normalized for tissue weight. (c) Phenotype of <i>irt1-/-</i> and <i>irt1-/-</i> expressing LIR1 (<i>irt1-/-</i> + LIR1) <i>A</i>. <i>thaliana</i> plants grown in soil irrigated with either water (Control) or 0.5 g/L Sequestrene (+ Iron) for 20, 23, 26 and 30 days post germination (dpg). (d) ICP-MS quantification of the iron total content from leaves of <i>irt1-/-</i> and <i>irt1-/-</i> expressing LIR1 (<i>irt1-/-</i> + LIR1) <i>A</i>. <i>thaliana</i> irrigated with either water (control) or 0.5 g/L Sequestrene (+ Iron). Plant tissue was collected 30 days post germination (dpg). The data represent the mean ± SD of 3 leave samples for each strain, normalized for tissue weight. * p = 0.0147 (<i>irt1-/-</i> + Iron vs. <i>irt1-/-</i> + LIR1 + Iron).</p

LIR1 deficiency markedly reduces <i>L</i>. <i>amazonensis</i> virulence.

<p>(a) Microscopic quantification of intracellular parasites in BMM infected with purified metacyclic forms of wild type (WT), <i>LIR1</i> double knockout (KO), <i>LIR1</i> single knockout (SKO) and add-back (AB) <i>L</i>. <i>amazonensis</i> (multiplicity of infection = 3) for 3, 24, 48, 72 and 96 h. The values represent the mean ± SD of triplicate determinations in one experiment that is representative of 3 independent experiments. * p = 0.031 (KO vs. AB 24 h), ** p = 0.0063 (KO vs. AB 72 h); p = 0.0019 (KO vs. AB 96 h); *** p = 0.0007 (KO vs. AB 48 h). (b) AlamarBlue fluorescence viability assay of 10⁶, 2x10⁶ and 4x10⁶ purified metacyclics purified from WT and KO promastigote cultures. The values represent the mean ± SD of 3 independent experiments. (c) Immunofluorescence deconvolution images of BMM infected for 3 h with WT and KO metacyclic promastigotes. Blue, DAPI DNA staining; green, lysosomes and PV membranes stained with anti-Lamp-1 antibodies. Scale bars = 11 μm. (d) Cutaneous lesion development in C57BL/6 mice inoculated with 10⁶ purified metacyclic forms of wild type (WT), <i>LIR1</i> double knockout (KO), <i>LIR1</i> single knockout (SKO) and add-back (AB) <i>L</i>. <i>amazonensis</i>. The data represent the mean ± SEM of measurements from 5 different mice in each group. (e) Parasite load in footpad tissues determined at 10.5 weeks after infection (n = 5).</p