Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1

Abstract

Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.

Figure 1. Identification and predicted topology of LHR1, a Leishmania transmembrane protein similar to the C. elegans heme importer HRG-4.

(A) Amino acid alignment of L. amazonensis LHR1 and C. elegans HRG-4 (ClustalW PAM 250 Lasergene MegAlign software) shows that LHR1 also has four predicted transmembrane domains (TMD 1–4, boxed regions). Identical and conserved amino acids are highlighted in black and gray, respectively, and a histidine shown to be important for heme uptake by CeHRG-4 is indicated by an asterisk. (B) Proposed membrane topology of Leishmania LHR1, illustrating the four transmembrane domains and the conserved histidine, based on the CeHRG-4 .

Figure 2. Heme deprivation increases LHR1 expression and promotes uptake of the heme analog ZnMP by L. amazonensis promastigotes.

(A) qPCR quantification of LHR1 transcripts relative to the reference gene GAPDH, in log phase L. amazonensis promastigotes grown in medium with or without heme for 15 h. The results represent the mean+/−standard deviation (SD) of three independent experiments. (B) Confocal fluorescence images of L. amazonensis promastigotes cultured for 15 h with or without heme, incubated with ZnMP for 3 or 6 h, and imaged live under identical conditions. ZnMP accumulation within the parasites is shown in red, and the viability dye FDA is shown in green. (C) Immunoblot of extracts of L. amazonensis promastigotes expressing LHR1 tagged or not with the 3xFLAG epitope, and probed with anti-FLAG M2 monoclonal antibodies. (D) Flow cytometry quantification of ZnMP uptake by promastigotes untransfected (top) or transfected with LHR1 (bottom), after 15 h pre-incubation in the presence or absence of heme, followed by uptake under the same conditions. The numbers indicate the percentage of cells with fluorescence levels above the gate value (vertical line on horizontal bar, determined from measurements on parasites not incubated with ZnMP – grey shaded profile).

Figure 3. Transfection with LHR1 increases the intracellular heme content of L. amazonensis promastigotes.

(A) Absorption spectra of the hemochrome content of lysates of increasing numbers of L. amazonensis promastigotes transfected with vector alone or with LHR1-3xFLAG, and grown in heme-containing medium. (B) Heme concentrations in (A) calculated based on the heme millimolar extinction coefficient of 20.7. The data represents the mean+/−SD of triplicate determinations. p = 0.199 (1×10⁹), ** p = 0.001 (2×10⁹), * p = 0.054 (4×10⁹) (two-tailed Student's t test).

Figure 4. GFP-LHR1 increases the heme content, and localizes to the plasma membrane and acidic endocytic compartments of promastigotes.

(A) Absorption spectra of the hemochrome content of lysates of L. amazonensis promastigotes (4×10⁹) transfected with vector alone (GFP or 3xFLAG) or LHR1 (GFP-LHR1 or LHR1-3xFLAG), and grown in heme-containing medium. (B) Heme concentrations in (A) calculated based on the heme milimolar extinction coefficient of 20.7. Data are represented as the mean ± standard error of three independent experiments. * p = 0.020 (GFP-LHR1 vs. GFP), p = 0.031 (LHR1-3xFLAG vs. 3xFLAG) (two-tailed Student's t test). (C) Spinning disk confocal microscopy images of live L. amazonensis promastigotes transfected with GFP vector alone or GFP-LHR1 and grown in heme-deficient medium. GFP-LHR1 is localized on the plasma membrane of promastigotes (arrowheads) and in intracellular compartments that colocalize with lysotracker (arrows). Bars = 3 µm.

Figure 5. LHR1 localizes to the plasma membrane and late endosomes in intracellular amastigotes and in mammalian cells.

(A) Confocal fluorescence images of bone marrow macrophages infected with axenic amastigotes transfected with GFP vector alone or GFP-LHR1, and incubated for 48 h. Green, GFP or GFP-LHR1; Red, propidium iodide DNA stain. LHR1 is localized on the plasma membrane of intracellular amastigotes (arrows) and in a large intracellular compartment. Bars = 10 µm. (B) Confocal fluorescence imaging of HeLa cells transfected with GFP-LHR1 and incubated for 1 h with Texas Red dextran followed by a 2 h chase to label lysosomes. GFP-LHR1 (green) is localized on the plasma membrane (arrows) and in dextran-containing lysosomes (red). Bars = 10 µm.

Figure 6. LHR1 rescues heme deficiency phenotypes in hem1Δ S. cerevisiae.

(A) Spot growth assay of the heme biosynthesis deficient yeast strain hem1Δ in heme limiting conditions. hem1Δ were transformed with pYes-DEST52 vector alone, CeHRG-4 or LHR1, spotted in serial dilutions on plates supplemented with indicated concentration of hemin, and incubated at 30°C for 3 days. (B) β-galactosidase activity in the hem1Δ strain co-transformed with pCYC1-LacZ and empty vector pYes-DEST52, CeHRG-4 or LHR1, determined after cultivation with the indicated concentrations of hemin for 12 h. The results represent the mean+/−standard error of the mean (SEM) from three independent experiments. *** p<0.001 (Student's t test). (C) Epifluorescence images of hem1Δ yeast transformed with pYes-DEST52 vector alone, CeHRG-4-HA or LHR1-HA and subjected to immunofluorescence with anti-HA rabbit antibodies. Bar = 5 µm.

Figure 7. LHR1 promotes heme uptake in S. cerevisiae.

Wild-type yeast transformed with yeast-optimized LHR1 (yLHR1) or the empty vector (vector) were grown in SC-Ura, 2% raffinose, 0.4% galactose medium and uptake of [⁵⁵Fe] hemin was measured. Assays were performed with [⁵⁵Fe] hemin at 1.2 µM and uptake was measured for the indicated number of minutes at 30°C. Assays were performed in triplicate and the experiment was replicated twice. The data represents the mean+/−SD of triplicate determinations.

Figure 8. LHR1 is required for maintaining heme homeostasis in L. amazonensis.

(A) Southern blot of genomic DNA from wild type (LHR1/LHR1) and heterozygous (LHR1/Δlhr1) promastigotes digested with XhoI and hybridized with LHR1 or HYG ORFs. Two independent hygromycin B resistant clones were used for genomic DNA isolation (LHR1/Δlhr1 C#1 and C#2). (B) Growth curves of LHR1/LHR1 and LHR1/Δlhr1 promastigotes expressing or not episomal LHR1-3xFLAG in promastigote growth medium containing hemin. (C) Flow cytometry quantification of ZnMP uptake by LHR1/LHR1 and LHR1/Δlhr1 promastigotes kept in heme-deficient medium during the 15 h pre-incubation and the 3 h assay. The numbers indicate the percentage of cells with fluorescence levels above the gate value (vertical line on horizontal bar, determined from measurements on parasites not incubated with ZnMP – grey shaded profile). (D) Absorption spectra of the hemochrome content of lysates of 4×10⁹ LHR1/LHR1 (red), LHR1/Δlhr1 expressing LHR1-3xFLAG (blue) or LHR1/Δlhr1 (green) L. amazonensis promastigotes grown in heme-containing medium. (E) Heme concentrations in (D) calculated based on the heme millimolar extinction coefficient of 20.7. The results correspond to the mean+/−SD of triplicate determinations. ** p<0.0004; * p<0.022 (two-tailed Student's t test).