Functional characterization of LIT1, the Leishmania amazonensis ferrous iron transporter

Leishmania amazonensis LIT1 was identified based on homology with IRT1, a ferrous iron transporter from Arabidopsis thaliana. Δlit1L. amazonensis are defective in intracellular replication and lesion formation in vivo, a virulence phenotype attributed to defective intracellular iron acquisition. Here we functionally characterize LIT1, directly demonstrating that it functions as a ferrous iron membrane transporter from the ZIP family. Conserved residues in the predicted transmembrane domains II, IV, V and VII of LIT1 are essential for iron transport in yeast, including histidines that were proposed to function as metal ligands in ZIP transporters. LIT1 also contains two regions within the predicted intracellular loop that are not found in Arabidopsis IRT1. Deletion of region I inhibited LIT1 expression on the surface of Leishmania promastigotes. Deletion of region II did not interfere with LIT1 trafficking to the surface, but abolished its iron transport capacity when expressed in yeast. Mutagenesis revealed two motifs within region II, HGHQH and TPPRDM, that are independently required for iron transport by LIT1. D263 was identified as a key residue required for iron transport within the TPPRDM motif, while P260 and P261 were dispensable. Deletion of proline-rich regions within region I and between regions I and II did not affect iron transport in yeast, but in L. amazonensis were not able to rescue the intracellular growth of Δlit1 parasites, or their ability to form lesions in mice. These results are consistent with a potential role of the unique intracellular loop of LIT1 in intracellular regulation by Leishmania-specific factors.

Graphical abstractFunctional characterization of the Leishmania amazonensis iron transporter LIT1. The residues in red (H108, E112, D263, H283, S284, H309 and E313) are conserved between LIT1 and Arabidopsis IRT1, and were shown essential for iron acquisition when LIT1 is expressed in the iron transport-deficient Δfet3fet4Saccharomyces cerevisiae. In green (Y382, D263 and D391) are the amino acids essential for iron transport in LIT1, but not in IRT1. In orange (D148, D153, R157 P259, P260 and S319) are the residues not required for iron transport in LIT1. Blue residues outlined in red were designated as regions I and II of the LIT1 unique intracellular loop, and targeted for deletion in this study. HxHxH indicates the histidine-rich motif within region II found to be essential for iron transport.Figure optionsDownload full-size imageDownload high-quality image (192 K)Download as PowerPoint slide