The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis.

Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists. Author Summary: Leishmania are protozoan parasites that infect humans and replicate intracellularly in macrophages, cells normally engaged in protecting the host from pathogens. These parasites have several strategies to survive inside the hostile environment of the host macrophage, and one of these strategies involves heme acquisition. Heme is an iron-containing molecule that is essential for many cellular functions. Unlike mammalian cells, Leishmania parasites cannot synthesize heme, so must acquire it from the host cell. In earlier work we found that the parasites express a surface protein, LHR1, which transports heme into the parasites. In this study we identified specific amino acids in LHR1 that are required for heme transport. When expressed in yeast cells, LHR1 carrying these mutations had defects in heme transport that were equivalent to the inhibition in virulence observed when these proteins were expressed in Leishmania and tested in macrophage and mouse infection assays. These critical amino acids do not exist in the human heme transporter, indicating that LHR1 is a promising target for the development of specific drugs for the treatment of leishmaniasis and possibly other serious parasitic diseases, such as Chagas' disease and sleeping sickness. [ABSTRACT FROM AUTHOR]