Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence

The protozoan parasite Leishmania amazonensis is a heme auxotroph and must acquire this essential factor from the environment. Previous studies showed that L. amazonensis incorporates heme through the transmembrane protein LHR1 (Leishmania Heme Response 1). LHR1-null promastigotes were not viable, suggesting that the transporter is essential for survival. Here, we compared the growth, differentiation, and infectivity for macrophages and mice of wild-type, LHR1-single-knockout (LHR1/Δlhr1), and LHR1-complemented (LHR1/Δlhr1 plus LHR1) L. amazonensis strains. LHR1/Δlhr1 promastigotes replicated poorly in heme-deficient media and had lower intracellular heme content than wild-type parasites. LHR1/Δlhr1 promastigotes were also less effective in reducing ferric iron to ferrous iron, a reaction mediated by the heme-containing parasite enzyme LFR1 (Leishmania Ferric Reductase 1). LHR1/Δlhr1 parasites differentiated normally into aflagellated forms expressing amastigote-specific markers but were not able to replicate intracellularly after infecting macrophages. Importantly, the intracellular growth of LHR1/Δlhr1 amastigotes was fully restored when macrophages were allowed to phagocytose red blood cells prior to infection. LHR1/Δlhr1 parasites were also severely defective in the development of cutaneous lesions in mice. All phenotypes observed in LHR1/Δlhr1 L. amazonensis were rescued by expression of episomal LHR1. Our results reveal the importance of efficient heme uptake for L. amazonensis replication and vertebrate host infectivity, reinforcing the potential usefulness of LHR1 as a target for new antileishmanial drugs.