Heme Uptake Mediated by LHR1 Is Essential for Leishmania amazonensis Virulence

Abstract

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/ ⌬lhr1), and LHR1-complemented (LHR1/⌬lhr1 plus LHR1) L. amazonensis strains. LHR1/⌬lhr1 promastigotes replicated poorly in heme-deficient media and had lower intracellular heme content than wild-type parasites. LHR1/⌬lhr1 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/⌬lhr1 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/⌬lhr1 amastigotes was fully restored when macrophages were allowed to phagocytose red blood cells prior to infection. LHR1/⌬lhr1 parasites were also severely defective in the development of cutaneous lesions in mice. All phenotypes observed in LHR1/⌬lhr1 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.

Isolation of Novel Trypanosomatid, Zelonia australiensis sp. nov. (Kinetoplastida: Trypanosomatidae) Provides Support for a Gondwanan Origin of Dixenous Parasitism in the Leishmaniinae

“…The rigorous growth of Z . australiensis in high haemoglobin concentrations and on chocolate agar is consistent with a haemoprotozoan (Fig 2, S1 File) [88] and/or adaptation to life as a parasite of hematophagous insects, which probably represents the first step in the transition to a dixenous life cycle. While Z .…”

Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters

“…Control and TbHRG‐expressing hem1 Δ yeasts were then plated in an ALA‐free medium with increasing concentrations of hemin. Figure B (upper panel) shows that TbHRG was able to rescue the growth of the mutant yeast at low hemin concentrations (0.25 μM), as described for LaHR1 (Miguel et al ., ) and other HRG proteins (Yuan et al ., ), strongly suggesting that TbHRG is indeed a heme transporter.…”

“…This possibility remains to be proven, but the essential nature of TbHRG suggests that this putative alternative mechanism for heme scavenging also relies on the traffic of any form of heme to the parasite's endolysosome, requiring TbHRG for heme export to the cytoplasm. LHR1 is also essential for L. amazonensis (Huynh et al ., ) (and probably for L. major ) and required for the parasite's virulence in animal models (Miguel et al ., ; Renberg et al ., ). Although we have not analyzed the role of the syntenic HRG gene of T. cruzi , an essential role could also be expected for TcHRG, as T. cruzi , responsible for Chagas disease, is also auxotrophic for heme and performs Hb endocytosis (Lara et al ., ).…”

“…Whether lysosomal LHR1 is physiologically relevant or is a product of its forced overexpression remains to be seen. Besides, LHR1 is essential for the parasite, and even the ablation of one allele strongly prevents the virulence of the parasite in animal models of cutaneous leishmaniasis, underlining its potential as a new drug target (Huynh et al, 2012;Miguel et al, 2013;Renberg et al, 2015).…”

Isolation, Derivative Synthesis, and Structure–Activity Relationships of Antiparasitic Bromopyrrole Alkaloids from the Marine Sponge Tedania brasiliensis

“…47 Leishmania (L.) infantum (MHOM/BR/1972/LD) was cultured as described above, with the exception that 5% human sterile urine was added to the culture medium. Trypanosoma cruzi epimastigotes (Y strain) were grown in liver infusion tryptose (LIT) medium supplemented with 20 mg/L hemin and 10% fetal calf serum at 28 °C as previously described.…”