Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Infect Immun. 2013 Oct;81(10):3620-6. doi: 10.1128/IAI.00687-13. Epub 2013 Jul 22.
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.
原生动物寄生虫利什曼原虫是一种血红素营养缺陷型生物，必须从环境中获取这种必需因子。先前的研究表明，利什曼原虫通过跨膜蛋白 LHR1（利什曼血红素反应 1）摄取血红素。LHR1 缺失的前鞭毛体无法存活，这表明该转运蛋白对生存至关重要。在这里，我们比较了野生型、LHR1 单敲除（LHR1/Δlhr1）和 LHR1 互补（LHR1/Δlhr1 加 LHR1）利什曼原虫菌株的生长、分化和对巨噬细胞及小鼠的感染力。LHR1/Δlhr1 前鞭毛体在血红素缺乏的培养基中繁殖不良，细胞内血红素含量低于野生型寄生虫。LHR1/Δlhr1 前鞭毛体还原铁离子为亚铁离子的效率也较低，该反应由含血红素的寄生虫酶 LFR1（利什曼铁还原酶 1）介导。LHR1/Δlhr1 寄生虫正常分化为鞭毛缺失的形式，表达无鞭毛体特异性标记物，但在感染巨噬细胞后无法在细胞内复制。重要的是，在感染前允许巨噬细胞吞噬红细胞，LHR1/Δlhr1 无鞭毛体的细胞内生长完全恢复。LHR1/Δlhr1 寄生虫在小鼠皮肤病变的发展中也严重缺陷。在 LHR1/Δlhr1 利什曼原虫中观察到的所有表型都通过表达附加体 LHR1 得到挽救。我们的研究结果揭示了高效摄取血红素对利什曼原虫复制和脊椎动物宿主感染力的重要性，加强了 LHR1 作为新抗利什曼药物靶点的潜在用途。
