Abstract
Infection of mammalian hosts with Leishmania amazonensis depends on the remarkable ability of these parasites to replicate within macrophage phagolysosomes. A critical adaptation for survival in this harsh environment is an efficient mechanism for gaining access to iron. In this study, we identify and characterize LIT1, a novel L. amazonensis membrane protein with extensive similarity to IRT1, a ZIP family ferrous iron transporter from Arabidopsis thaliana. The ability of LIT1 to promote iron transport was demonstrated after expression in yeast and in L. amazonensis LIT1-null amastigotes. Endogenous LIT1 was only detectable in amastigotes replicating intracellularly, and its intracellular expression was accelerated under conditions predicted to result in iron deprivation. Although L. amazonensis lacking LIT1 grew normally in axenic culture and had no defects differentiating into infective forms, replication within macrophages was abolished. Consistent with an essential role for LIT1 in intracellular growth as amastigotes, Δlit1 parasites were avirulent. After inoculation into highly susceptible mice, no lesions were detected, even after extensive periods of time. Despite the absence of pathology, viable Δlit1 parasites were recovered from the original sites of inoculation, indicating that L. amazonensis can persist in vivo independently of the ability to grow in macrophages. Our findings highlight the essential role played by intracellular iron acquisition in Leishmania virulence and identify this pathway as a promising target for therapeutic intervention.
Highlights
The Leishmania genome encodes two copies of LIT1, a homologue of the IRT1 iron transporter–encoding gene of Arabidopsis thaliana Homology searches of the L. major database identified two identical genes on chromosome 31 in tandem, LmjF31.3060 and LmjF31.3070
There is complete conservation of all five residues shown to be essential for divalent metal transport by Arabidopsis IRT1 (Fig. 1 A, residues boxed in red) [17], reinforcing the possibility that the Leishmania LIT1 genes encode an iron transporter
We make an important step toward understanding the molecular mechanisms involved in adaptation to this harsh intracellular environment by identifying the first Leishmania membrane protein that functions intracellularly as a ferrous iron transporter
Summary
The critical role of iron for the survival of microbes within endocytic vesicles is highlighted by the importance of Nramp ( known as Slc11a1) in natural resistance to infections [2]. Recent investigations into the role of Nramp revealed that it functions as a pH-dependent transporter that can extrude Mn2+, and possibly other divalent cations, from phagolysosomes [3]. This has not yet been directly demonstrated, Nramp may promote resistance to infection by removing iron from intracellular compartments, starving pathogens. The uptake of iron for these essential enzymatic functions poses a particular problem for both pathogens and host cells [6].
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