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Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of <em>Leishmania</em> Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

University of Maryland

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.

LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms.

During its life cycle, Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. Elevated temperature and decreased pH, conditions encountered after macrophage invasion, can induce axenic differentiation of avirulent promastigotes into virulent amastigotes. Here we show that iron uptake is a major trigger for the differentiation of Leishmania amazonensis amastigotes, independently of temperature and pH changes. We found that iron depletion from the culture medium triggered expression of the ferrous iron transporter LIT1 (Leishmania iron transporter 1), an increase in iron content of the parasites, growth arrest, and differentiation of wild-type (WT) promastigotes into infective amastigotes. In contrast, LIT1-null promastigotes showed reduced intracellular iron content and sustained growth in iron-poor media, followed by cell death. LIT1 up-regulation also increased iron superoxide dismutase (FeSOD) activity in WT but not in LIT1-null parasites. Notably, the superoxide-generating drug menadione or H(2)O(2) was sufficient to trigger differentiation of WT promastigotes into fully infective amastigotes. LIT1-null promastigotes accumulated superoxide radicals and initiated amastigote differentiation after exposure to H(2)O(2) but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species in orchestrating the differentiation of virulent Leishmania amastigotes in a process regulated by iron availability.

Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels.

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 this transporter is essential for survival. Here we compared the growth, differentiation, and infectivity for macrophages and mice of wild type, LHR1-single knockout (LHR1/Î”lhr1) and LHR1-complemented (LHR1/Î”lhr1 + LHR1) L. amazonensis strains. LHR1/Î”lhr1 promastigotes replicated poorly in heme-deficient media and had lower intracellular heme content when compared to wild type parasites. LHR1/Î”lhr1 promastigotes were also less effective in reducing ferric to ferrous iron, a reaction mediated by the heme-containing parasite enzyme LFR1 (Leishmania Ferric Reductase 1). LHR1/Î”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/Î”lhr1 amastigotes was fully restored when macrophages were allowed to phagocytose red blood cells prior to infection. LHR1/Î”lhr1 parasites were also severely defective in the development of cutaneous lesions in mice. All phenotypes observed in LHR1/Î”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 anti-leishmanial drugs.

Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence.

The protozoan parasite Leishmania experiences extreme environmental changes as it alternates between insect and mammalian hosts. In some species, differentiation of insect promastigotes into mammalian-infective amastigotes is induced by elevated temperature and low pH, conditions found within macrophage parasitophorous vacuoles (PVs). However, the signaling events controlling amastigote differentiation remain poorly understood. Recent studies revealed a novel role for iron uptake in orchestrating the differentiation of amastigotes, through a mechanism that involves production of reactive oxygen species (ROS) and is independent from pH and temperature changes. ROS are generally thought to be deleterious for pathogens, but it is becoming increasingly apparent that they can also function as signaling molecules regulating Leishmania differentiation, in a process that is tightly controlled by iron availability.

Irony of fate: role of iron-mediated ROS in Leishmania differentiation.

Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.

A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

Leishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide.infection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role,mitochondrial SOD may also initiate HO-mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform inOur inability to generatenull mutants and the lethal phenotype observed following RNAi-mediated silencing of theortholog suggests that SODA is essential for trypanosomatid survival.metacyclic promastigotes lacking oneallele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure of/Δpromastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, as/Δcultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotesvirulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms.

Bidyottam Mittra

Department of Cell Biology and Molecular Genetics

University of Maryland

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

Bidyottam Mittra

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology and Molecular Genetics

University of Maryland

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

Department of Cell Biology and Molecular Genetics