Leishmania amazonensis: biosynthesis of polyprenols of 9 isoprene units by amastigotes

The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol.     

Leishmania species: evidence for transglutaminase activity and its role in parasite proliferation

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.     

Mitochondrial damage contribute to epigallocatechin-3-gallate induced death in Leishmania amazonensis

Epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi however, the mechanism of protozoan action of EGCG has not been studied. In the present study, we demonstrate the mechanism for the antileishmanial activity of EGCG against Leishmania amazonensis promastigotes. Incubation with EGCG significantly inhibited L. amazonensis promastigote proliferation in a time- and dose-dependent manner. The IC(50) for EGCG at 120h was 0.063mM. Ultrastructural alterations of the mitochondria were observed in promastigote treated with EGCG, being the organelle injury reinforced by the decrease in rhodamine 123 fluorescence. The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. These data suggest mitochondrial collapse as a part of the EGCG mechanism of action and demonstrate the leishmanicidal effect of EGCG.     

Sterol methenyl transferase inhibitors alter the ultrastructure and function of the Leishmania amazonensis mitochondrion leading to potent growth inhibition

We describe here the effects of Delta(24(25)) sterol methenyl transferase inhibitors (SMTI) on promastigote and axenic amastigote forms of Leishmania amazonensis. When these cells were exposed to 20-piperidin-2-yl-5alpha-pregnan-3beta-20-diol (22,26-azasterol; AZA), hydrazone-imidazol-2-yl-5alpha-pregnan-3beta-ol (IMI), 20-hydrazone-pyridin-2-yl-5alpha-pregnan-3beta-ol (PYR) or 24(R,S),25-epiiminolanosterol (EIL), a concentration- and time-dependent inhibition of growth was observed, with IC(50) values in the sub-micromolar range. Ultrastructural alterations in treated cells were mainly observed in the mitochondrion, which displayed an intense swelling and a reduction of the electron density of the matrix with remarkable changes in the inner mitochondrial membranes. Mitochondrial transmembrane electric potential (DeltaPsi) was measured using spectrophotometric methods in control and treated promastigotes permeabilized with digitonin. After energization with the substrates for complexes I, II or IV of the respiratory chain, it was possible to detect marked changes of DeltaPsi in promastigotes treated with 1 microM of the SMTI for 48 or 72 h when compared with normal cells, indicating that these compounds led to the loss of the energy-transducing properties of the mitochondrial inner membrane, probably related to the alteration of its lipid composition. The present study confirms these findings, showing that in Leishmania amazonensis the mitochondrial complex appears to be the first organelle affected after treatment with different SMTI.     

Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies

Several (glyco)(sphingo)lipids from different human pathogens have been characterized, and frequently many of these molecules are participating in host-pathogen interaction. In Leishmania (Leishmania) amazonensis, for example, amastigotes present on their surface glycosphingolipids (GSLs) with the structure Galbeta1-3Galalpha, which is recognized by 30 kDa receptor of macrophages. Furthermore, other Leishmania species, such as Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis present glycosylinositolphospholipids (GIPLs) which are involved in Leishmania-macrophage interaction. It is worth to mention that these antigens are not expressed in mammalian cells. Leishmania promastigotes also present inositol phosphorylceramide (IPC), a unique sphingolipid characteristic of fungi and plants. It was observed that IPC synthesis is essential for parasite division, since Aureobasidin A, an inhibitor of IPC synthase, inhibited significantly promastigote and amastigote growths. Recently, it was also demonstrated that GIPLs, IPC and sterols are preferentially present in the parasite membrane microdomains resistant to Triton X-100 at 4 degrees C. The disruption of these microdomains by incubating parasites with methyl-beta-cyclodextrin inhibited significantly macrophage infectivity by Leishmania. Other pathogens, such as fungi, also present unique glycolipids which may have an important role for the fungal development and/or disease establishment. Taking together these results, this review will discuss different biological roles for (glyco)(sphingo)lipids of different pathogens.     

Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania Spp

The World Health Organization has identified leishmania sis as a major public health problem, particularly in Africa, Asia, and Latin America. About 1.5 to 2 million people are affected annually by this parasitic infection. As there is no vaccine, there is still a strong need for sufficient drugs. In a preliminary screening, extracts of 50 different plants were evaluated for their possible leishmanicidal activity against the promastigote form of Leishmania mexicana amazonensis. Eighteen extracts showed at least 50% inhibition at 100 microg/ml. The ethanolic extract from Yucca filamentosa L. showed the strongest leishmanicidal activity (100% inhibition at 5 microg/ml). The bioactivity-guided fractionation of this extract led to the isolation of three main components (Yuccasaponins MC 1--3). In further experiments, the effect of Yuccasaponin MC 3 on the promastigote form of L. mexicana amazonensis was quantified and characterized using flow cytometry and specific fluorescent dyes [propidium iodide, Syto 9, and DiBAC(4)(3)]. The data revealed that the membrane of the promastigote is attacked. The effect of Yuccasaponin MC 3 on intracellular forms (amastigote) was also characterized; green fluorescent protein-transfected Leishmania major were used. By this method, an inhibition of intracellular growth of L. major was demonstrated. This paper shows that, together, flow cytometry and microscopy are quick, sensitive, and easily reproducible methods to describe the effects of drugs on parasites.     

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.     

Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.     

Antileishmanial activity of Eugenol-rich essential oil from Ocimum gratissimum

Leishmaniasis is a group of diseases with a large spectrum of clinical manifestations caused by protozoans of the genus Leishmania. Here we demonstrate the leishmanicidal activity of the essential oil of Ocimum gratissimum as well as its main constituent, eugenol. The eugenol-rich essential oil of O. gratissimum progressively inhibited Leishmania amazonensis growth at concentrations ranging from 100 to 1000 microg/ml. The IC50 (sub-inhibitory concentration) of the essential oil for promastigotes and amastigotes were respectively 135 and 100 microg/ml and the IC50 of eugenol was 80 microg/ml for promastigote forms. L. amazonensis exposed to essential oil at concentrations corresponding to IC50 for promastigotes and for amastigotes underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Two or more nuclei or flagella were observed in 31% and 23.3% of treated amastigote and promastigote forms, respectively, suggesting interference in cell division. Considerable mitochondrial swelling was observed in essential oil-treated promastigotes and amastigotes, which had the inner mitochondrial membrane altered, with a significant increase in the number of cristae; in some amastigotes the mitochondrial matrix became less electron-dense. The minimum inhibitory concentration for both promastigotes and amastigotes was 150 microg/ml. Pretreatment of mouse peritoneal macrophages with 100 and 150 microg/ml essential oil reduced the indices of association between promastigotes and the macrophages, followed by increased in nitric oxide production by the infected macrophages. The essential oil showed no cytototoxic effects against mammalian cells. This set of results suggests that O. gratissimum essential oil and its compounds could be used as sources for new antileishmanial drugs.     

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD?? of 0.9 μM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.     

Characterisation of atovaquone resistance in Leishmania infantum promastigotes

Atovaquone, an antiparasitic agent, could possibly represent an alternative therapy after relapse following classical treatment for visceral leishmaniasis. Atovaquone-resistant strains were selected in vitro by stepwise drug pressure to study the mechanism of resistance in Leishmania. Characteristics of a promastigote strain resistant to 250 microg/ml of atovaquone were compared with those of the wild type (WT) strain. Resistant strains were shown to have a high level of resistance (45 times). They were stable in drug-free medium for 6 months, and showed no cross-resistance with other antileishmanial drugs. Rhodamine uptake and efflux were studied. They were not modified in the resistant strain, indicating the absence of P-glycoprotein overexpession. The effect of atovaquone on membrane lipidic composition was determined in both WT and atovaquone-resistant promastigotes. Analysis of lipid composition of the atovaquone-resistant strain showed that sterol biosynthesis was decreased in atovaquone-resistant parasites. Cholesterol was found to be the major membrane sterol as opposed to the WT strain. Cholesterol, due to its ordering effect, could decrease membrane fluidity and subsequently block the passage of atovaquone through the membrane. Increased membrane cholesterol content and altered drug membrane fluidity resulted from possible decrease of ergosterol biosynthesis by atovaquone, incorporation of cholesterol by promastigotes in the culture medium, solubilisation of atovaquone by cholesterol and co-passage of the two compounds or influence of dimethylsulfoxide. These results indicate that different cellular alterations may participate in the resistant phenotype, by altering drug membrane permeability.     

Characterization of the species- and stage-specificity of two monoclonal antibodies against Leishmania amazonensis

Leishmania metacyclogenesis is associated with changes in morphology, gene expression, and structural alterations of the lipophosphoglycan (LPG), the promastigote most abundant surface glycolipid. Purification of metacyclics is accomplished using lectins or monoclonal antibodies (MAbs) that exploit stage-specific differences in the LPG. Besides, LPG displays extensive interspecies polymorphisms and is synthesized by promastigotes of all species investigated to date. In this work we studied the species- and stage-specificity of two MAbs (3A1-La and LuCa-D5) used to purify metacyclics of Leishmania amazonensis. Their ability to recognize different members of the Trypanosomatidae family was tested by direct agglutination, indirect immunofluorescence, and dot-blot analysis of LPG. We found that both MAbs were highly selective for L. amazonensis: 3A1-La recognized only promastigotes and LuCa-D5 labeled amastigote and promastigote stages of this species. These MAbs might be useful for Leishmania typing.     

Axenic promastigote forms of Leishmania (Viannia) lainsoni as an alternative source for Leishmania antigen production

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.     

Destiny and intracellular survival of Leishmania amazonensis in control and dexamethasone-treated glial cultures: protozoa-specific glycoconjugate tagging and TUNEL staining.

Leishmania amazonensis, an obligatory intracellular parasite, survives internalization by macrophages, but no information is available on the involvement of microglia. We have investigated microglia-protozoa interactions in mixed glial cultures infected with promastigote forms of L. amazonensis after lipopolysaccharide (LPS) or dexamethasone (DM) treatment. After 2 hr of exposure to parasites in control cultures, there was a small number of infected microglia (1%). Preincubation with LPS or DM led to 14% or 60% of microglial cells with attached parasites, respectively. DM treatment resulted in 39% of microglial cells with internalized parasites (controls or LPS-treated cells had < or =1%). Scanning electron micrographs showed numerous filopodia in DM-treated cells, whereas these projections were rarely observed in LPS-treated or control cells. DM treatment also affected the intramicroglial survival of Leishmania. In control cultures, internalized parasites, tagged with an anti-lipophosphoglycan (anti-LPG) antibody, showed fragmented DNA [terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL+)] after 4 hr of interaction, but changes seemed slightly delayed in DM-treated cultures. After 12 hr, there were no LPG+/TUNEL+ profiles in controls, whereas rare LPG+ profiles still persisted in DM-treated cells. Our results suggest that microglia are highly effective in the elimination of Leishmania and that the process can be effectively studied by LPG/TUNEL double labeling.     

The 3A1-La monoclonal antibody reveals key features of Leishmania (L) amazonensis metacyclic promastigotes and inhibits procyclics attachment to the sand fly midgut

In this work, we characterise metacyclic promastigotes of Leishmania amazonensis, the causative agent of cutaneous and diffuse cutaneous leishmaniasis in the New World. To purify metacyclics from stationary culture by negative selection, we used the monoclonal antibody 3A1-La produced against procyclic promastigotes. The purified forms named 3A1-La(-) promastigotes, present key metacyclic characteristics: slender cell body and long flagella, ultrastructural features, resistance to complement lysis, high infectivity for macrophages and mice and reduced capacity for binding to the sand fly midgut. Moreover, the epitope recognised by 3A1-La is important for the promastigote attachment to the insect vector midgut epithelium. These results further characterise 3A1-La(-) promastigotes as metacyclic forms of L. amazonensis.     

Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.     

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

Alterations Induced by the Antifungal Compounds Ketoconazole and Terbinafine in Leishmania

ABSTRACT. The antiproliferative effects and ultrastructural alterations induced in vitro by two antifungal compounds, the azole ketoconazole and the allylamine terbinafine on Leishmania amazonensis are reported. Promastigotes treatment with ketoconazole and terbinafine induced growth arrest and cell lysis in 72 hours. Combination of the two agents produced additive effects on promastigote axenic growth and synergistic effects on intracellular amastigote proliferation. The amastigotes, either axenically grown or infecting murine macrophages, were about 100-fold more sensitive to the drugs. These compounds induced the appearance of large multivesicular bodies, especially after ketoconazole treatment, increased amount of lipid inclusions as well as numerous, polymorphic volutin granules, particularly in terbinafine-treated cells. Multivesicular bodies were observed in close apposition with organelles such as mitochondria, which also showed alterations in the distribution and appearance of cristae, and the formation of paracrystalline arrays within the matrix. Some cells presented large portions of cytoplasm wrapped by endoplasmic reticulum and many parasites also presented myelin-like endoplasmic reticulum profiles. Such alterations together with the strong acid phosphatase activity observed in the multivesicular bodies and volutin granules may indicate the existence of an unusual autophagic process in cells treated with ergosterol biosynthesis inhibitors.     

Effect of L-arginine analogs and a calcium chelator on nitric oxide (NO) production by Leishmania sp

Leishmania amazonensis, L. braziliensis and L. chagasi promastigotes were grown in the presence of L-arginine analogs such as Nomega-nitro-L-arginine methyl ester (L-NAME), NG-nitro-L-arginine (L-NNA) and D-arginine (an inactive L-arginine isomer), besides an intracellular calcium chelator [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid; EGTA] to verify the importance of L-arginine metabolism and the cofactors for these parasites. The parasite's growth curve was followed up and the culture supernatants were used to assay nitric oxide (NO) production by the Griess reaction. The results showed a significant effect of L-arginine analogs on NO production by all Leishmania species studied, especially L-NAME, an irreversible inhibitor of the constitutive nitric oxide synthase (cNOS). When L. amazonensis promastigotes were pre-incubated with L-NAME, the infection range of the murine macrophages was lowered to 61% in 24 h and 19% after 48 h. These data demonstrated that the parasite NO pathway is important to the establishment of the infection.     

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

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.