Heme metabolism in Strigomonas culicis: Implications of H2O2 resistance induction and symbiont elimination.

Monoxenous trypanosomatid Strigomonas culicis harbors an endosymbiotic bacterium, which enables the protozoa to survive without heme supplementation. The impact of H2O2 resistance and symbiont elimination on intracellular heme and Fe2+ availability was analyzed through a comparison of WT strain with both WT H2O2-resistant (WTR) and aposymbiotic (Apo) protozoa. The relative quantification of the heme biosynthetic pathway through label-free parallel reaction monitoring targeted mass spectrometry revealed that H2O2 resistance does not influence the abundance of tryptic peptides. However, the Apo strain showed increased coproporphyrinogen III oxidase and ferrochelatase levels. A putative ferrous iron transporter, homologous to LIT1 and TcIT from Leishmania major and Trypanosoma cruzi, was identified for the first time. Label-free parallel reaction monitoring targeted mass spectrometry also showed that S. culicis Iron Transporter (ScIT) increased 1.6- and 16.4-fold in WTR and Apo strains compared to WT. Accordingly, antibody-mediated blockage of ScIT decreased by 28.0% and 40.0% intracellular Fe2+concentration in both WTR and Apo strains, whereas no effect was detected in WT. In a heme-depleted medium, adding 10 µM hemin decreased ScIT transcript levels in Apo, whereas 10 µM PPIX, the substrate of ferrochelatase, increased intracellular Fe2+ concentration and ferric iron reduction. Overall, the data suggest mechanisms dependent on de novo heme synthesis (and its substrates) in the Apo strain to overcome reduced heme availability. Given the importance of heme and Fe2+ as cofactors in metabolic pathways, including oxidative phosphorylation and antioxidant systems, this study provides novel mechanistic insights associated with H2O2 resistance in S. culicis.

Interleukin-27 Promotes Divergent Effects on HIV-1 Infection in Peripheral Blood Mononuclear Cells through BST-2/Tetherin.

Interleukin-27 (IL-27) is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), macrophages, and dendritic cells. Here, we identify that IL-27 can produce opposing effects on HIV-1 replication in PBMCs and that the HIV-1 restriction factor BST-2/Tetherin is involved in both inhibitory and enhancing effects on HIV-1 infection induced by IL-27. IL-27 inhibited HIV-1 replication when added to cells 2 h after infection, promoting the prototypical BST-2/Tetherin-induced virion accumulation at the cell membrane of HIV-1-infected PBMCs. BST-2/Tetherin gene expression was significantly upregulated in the IL-27-treated PBMCs, with a simultaneous increase in the number of BST-2/Tetherin+ cells. The silencing of BST-2/Tetherin diminished the anti-HIV-1 effect of IL-27. In contrast, IL-27 increased HIV-1 production when added to infected cells 4 days after infection. This enhancing effect was prevented by BST-2/Tetherin gene knockdown, which also permitted IL-27 to function again as an HIV-1 inhibitory factor. These contrasting roles of IL-27 were associated with the dynamic of viral production, since the IL-27-mediated enhancement of virus replication was prevented by antiretroviral treatment of infected cells, as well as by keeping cells under agitation to avoid cell-to-cell contact. Likewise, inhibition of CD11a, an integrin associated with HIV-1 cell-to-cell transmission, abrogated the IL-27 enhancement of HIV-1 production. Our findings illustrate the complexity of the HIV-1-host interactions and may impact the potential therapeutic use of IL-27 and other soluble mediators that induce BST-2/Tetherin expression for HIV-1 infection. IMPORTANCE Here, we describe new findings related to the ability of the cytokine IL-27 to regulate the growth of HIV-1 in CD4+ T lymphocytes. IL-27 has long been considered a potent inhibitor of HIV-1 replication, a notion based on several reports showing that this cytokine controls HIV-1 infection in peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages, and dendritic cells. However, our present results are contrary to the current knowledge that IL-27 acts only as a powerful downregulator of HIV-1 replication. We observed that IL-27 can either prevent or enhance viral growth in PBMCs, an outcome dependent on when this cytokine is added to the infected cells. We detected that the increase of HIV-1 dissemination is due to enhanced cell-to-cell transmission with the involvement of the interferon-induced HIV-1 restriction factor BST-2/Tetherin and CD11a (LFA-1), an integrin that participates in formation of virological synapse.

Is the mitochondrion a promising drug target in trypanosomatids?

The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are etiological agents of important neglected tropical diseases, affecting millions of people worldwide, and the drugs available for these diseases present several limitations. Novel efficient and nontoxic drugs are necessary as an alternative to the current chemotherapy. The unique mitochondrion of trypanosomatids and its peculiar features turn this organelle a potential drug target. Several phenotypic studies describe the damage in the parasite mitochondrial ultrastructure, but the molecular target is unknown. Few reports demonstrated the electron transport system (ETS) as a target due to the high similarities to mammalian orthologues, hence ETS is not a good candidate for drug intervention. On the other hand, antioxidant enzymes, such as trypanothione reductase, and an alternative oxidase (AOX) seem to be interesting targets; however no high active inhibitors were developed up to now. Finally, due to the remarkable differences to mammalian machinery, together with the high biological importance for the parasite survival, the mitochondrial import system stands out as a very promising target in trypanosomatids. Archaic translocase of the outer membrane (ATOM) and translocase of the inner membrane (TIM) complexes, which mediate both protein and tRNA import, composed by specific subunits of these parasites, could be excellent candidates, deserving studies focused on the development of specific drugs.

Host cholesterol influences the activity of sterol biosynthesis inhibitors in Leishmania amazonensis.

A significant percentage of exogenous cholesterol was found in promastigotes and amastigotes of all studied species of Leishmania, suggesting a biological role for this molecule. Previous studies have shown that promastigotes of Leishmania uptake more low-density lipoprotein (LDL) particles under pharmacological pressure and are more susceptible to ergosterol inhibition in the absence of exogenous sources of cholesterol. This work shows that the host's LDL is available to intracellular amastigotes and that the absence of exogenous cholesterol enhances the potency of sterol biosynthesis inhibitors in infected macrophages. A complete understanding of cholesterol transport to the parasitophorous vacuole can guide the development of a new drug class to be used in combination with sterol biosynthesis inhibitors for the treatment of leishmaniases.

Ruthenium(II)- and Palladium(II)-catalyzed position-divergent CH oxygenations of arylated quinones: Identification of hydroxylated quinonoid compounds with potent trypanocidal activity.

A diversity-oriented synthesis of hydroxylated aryl-quinones via CH oxygenation reactions and their evaluation against Trypanosoma cruzi, the etiological agent of Chagas disease, was accomplished. With the use of ruthenium(II)- or palladium(II)-based catalysts, complementary regioselectivities were observed in the hydroxylation reactions and we have identified 9 compounds more potent than benznidazole (Bz) among these novel arylated and hydroxylated quinones. For instance, 5-hydroxy-2-[4-(trifluoromethyl)phenyl]-1,4-naphthoquinone (4h) with an IC50/24 h value of 22.8 µM is 4.5-fold more active than the state-of-the-art drug Bz. This article provides the first example of the application of CH activation for the position-selective hydroxylation of arylated quinones and the identification of these compounds as trypanocidal drug candidates.

Strategies towards potent trypanocidal drugs: Application of Rh-catalyzed [2 + 2 + 2] cycloadditions, sulfonyl phthalide annulation and nitroalkene reactions for the synthesis of substituted quinones and their evaluation against Trypanosoma cruzi.

Rhodium-catalyzed [2 + 2 + 2] cycloadditions, sulfonyl phthalide annulations and nitroalkene reactions have been employed for the synthesis of 56 quinone-based compounds. These were evaluated against Trypanosoma cruzi, the parasite that causes Chagas disease. The reactions described here are part of a program that aims to utilize modern, versatile and efficient synthetic methods for the one or two step preparation of trypanocidal compounds. We have identified 9 compounds with potent activity against the parasite; 3 of these were 30-fold more potent than benznidazole (Bz), a drug used for the treatment of Chagas disease. This article provides a comprehensive outline of reactions involving over 120 compounds aimed at the discovery of new quinone-based frameworks with activity against T. cruzi.

Expression and cellular localisation of Trypanosoma cruzi calpains.

BACKGROUND: Calpains are present in almost all organisms and comprise a family of calcium-dependent cysteine peptidases implicated in crucial cellular functions. Trypanosoma cruzi, the causative agent of Chagas disease, presents an expansion on this gene family with unexplored biological properties. OBJECTIVES: Here, we searched for calpains in the T. cruzi genome, evaluated the mRNA levels, calpain activity and the protein expression and determined the cellular localisation in all three parasite life cycle forms. METHODS/FINDINGS: Sixty-three calpain sequences were identified in T. cruzi CL Brener genome, with fourteen domain arrangements. The comparison of calpain mRNA abundance by quantitative polymerase chain reaction (qPCR) revealed seven up-regulated sequences in amastigotes and/or bloodstream trypomastigotes and five in epimastigotes. Western Blotting analysis revealed seven different molecules in the three parasite forms, and one amastigote-specific, while no proteolytic activity could be detected. Flow cytometry assays revealed a higher amount of intracellular calpains in amastigotes and/or trypomastigotes in comparison to epimastigotes. Finally, ultrastructural analysis revealed the presence of calpains in the cytoplasm, vesicular and plasma membranes of the three parasite forms, and in the paraflagellar rod in trypomastigotes. CONCLUSION: Calpains are differentially expressed and localised in the T. cruzi life cycle forms. This study adds data on the calpain occurrence and expression pattern in T. cruzi.

Antiparasitic and anti-inflammatory activities of ß-lapachone-derived naphthoimidazoles in experimental acute Trypanosoma cruzi infection.

BACKGROUND: Chagas disease, which is caused by the protozoan Trypanosoma cruzi, is endemic to Latin America and mainly affects low-income populations. Chemotherapy is based on two nitrocompounds, but their reduced efficacy encourages the continuous search for alternative drugs. Our group has characterised the trypanocidal effect of naphthoquinones and their derivatives, with naphthoimidazoles derived from ß-lapachone (N1, N2 and N3) being the most active in vitro. OBJECTIVES: In the present work, the effects of N1, N2 and N3 on acutely infected mice were investigated. METHODS: in vivo activity of the compounds was assessed by parasitological, biochemical, histopathological, immunophenotypical, electrocardiographic (ECG) and behavioral analyses. FINDINGS: Naphthoimidazoles led to a decrease in parasitaemia (8 dpi) by reducing the number of bloodstream trypomastigotes by 25-50% but not by reducing mortality. N1 protected mice from heart injury (15 dpi) by decreasing inflammation. Bradycardia was also partially reversed after treatment with N1 and N2. Furthermore, the three compounds did not reverse hepatic and renal lesions or promote the improvement of other evaluated parameters. MAIN CONCLUSION: N1 showed moderate trypanocidal and promising immunomodulatory activities, and its use in combination with benznidazole and/or anti-arrhythmic drugs as well as the efficacy of its alternative formulations must be investigated in the near future.

Participation of Trypanosoma cruzi gp63 molecules on the interaction with Rhodnius prolixus.

Trypanosoma cruzi is the causative agent of Chagas disease, a vector-borne disease. The parasite molecules involved in vector interaction have been little investigated. Metallopeptidases and gp63 molecules have been implicated in parasite adhesion of several trypanosomatids to the insect midgut. Although gp63 homologues are highly expanded in the T. cruzi genome, and are implicated in parasite-mammalian host interaction, its role in the insect vector has never been explored. Here, we showed that divalent metal chelators or anti-Tcgp63-I antibodies impaired T. cruzi adhesion to Rhodnius prolixus midgut. Parasites isolated after insect colonization presented a drastic enhancement in the expression of Tcgp63-I. These data highlight, for the first time, that Tcgp63-I and Zn-dependent enzymes contribute to the interaction of T. cruzi with the insect vector.

Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis.

BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Therapeutic effects of adipose-tissue-derived mesenchymal stromal cells and their extracellular vesicles in experimental silicosis.

BACKGROUND: Silicosis is an occupational disease that affects workers who inhale silica particles, leading to extensive lung fibrosis and ultimately causing respiratory failure. Mesenchymal stromal cells (MSCs) have been shown to exert therapeutic effects in lung diseases and represent an alternative treatment for silicosis. Recently, it has been suggested that similar effects can be achieved by the therapeutic use of extracellular vesicles (EVs) obtained from MSCs. The aim of this study was to investigate the effects of adipose-tissue-derived MSCs (AD-MSCs) or their EVs in a model of silicosis. METHODS: Silicosis was induced by intratracheal instillation of silica in C57BL/6 mice. After the onset of disease, animals received saline, AD-MSCs, or EVs, intratracheally. RESULTS: At day 30, AD-MSCs and EVs led to a reduction in collagen fiber content, size of granuloma, and in the number of macrophages inside granuloma and in the alveolar septa. In addition, the expression levels of interleukin 1ß and transforming growth factor beta in the lungs were decreased. Higher dose of EVs also reduced lung static elastance when compared with the untreated silicosis group. CONCLUSIONS: Both AD-MSCs and EVs, locally delivered, ameliorated fibrosis and inflammation, but dose-enhanced EVs yielded better therapeutic outcomes in this model of silicosis.

Combination of Aryl Diselenides/Hydrogen Peroxide and Carbon-Nanotube/Rhodium Nanohybrids for Naphthol Oxidation: An Efficient Route towards Trypanocidal Quinones.

This work reports a combination of aryl diselenides/hydrogen peroxide and carbon-nanotube (CNT)/rhodium nanohybrids (RhCNT) for naphthol oxidation towards the synthesis of 1,4-naphthoquinones and evaluation of their relevant trypanocidal activity. Under a combination of (PhSe)2 /H2 O2 in the presence of O2 in iPrOH/hexane, several benzenoid (A-ring)-substituted quinones were prepared in moderate to high yields. We also studied the contribution of RhCNT as co-catalyst in this process and, in some cases, yields were improved. This method provides an efficient and versatile alternative for preparing A-ring-modified naphthoquinonoid compounds with relevant biological profile.

Direct sequential C-H iodination/organoyl-thiolation for the benzenoid A-ring modification of quinonoid deactivated systems: a new protocol for potent trypanocidal quinones.

We report a sequential C-H iodination/organoyl-thiolation of naphthoquinones and their relevant trypanocidal activity. Under a combination of AgSR with a copper source, sulfur-substituted benzenoid quinones were prepared in high yields (generally >90%). This provides an efficient and general method for preparing A-ring modified naphthoquinoidal systems, recognized as a challenge in quinone chemistry.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps.

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade.

BACKGROUND: Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES: We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS: The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS: In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS: This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

TrypanocidalActivity of Natural Sesquiterpenoids Involves Mitochondrial Dysfunction, ROS Production and Autophagic Phenotype in Trypanosomacruzi.

Chagas disease is a neglected tropical disease that is caused by the protozoan Trypanosomacruzi and represents a serious health problem, especially in Latin America. The clinical treatment of Chagas disease is based on two nitroderivatives that present severe side effects and important limitations. In folk medicine, natural products, including sesquiterpenoids, have been employed for the treatment of different parasitic diseases. In this study, the trypanocidal activity of compounds isolated from the Chilean plants Drimys winteri, Podanthus mitiquiand Maytenus boaria on three T. cruzi evolutive forms (epimastigote, trypomastigote and amastigote) was evaluated. Total extracts and seven isolated sesquiterpenoids were assayed on trypomastigotes and epimastigotes. Polygodial (Pgd) from D. winteri, total extract from P. mitiqui (PmTE) and the germacrane erioflorin (Efr) from P. mitiqui were the most bioactive substances. Pgd, Efr and PmTE also presented strong effects on intracellular amastigotes and low host toxicity. Many ultrastructural effects of these substances, including reservosome disruption, cytosolic vacuolization, autophagic phenotype and mitochondrial swelling (in the case of Pgd), were observed. Flow cytometric analysis demonstrated a reduction in mitochondrial membrane potential in treated epimastigotes and an increase in ROS production and high plasma membrane permeability after treatment with Pgd. The promising trypanocidal activity of these natural sesquiterpenoids may be a good starting point for the development of alternative treatmentsforChagas disease.

Why calpain inhibitors are interesting leading compounds to search for new therapeutic options to treat leishmaniasis?

Leishmaniasis is a neglected disease, which needs improvements in drug development, mainly due to the toxicity, parasite resistance and low compliance of patients to treatment. Therefore, the development of new chemotherapeutic compounds is an urgent need. This opinion article will briefly highlight the feasible use of calpain inhibitors as leading compounds to search for new therapeutic options to treat leishmaniasis. The milestone of this approach is to take advantage on the myriad of inhibitors developed against calpains, some of which are in advanced clinical trials. The deregulated activity of these enzymes is associated with several pathologies, such as strokes, diabetes and Parkinson's disease, to name a few. In Leishmania, calpain upregulation has been associated to drug resistance and virulence. Whereas the difficulties in developing new drugs for neglected diseases are more economical than biotechnological, repurposing approach with compounds already approved for clinical use by the regulatory agencies can be an interesting shortcut to a successful chemotherapeutic treatment for leishmaniasis.

Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host.

A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites' physiology.

Iron-modulated pseudocyst formation in Tritrichomonas foetus.

Iron is an essential element for the survival of trichomonads during host-parasite interaction. The availability of this metal modulates several metabolic pathways of the parasites and regulates the expression of virulence factors such as adhesins and proteolytic enzymes. In this study, we investigated the effect of iron depletion on the morphology and life cycle of Tritrichomonas foetus. Scanning and transmission electron microscopy analyses revealed that depletion of iron from the culture medium (named TYM-DIP inducer medium) induces morphological transformation of typical pear-shaped trophozoites into spherical and non-motile pseudocysts. Remarkably, inoculation of pseudocysts into an iron-rich medium (standard TYM medium), or addition of FeSO4 to a TYM-DIP inducer medium reverted the morphological transformation process and typical trophozoites were recovered. These results show that pseudocysts are viable forms of the parasite and highlight the role of iron as a modulator of the parasite phenotype. Although iron is required for the survival of T. foetus, iron depletion does not cause a cellular collapse of pseudocysts, but instead induces phenotypic alterations, probably in order to allow the parasite to survive conditions of nutritional stress. Together, these findings support previous studies that suggest pseudocysts are a resistance form in the life cycle of T. foetus and enable new approaches to understanding the multifactorial role of iron in the cell biology of this protozoan parasite.