Robert Sutak

Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation.

Giardia intestinalis (syn. lamblia) is one of the most widespread intestinal protozoan pathogens worldwide, causing hundreds of thousands of cases of diarrhoea each year. Giardia is a member of the diplomonads, often described as an ancient protist group whose primitive nature is suggested by the lack of typical eukaryotic organelles (for example, mitochondria, peroxisomes), the presence of a poorly developed endomembrane system and by their early branching in a number of gene phylogenies. The discovery of nuclear genes of putative mitochondrial ancestry in Giardia and the recent identification of mitochondrial remnant organelles in amitochondrial protists such as Entamoeba histolytica and Trachipleistophora hominis suggest that the eukaryotic amitochondrial state is not a primitive condition but is rather the result of reductive evolution. Using an in vitro protein reconstitution assay and specific antibodies against IscS and IscU—two mitochondrial marker proteins involved in iron–sulphur cluster biosynthesis—here we demonstrate that Giardia contains mitochondrial remnant organelles (mitosomes) bounded by double membranes that function in iron–sulphur protein maturation. Our results indicate that Giardia is not primitively amitochondrial and that it has retained a functional organelle derived from the original mitochondrial endosymbiont.

Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence

The effective acquisition of iron is a pre-requisite for survival of all organisms, especially parasites that have a high iron requirement. In mammals, iron homeostasis is meticulously regulated; extracellular free iron is essentially unavailable and host iron availability has a crucial role in the host-pathogen relationship. Therefore, pathogens use specialized and effective mechanisms to acquire iron. In this review, we summarize the iron-uptake systems in eukaryotic unicellular organisms with particular focus on the pathogenic species: Candida albicans, Tritrichomonas foetus, Trypanosoma brucei and Leishmania spp. We describe the diversity of their iron-uptake mechanisms and highlight the importance of the process for virulence.

Hepcidin, the hormone of iron metabolism, is bound specifically to α-2-macroglobulin in blood

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

The MCK mouse heart model of Friedreich's ataxia: Alterations in iron-regulated proteins and cardiac hypertrophy are limited by iron chelation

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreichs ataxia (FA). The identification of potentially toxic mitochondrial (MIT) iron (Fe) deposits in FA suggests that Fe plays a role in its pathogenesis. This study used the muscle creatine kinase conditional frataxin (Fxn) knockout (mutant) mouse model that reproduces the classical traits associated with cardiomyopathy in FA. We examined the mechanisms responsible for the increased cardiac MIT Fe loading in mutants. Moreover, we explored the effect of Fe chelation on the pathogenesis of the cardiomyopathy. Our investigation showed that increased MIT Fe in the myocardium of mutants was due to marked transferrin Fe uptake, which was the result of enhanced transferrin receptor 1 expression. In contrast to the mitochondrion, cytosolic ferritin expression and the proportion of cytosolic Fe were decreased in mutant mice, indicating cytosolic Fe deprivation and markedly increased MIT Fe targeting. These studies demonstrated that loss of Fxn alters cardiac Fe metabolism due to pronounced changes in Fe trafficking away from the cytosol to the mitochondrion. Further work showed that combining the MIT-permeable ligand pyridoxal isonicotinoyl hydrazone with the hydrophilic chelator desferrioxamine prevented cardiac Fe loading and limited cardiac hypertrophy in mutants but did not lead to overt cardiac Fe depletion or toxicity. Fe chelation did not prevent decreased succinate dehydrogenase expression in the mutants or loss of cardiac function. In summary, we show that loss of Fxn markedly alters cellular Fe trafficking and that Fe chelation limits myocardial hypertrophy in the mutant.

Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance

Ferredoxin, Fd, is often deficient in metronidazole‐resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by ∼2.6 and ∼2.0 kBp of the Fd 5′ and 3′ flanking regions (pKO‐FD‐NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO‐FD‐NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.

Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomes

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreichs ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe–S cluster‐containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin‐depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe–S cluster biogenesis and protection from reactive oxygen species.

Iron chelation by clinically relevant anthracyclines: alteration in expression of iron-regulated genes and atypical changes in intracellular iron distribution and trafficking.

Anthracyclines are effective anticancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron and to perturb iron metabolism (Mol Pharmacol 68:261-271, 2005). These effects on iron-trafficking remain poorly understood, but they are important to decipher because treatment for anthracycline cardiotoxicity uses the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the iron-regulated genes transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by iron depletion, because it was reversed by adding iron and it was prevented by saturating the anthracycline metal binding site with iron. However, DOX did not act like a typical chelator, because it did not induce cellular iron mobilization. In the presence of DOX and 59Fe-transferrin, iron-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic iron deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via hypoxia-inducible factor-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on iron metabolism were complex because of its multiple effector mechanisms.

NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

Evidence that yeast frataxin is not an iron storage protein in vivo

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Deltayfh1, Deltaggc1 and Deltassq1) displayed a nearly identical distribution. This was also the case for Deltaggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.

A comparative study of iron uptake mechanisms in marine microalgae: iron binding at the cell surface is a critical step.

We investigated iron uptake mechanisms in five marine microalgae from different ecologically important phyla: the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, the prasinophyceae Ostreococcus tauri and Micromonas pusilla, and the coccolithophore Emiliania huxleyi. Among these species, only the two diatoms were clearly able to reduce iron, via an inducible (P. tricornutum) or constitutive (T. pseudonana) ferrireductase system displaying characteristics similar to the yeast (Saccharomyces cerevisiae) flavohemoproteins proteins. Iron uptake mechanisms probably involve very different components according to the species, but the species we studied shared common features. Regardless of the presence and/or induction of a ferrireductase system, all the species were able to take up both ferric and ferrous iron, and iron reduction was not a prerequisite for uptake. Iron uptake decreased with increasing the affinity constants of iron-ligand complexes and with increasing ligand-iron ratios. Therefore, at least one step of the iron uptake mechanism involves a thermodynamically controlled process. Another step escapes to simple thermodynamic rules and involves specific and strong binding of ferric as well as ferrous iron at the cell surface before uptake of iron. Binding was paradoxically increased in iron-rich conditions, whereas uptake per se was induced in all species only after prolonged iron deprivation. We sought cell proteins loaded with iron following iron uptake. One such protein in O. tauri may be ferritin, and in P. tricornutum, Isip1 may be involved. We conclude that the species we studied have uptake systems for both ferric and ferrous iron, both involving specific iron binding at the cell surface.