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0-3 composites containing high permittivity ceramic particles and high breakdown strength polymer have aroused extensive concern for energy-storage application. In this research, flexible composite films were fabricated through incorporating of strontium titanate (SrTiO3) platelets (P-ST) and poly(vinylidene fluoride) (PVDF), and the effect of P-ST content on the dielectric and energy storage performances were researched systematically. It was found that at the relatively low electric field of 350 MV/m, the composite with 1 wt% P-ST exhibited an increased discharged energy density of 9.48 J/cm3, exceeding most of the current reports at the same electric field. And the distribution of local electric field and local polarization of composite films were simulated by COMSOL Multiphysics. This work provided a reference for polymer-based composite films featuring high energy storage property at relatively low applied electric field, which could reduce the possibility of failure for capacitors.

High-temperature dielectric composite films of all-organic PVDF/ABS were fabricated. The composites possessed excellent temperature stability. Ultrahigh Udischarged (11.42 J cm−2) and high efficiency (75.8%) at 100 °C were simultaneously achieved.

Polymer-based 0–3 composites filled with ceramic particles are identified as ideal materials for energy storage capacitors in electric systems. Herein, PVDF composite films filled with a small content (

The size of ceramic fillers is an important parameter influencing the energy storage performance of composite materials. Herein, composite films containing linear poly (methyl methacrylate) (PMMA) and ferroelectric polyvinylidene fluoride (PVDF) polymer matrices and BaTiO3 (BT) particles with different sizes were fabricated, and the dependences of their dielectric and energy storage properties on BT particle size were investigated systematically. The observed particle size effects on the PMMA/BT and PVDF/BT characteristics were almost identical. In particular, the dielectric permittivities of the composites gradually decreased while their breakdown electric fields (Eb) increased with an increase in BT particle size. Moreover, the composite films with small BT particles exhibited high discharged energy densities (Udischarged) at relatively low electric fields. The Udischarged of the PMMA/BT-60 nm sample was 5.68 J/cm3, which was 1.24 times higher than that of the PMMA/BT-500 nm sample at an electric field of 450 MV/m. Meanwhile, the Udischarged of PVDF/BT-60 nm was 10.06 J/cm3, which was 1.27 times greater than the Udischarged of PVDF/BT-500 nm at 350 MV/m. However, under high electric fields, the composite films with large BT particles produced higher Udischarged values. The maximum Udischarged magnitudes of the PMMA/BT-500 nm and PVDF/BT-500 nm samples were equal to 9.53 J/cm3 at 650 MV/m and 18.12 J/cm3 at 525 MV/m, respectively. Furthermore, the composite films filled with 500 nm BT particles demonstrated high cycling stability and fatigue endurance. The results of numerical simulations conducted to complement the experimental data revealed that the composite films with large BT particles possessed higher breakdown strengths.

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites.

Evolutionary pressure has pushed many extant plants and animals to develop micro/nanostructures on their surfaces to keep them clean. These structures have become ideal models for bio-inspired design. Although microstructures on biological surfaces have been widely studied, little attention has been paid to the combined role of microstructures and animal’s active cleaning behaviors in keeping their surfaces clean. In this study, we explored the relationship between these micro/nanostructures and wettability as well as the role of the housefly cleaning behaviors in keeping their wings clean. Hierarchical structures consisting of microscale macrotrichias with nanoscale grooves on the wings were observed under scanning electron microscope. The wings were hydrophobic (CA = 133.3°) but with high adhesion to water (CAH = 87.5°), indicating that they were non-self-cleaning surfaces. Macroscale droplets standing on the wings could be best described as being in a transitional wetting state between Wenzel and Cassie-Baxter states due to the presence of the nanoscale grooves, which increased the resistance to water penetration. The hydrophobicity decreased (CA = 109.9°) when the nanostructures were removed by coating the wings with a thick layer of polydimethylsiloxane (PDMS). The houseflies could highly efficiently remove the microscale droplets atop the macrotrichias, and reduce bacterial contamination on their wings through grooming and flutter activities. These active cleaning behaviors could offset the absence of self-cleaning properties and play a key role in keeping the wings clean. The results indicate that housefly wings could be used as a template for the design of special functional surfaces. The present findings not only improve our understanding of the wettability and cleaning properties of natural surfaces, but also provide important insights into the design of bio-inspired materials.

The Zr-1.0Sn-0.25Nb-0.2Fe-0.1Cr alloys are prepared by low temperature processing (LTP) and high temperature processing (HTP), respectively. The microstructure, aqueous corrosion behavior and hydrogen uptake of the two different samples are investigated. The results show that higher hot rolling temperature causes the growth of the second phase particles (SPPs), which remain in the final microstructure of the Zr matrix. Presence of large SPPs promotes the formation and propagation of cracks in the oxide scale, leading to the higher corrosion rate and higher hydrogen uptake fraction of the HTP samples than LTP samples in the later stage of the corrosion test.

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii , the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii .

Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites.

Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii's 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii. Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting.

Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron–sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe–2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron–sulfur protein assembly (CIA) pathway includes the essential Cfd1–Nbp35 scaffold complex that assembles a [4Fe–4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes., The functions of the highly reduced mitochondria (mitosomes) of microsporidians are not well-characterized. Here, the authors show that the Trachipleistophora hominis mitosome is the site of iron–sulfur cluster assembly and that its retention is likely linked to its role in cytosolic and nuclear iron–sulfur protein maturation.

In this work, Pb(In1/2Nb1/2)O3-Pb(Mg1/3Nb2/3)O3-PbTiO3 (PIN-PMN-PT) single crystal/epoxy 1–3 composites with different thicknesses (400 μm to 825 μm) were fabricated using the conventional dice-and-fill method. Their properties were compared with the corresponding lead zirconate titanate (PZT) ceramic 1–3 composites. Excellent properties for ultrasonic transducer applications have been achieved, such as high electromechanical coupling coefficient (kt ≈ 78% to 83%), high piezoelectric strain coefficient (d33 ≈ 1000 pm/V), and lower acoustic impedance (Z ≈ 20 Mrayl). The strain levels of PIN-PMN-PT composites were almost constant (1000 pm/V) with decreasing thickness, being much higher than those of PZT composites (650 pm/V). However, an increase in strain hysteresis was observed with decreasing thickness, reaching 25.3% for the 400-μm single-crystal 1–3 composite, which is lower than the corresponding PZT composites (44.1% for 350-μm PZT ceramic 1–3 composite). These results show that PIN-PMN-PT single-crystal 1–3 composites have great potential for use in advanced ultrasound transducer applications.

Summary Ferredoxins are highly conserved proteins that func- tion universally as electron transporters. They not only require Fe-S clusters for their own activity, but are also involved in Fe-S formation itself. We identified two homologues of ferredoxin in the genome of the parasitic protist Trypanosoma brucei and named them TbFdxA and TbFdxB. TbFdxA protein, which is homologous to other eukaryotic mitochondrial ferre- doxins, is essential in both the procyclic (= insect- transmitted) and bloodstream (mammalian) stage, but is more abundant in the active mitochondrion of the former stage. Depletion of TbFdxA caused disruption of Fe-S cluster biogenesis and lowered the level of intracellular haem. However,TbFdxB, which is present exclusively within kinetoplastid flagellates, was non-essential for the procyclic stage, and double knock-down with TbFdxA showed this was not due to functional redundancy between the two homologues. Heterologous expressions of human orthologues HsFdx1 and HsFdx2 fully rescued the growth and Fe-S-dependent enzymatic activities of TbFdxA knock-down. In both cases, the genuine human import signals allowed efficient import into theT. brucei mito- chondrion. Given the huge evolutionary distance between trypanosomes and humans, ferredoxins clearly have ancestral and highly conserved function in eukaryotes and both human orthologues have retained the capacity to participate in Fe-S cluster assembly.

Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.

Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.

Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei , its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasite’s cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress.

IscA/Isa proteins function as alternative scaffolds for the assembly of Fe-S clusters and/or provide iron for their assembly in prokaryotes and eukaryotes. Isa are usually non-essential and in most organisms are confined to the mitochondrion. We have studied the function of TbIsa1 and TbIsa2 in Trypanosoma brucei, where the requirement for both of them to sustain cell growth depends on the life cycle stage. The TbIsa proteins are abundant in the procyclic form, which contains an active organelle. Both proteins are indispensable for growth, as they are required for the assembly of Fe-S clusters in mitochondrial aconitase, fumarase and succinate dehydrogenase. Reactive oxygen species but not iron accumulate in the procyclic mitochondrion upon ablation of the TbIsa proteins, but their depletion does not influence the assembly of Fe-S clusters in cytosolic proteins. In the bloodstream form, which has a downregulated mitochondrion, the TbIsa proteins are non-essential. The Isa2 orthologue of the anaerobic protist Blastocystis partially rescued the growth and enzymatic activities of TbIsa1/2 knock-down. Rescues of single knock-downs as well as heterologous rescues with human Isa orthologues partially recovered the activities of aconitase and fumarase. These results show that the Isa1 and Isa2 proteins of diverse eukaryotes have overlapping functions.

Kinetoplastids encode a single nuclear tryptophanyl tRNA that contains a CCA anticodon able to decode the UGG codons used in cytoplasmic protein synthesis but cannot decode the mitochondrial UGA codons. Following mitochondrial import, this problem is circumvented in Trypanosoma brucei by specifically editing the tRNA(Trp) anticodon to UCA, which can now decode the predominant mitochondrial UGA tryptophan codons. This tRNA also undergoes an unusual thiolation at position 33 of the anticodon loop, the only known modification at U33 in any tRNA. In other organisms, tRNA thiolation is mediated by the cysteine desulfurase, Nfs1 (IscS). However, T. brucei encodes two Nfs homologues, one cytoplasmic and the other mitochondrial. We show by a combination of RNA interference and Northern and Western analyses that the mitochondria-targeted TbNfs, and not TbNfs-like protein, is essential for thiolation of both cytosolic and mitochondrial tRNAs. Given the exclusive mitochondrial localization of TbNfs, how it mediates thiolation in the cytoplasm remains unclear. Furthermore, thiolation specifically affects thiolated tRNA stability in the cytoplasm but more surprisingly acts as a negative determinant for the essential C to U editing in T. brucei. This provides a first line of evidence for mitochondrial C to U editing regulation in this system.

Frataxin is a conserved mitochondrial protein, almost universally present in prokaryotes and eukaryotes, where it is implicated in Fe-S cluster assembly and several other processes. Here we show that frataxins from the diatom Thalassiosira pseudonana and the plant Arabidopsis thaliana are efficiently targeted and processed in the mitochondrion of the evolutionary distant excavate kinetoplastid flagellate Trypanosoma brucei. Moreover, both heterologous frataxins are able to rescue a lethal deficiency for T. brucei frataxin.

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion., Author summary One of the most common motifs that binds calcium to transduce intracellular signals is called an EF hand- named after the globular domain structure first characterized in ovalbumin. A conserved cluster of four EF hands, each of which that binds one calcium atom, is a conserved feature of calmodulin, centrins, and calmodulin-like proteins, including myosin light chains. Although the presence of EF hands is predictive of calcium binding, it alone does not allow classification of biological function as this set of conserved proteins have very diverse functions. Here we used modified editing procedures based on CRISPR/Cas9 combined with a plant-like degradation system to define the roles of three calmodulin-like proteins in T. gondii. These proteins all localized to a specialized apical structure called the conoid where they overlap with the motor protein called MyoH. Additionally, biochemical and genetic studies suggest they coordinately regulate cell invasion. These new genomic editing tools, combined with an efficient system for protein degradation, expand the functional tool kit for an analysis of essential genes and proteins in T. gondii.

Ferredoxins are highly conserved proteins that function universally as electron transporters. They not only require Fe-S clusters for their own activity, but are also involved in Fe-S formation itself. We identified two homologues of ferredoxin in the genome of the parasitic protist Trypanosoma brucei and named them TbFdxA and TbFdxB. TbFdxA protein, which is homologous to other eukaryotic mitochondrial ferredoxins, is essential in both the procyclic (= insect-transmitted) and bloodstream (mammalian) stage, but is more abundant in the active mitochondrion of the former stage. Depletion of TbFdxA caused disruption of Fe-S cluster biogenesis and lowered the level of intracellular haem. However, TbFdxB, which is present exclusively within kinetoplastid flagellates, was non-essential for the procyclic stage, and double knock-down with TbFdxA showed this was not due to functional redundancy between the two homologues. Heterologous expressions of human orthologues HsFdx1 and HsFdx2 fully rescued the growth and Fe-S-dependent enzymatic activities of TbFdxA knock-down. In both cases, the genuine human import signals allowed efficient import into the T. brucei mitochondrion. Given the huge evolutionary distance between trypanosomes and humans, ferredoxins clearly have ancestral and highly conserved function in eukaryotes and both human orthologues have retained the capacity to participate in Fe-S cluster assembly.

IscA/Isa proteins function as alternative scaffolds for the assembly of Fe-S clusters and/or provide iron for their assembly in prokaryotes and eukaryotes. Isa are usually non-essential and in most organisms are confined to the mitochondrion. We have studied the function of TbIsa1 and TbIsa2 in Trypanosoma brucei, where the requirement for both of them to sustain cell growth depends on the life cycle stage. The TbIsa proteins are abundant in the procyclic form, which contains an active organelle. Both proteins are indispensable for growth, as they are required for the assembly of Fe-S clusters in mitochondrial aconitase, fumarase and succinate dehydrogenase. Reactive oxygen species but not iron accumulate in the procyclic mitochondrion upon ablation of the TbIsa proteins, but their depletion does not influence the assembly of Fe-S clusters in cytosolic proteins. In the bloodstream form, which has a downregulated mitochondrion, the TbIsa proteins are non-essential. The Isa2 orthologue of the anaerobic protist Blastocystis partially rescued the growth and enzymatic activities of TbIsa1/2 knock-down. Rescues of single knock-downs as well as heterologous rescues with human Isa orthologues partially recovered the activities of aconitase and fumarase. These results show that the Isa1 and Isa2 proteins of diverse eukaryotes have overlapping functions.

YCF45 belongs to a family of proteins of unknown function usually located in the chloroplast of plants. Its highly conserved homologues were found in the genomes of several Trypanosoma and Leishmania species. HA(3)-tagging of the YCF45 protein with the start codon as annotated in the Gene(DB) revealed its cytosolic localization in the cultured procyclic stage of Trypanosoma brucei. However, when a more upstream located start codon was used in another HA(3)-tagged construct, the resulting protein was targeted to the mitochondrion. We propose that YCF45 was acquired by an ancestral trypanosomatid by horizontal gene transfer and in the absence of a plastid was re-targeted to the mitochondrion.

Trypanosoma brucei , the agent of human sleeping sickness and ruminant nagana, is the most genetically tractable representative of the domain Excavata. It is evolutionarily very distant from humans, with a last common ancestor over 1 billion years ago. Frataxin, a highly conserved small protein involved in iron-sulfur cluster synthesis, is present in both organisms, and its deficiency is responsible for Friedreich's ataxia in humans. We have found that T. brucei growth-inhibition phenotype caused by down-regulated frataxin is rescued by means of human frataxin. The rescue is fully dependent on the human frataxin being imported into the trypanosome mitochondrion. Processing of the imported protein by mitochondrial processing peptidase can be blocked by mutations in the signal peptide, as in human cells. Although in human cells frataxin must be processed to execute its function, the same protein in the T. brucei mitochondrion is functional even in the absence of processing. Our results illuminate remarkable conservation of the mechanisms of mitochondrial protein import and processing.

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's 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.