Jorge Tovar

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.

The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica

Ultrastructural analysis of Entamoeba histolytica reveals that this intestinal human pathogen lacks recognizable mitochondria, but the presence in its genome of genes encoding proteins of mitochondrial origin suggests the existence of a mitochondrially derived compartment. We have cloned the full‐length E. histolytica gene encoding one such protein, chaperonin CPN60, and have characterized its structure and expression. Using an affinity‐purified antibody raised against recombinant protein, we have localized native E. histolytica CPN60 to a previously undescribed organelle of putative mitochondrial origin, the mitosome. Most cells contain only one mitosome, as determined by immunofluorescence studies. Entamoeba histolytica CPN60 has an amino‐terminal extension reminiscent of known mitochondrial and hydrogenosomal targeting signals. Deletion of the first 15 amino acids of CPN60 leads to an accumulation of the truncated protein in the cytoplasm. However, this mutant phenotype can be reversed by replacement of the deleted amino acids with a mitochondrial targeting signal from Trypanosoma cruzi HSP70. The observed functional conservation between mitochondrial import in trypanosomes and mitosome import in Entamoeba is strong evidence that the E. histolytica organelle housing chaperonin CPN60 represents a mitochondrial remnant.

Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages

Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR‐knockout mutants by gene disruption in L.donovani and L.major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L.donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR‐containing region to a larger chromosome. Mutants of L.donovani and L.major possessing only one wild‐type TR allele express less TR mRNA and have lower TR activity compared with wild‐type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates.

Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol‐redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine‐activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti‐leishmanial agents for chemotherapeutic use.

Protein import, replication and inheritance of a vestigial mitochondrion

Mitochondrial remnant organelles (mitosomes) that exist in a range of “amitochondrial” eukaryotic organisms represent ideal models for the study of mitochondrial evolution and for the establishment of the minimal set of proteins required for the biogenesis of an endosymbiosis-derived organelle. Giardia intestinalis, often described as the earliest branching eukaryote, contains double membrane-bounded structures involved in iron-sulfur cluster biosynthesis, an essential function of mitochondria. Here we present evidence that Giardia mitosomes also harbor Cpn60, mtHsp70, and ferredoxin and that despite their advanced state of reductive evolution they have retained vestiges of presequence-dependent and -independent protein import pathways akin to those that operate in mammalian mitochondria. Although import of IscU and ferredoxin is still reliant on their amino-terminal presequences, targeting of Giardia Cpn60, IscS, or mtHsp70 into mitosomes no longer requires cleavable presequences, a derived feature from their mitochondrial homologues. In addition, we found that division and segregation of a single centrally positioned mitosome tightly associated with the microtubular cytoskeleton is coordinated with the cell cycle, whereas peripherally located mitosomes are inherited into daughter cells stochastically.

Mitochondrion-Derived Organelles in Protists and Fungi

The mitochondrion is generally considered to be a defining feature of eukaryotic cells, yet most anaerobic eukaryotes lack this organelle. Many of these were previously thought to derive from eukaryotes that diverged prior to acquisition of the organelle through endosymbiosis. It is now known that all extant eukaryotes are descended from an ancestor that had a mitochondrion and that in anaerobic eukaryotes the organelle has been modified into either hydrogenosomes, which continue to generate energy for the host cell, or mitosomes, which do not. These organelles have each arisen independently several times. Recent evidence suggests a shared derived characteristic that may be responsible for the retention of the organelles in the absence of the better-known mitochondrial functions--iron-sulfur cluster assembly. This review explores the events leading to this new understanding of mitochondrion-derived organelles in amitochondriate eukaryotes, the current state of our knowledge, and future areas for investigation.

A Novel ADP/ATP Transporter in the Mitosome of the Microaerophilic Human Parasite Entamoeba histolytica

Recent data suggest that microaerophilic and parasitic protozoa, which lack oxidative phosphorylation, nevertheless contain mitochondrial homologs [1-6], organelles that share common ancestry with mitochondria. Such widespread retention suggests there may be a common function for mitochondrial homologs that makes them essential for eukaryotic cells. We determined the mitochondrial carrier family (MCF) complement of the Entamoeba histolytica mitochondrial homolog, also known as a crypton [5] or more commonly as a mitosome [3]. MCF proteins support mitochondrial metabolic energy generation, DNA replication, and amino-acid metabolism by linking biochemical pathways in the mitochondrial matrix with those in the cytosol [7]. MCF diversity thus closely mirrors important facets of mitochondrial metabolic diversity. The Entamoeba histolytica mitosome has lost all but a single type of MCF protein, which transports ATP and ADP via a novel mechanism that is not reliant on a membrane potential. Phylogenetic analyses confirm that the Entamoeba ADP/ATP carrier is distinct from archetypal mitochondrial ADP/ATP carriers, an observation that is supported by its different substrate and inhibitor specificity. Because many functions of yeast and human mitochondria rely on solutes transported by specialized members of this family, the Entamoeba mitosome must contain only a small subset of these processes requiring adenine nucleotide exchange.

The iron-sulfur cluster assembly genes iscS and iscU of Entamoeba histolytica were acquired by horizontal gene transfer

BackgroundIron-sulfur (FeS) proteins are present in all living organisms and play important roles in electron transport and metalloenzyme catalysis. The maturation of FeS proteins in eukaryotes is an essential function of mitochondria, but little is known about this process in amitochondriate eukaryotes. Here we report on the identification and analysis of two genes encoding critical FeS cluster (Isc) biosynthetic proteins from the amitochondriate human pathogen Entamoeba histolytica.ResultsE. histolytica IscU and IscS were found to contain all features considered essential for their biological activity, including amino acid residues involved in substrate and/or co-factor binding. The IscU protein differs significantly from other eukaryotic homologs and resembles the long type isoforms encountered in some bacteria. Phylogenetic analyses of E. histolytica IscS and IscU showed a close relationship with homologs from Helicobacter pylori and Campylobacter jejuni, to the exclusion of mitochondrial isoforms.ConclusionsThe bacterial-type FeS cluster assembly genes of E. histolytica suggest their lateral acquisition from epsilon proteobacteria. This is a clear example of horizontal gene transfer (HGT) from eubacteria to unicellular eukaryotic organisms, a phenomenon known to contribute significantly to the evolution of eukaryotic genomes.

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria.

The assembly of vital reactive iron‐sulfur (Fe‐S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial‐type Fe‐S cluster assembly proteins and possess instead an analogous bacterial‐type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial‐type Fe‐S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10‐fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe‐S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe‐S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe‐S protein‐mediated oxygen detoxification.

Evidence from bioinformatics, expression and inhibition studies of phosphoinositide-3 kinase signalling in Giardia intestinalis

BackgroundGiardia intestinalis is a parasitic protozoan and major cause of diarrhoeal disease. Disease transmission is dependent on the ability of the parasite to differentiate back and forth between an intestine-colonising trophozoite and an environmentally-resistant infective cyst. Our current understanding of the intracellular signalling mechanisms that regulate parasite replication and differentiation is limited, yet such information could suggest new methods of disease control. Phosphoinositide-3 kinase (PI3K) signalling pathways have a central involvement in many vital eukaryotic processes, such as regulation of cell growth, intracellular membrane trafficking and cell motility. Here we present evidence for the existence of functional PI3K intracellular signalling pathways in G. intestinalis.ResultsWe have identified and characterised two genes, Gipi3k1 and Gipi3k2, which encode putative PI3Ks. Both genes are expressed in trophozoites and encysting cells, suggesting a possible role of GiPI3K1 and GiPI3K2 in regulating giardial growth and differentiation. Extensive nucleotide and amino acid sequence characterisation predicts that both encoded PI3Ks are functional as indicated by the presence of highly conserved structural domains and essential catalytic residues. The inhibitory effect of the PI3K inhibitor LY294002 on trophozoite proliferation also supports their functionality. Phylogenetic analysis supports the identity of GiPI3K1 as a Class I isoform and GiPI3K2 as a Class III isoform. In addition, giardial genes encoding putative homologues of phosphoinositide-metabolising enzymes such as PTEN, MTM, PIPkin and PI 5-phosphatase as well as downstream effectors with phosphoinositide-binding domains have been identified, placing GiPI3K1 and GiPI3K2 in a broader signalling context. Compared with twenty-six PI3Ks from other organisms, GiPI3K1 and GiPI3K2 are unique in that they contain large insertions within their highly conserved kinase domains. The function of these insertions is unknown; however we show here that they are not intron-derived and would probably not hinder substrate binding. These insertions may represent a plausible drug target.ConclusionG. intestinalis encodes and expresses two putative PI3Ks, at least one of which appears to be required during normal parasite proliferation. The identification of Class I and Class III but not Class II isoforms suggests that both extracellularly-initiated signalling (Class I-regulated) and intracellular vesicle trafficking (Class III-regulated) might be controlled by PI3Ks in G. intestinalis. The presence of genes encoding putative homologues of phosphoinositide-metabolising enzymes and downstream effectors in the G. intestinalis genome further suggests that the overall architecture of PI3K signalling may be comparable with pathways present in other better-studied organisms.