Glycogen phosphorylase sequences from the amitochondriate protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis

Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.     

In vitro effect of nitazoxanide against Entamoeba histolytica,Giardia intestinalis and Trichomonas vaginalis trophozoites

Nitazoxanide, a 5-nitrothiazolyl derivative, is effective in the treatment of a broad range of parasitic infections. In vitro, it is active against several protozoa, including Cryptosporidium parvum, Blastocystis hominis, and Giardia intestinalis. The objective of this study was to determine the in vitro effect of nitazoxanide on the growth and morphology of three anaerobic protozoa (Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis) and to compare these effects with those of metronidazole and albendazole. A subculture method was used to determine the concentrations required to inhibit growth by 50% or 90% (IC50 and IC90,). Nitazoxanide exhibited IC50, and IC90 values of 0.017 and 0.776 microg/ml respectively, against E. histolytica, 0.004 and 0.067 microg/ml against G. intestinalis, and 0.034 and 2.04 6 microg/ml against T. vaginalis. Based on the IC90 values, nitazoxanide was more toxic than metronidazole and albendazole against E. histolytica; albendazole and nitazoxanide were more toxic than metronidazole against G. intestinalis; and metronidazole was the most toxic drug against T. vaginalis. The effects of nitazoxanide on trophozoite ultrastructure of all three parasites included cell swelling and distorted cell shape, a redistribution of vacuoles, plasma membrane damage, and the formation of extensive empty areas in the cytoplasm of the protozoa.     

Evolutionary relationships of the glucokinase from the amitochondriate protist, Trichomonas vaginalis

Two genes coding for Trichomonas vaginalis glucokinase were isolated and sequenced. The putative translation products have molecular masses of 41,584 and 41,772 Da, corresponding to 375 and 377 amino acids, respectively. These values agree with data determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the enzyme purified from the organism. The two sequences showed 78% amino acid identity. The sequences and their phylogenetic reconstruction show that they are members of a glucokinase/fructokinase protein family found in eubacteria and also in the eukaryote Giardia lamblia and are only distantly related to typical eukaryotic hexokinases. The results indicate that the evolutionary past of this enzyme, catalyzing the first step of glycolysis in T. vaginalis, is different from that of the enzyme performing this key role in almost all other eukaryotes.     

Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma brucei

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.     

YI-S, a Casein-free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia , and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica . In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.     

A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote,Trichomonas vaginalis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD⁺ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica—Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium. Correspondence to: M. Müller     

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.     

The Giardia lamblia actin gene and the phylogeny of eukaryotes

The single-copy actin gene of Giardia lamblia lacks introns; it has an average of 58% amino acid identity with the actin of other species; and 49 of its amino acids can be aligned with the amino acids of a consensus sequence of heat shock protein 70. Analysis of the potential RNA secondary structure in the transcribed region of the G. lamblia actin gene and of the single-copy actin gene of nine other species did not reveal any conserved structures. The G. lamblia actin sequence was used to root the phylogenetic trees based on 65 actin protein sequences from 43 species. This tree is congruent with small-subunit rRNA trees in that it shows that oomycetes are not related to higher fungi; that kinetoplatid protozoans, green plants, fungi and animals are monophyletic groups; and that the animal and fungal lineages share a more recent common ancestor than either does with the plant lineage. In contrast to smalls-ubunit rRNA trees, this tree shows that slime molds diverged after the plant lineage. The slower rate of evolution of actin genes of slime molds relative to those of plants, fungi, and animals species might be responsible for this incongruent branching. Correspondence to: G. Drouin     

Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.     

Sequence analysis of genes encoding ribosomal proteins of amitochondriate protists: L1 of Trichomonas vaginalis and L29 of Giardia lamblia

Two genes encoding the ribosomal proteins were cloned and sequenced from amitochondriate protists, L1 (L10a in mammalian nomenclature) from Trichomonas vaginalis and L29 (L35 in mammalian nomenclature) from Giardia lamblia. The deduced amino acid sequences were analyzed by sequence alignments and phylogenetic reconstructions. Both the T. vaginalis L1 and the G. lamblia L29 displayed eukaryotic sequence features, when compared with all the homologs from the three primary kingdoms.     

Methyl jasmonate induces cell cycle block and cell death in the amitochondriate parasite Trichomonas vaginalis

Jasmonates are a group of small lipids produced in plants and function as stress hormones. They are selectively cytotoxic against cancer cells. Methyl jasmonate (MJ), one of the naturally occurring jasmonates, has direct mitochondriotoxic effects, strongly suggesting that mitochondria are target organelles of jasmonates. We have previously shown that jasmonates are cytotoxic to two human parasites -Schistosoma mansoni and Plasmodium falciparum. Both the cancer cells and the parasites mentioned above possess mitochondria. The present work aimed to examine whether jasmonates are able to damage cells lacking mitochondria, e.g., some unicellular human parasitic flagellates. We found that MJ induced death of the amitochondriate Trichomonas vaginalis parasites. MJ caused fragmentation and condensation of the DNA of T. vaginalis, resembling phenomena associated with apoptotic death. However, DNA laddering, a sub-G(1) cell cycle stage peak and caspase-3 activation were not observed. Thus, MJ-induced T. vaginalis cell death appears to be non-apoptotic. We found that MJ induced cell cycle block at the G(2)/M phase in T. vaginalis, similar to the effect of metronidazole. We examined the influence of MJ on the bioenergetic pathways of T. vaginalis, and found that depletion of ATP did not precede death of the parasites, but rather reflected it. Nevertheless, 2-deoxy-d-glucose, a glycolysis blocker, was synergistic with MJ in causing death of T. vaginalis cells, suggesting that MJ does perturb the bioenergetic homeostasis of the parasites. Finally, MJ was found to be cytotoxic towards a metronidazole-resistant strain of T. vaginalis (ATCC 50143), suggesting that it may be effective for the treatment of nitroimidazole-refractory trichomoniasis. In conclusion, MJ was found to exhibit mitochondria-independent cytotoxicity and presents a potentially novel agent against T. vaginalis.     

Lycorine induces cell death in the amitochondriate parasite, Trichomonas vaginalis, via an alternative non-apoptotic death pathway

In this study, the mechanism of action of the pro-apoptotic alkaloid lycorine on an amitochondriate cell, the parasite Trichomonas vaginalis, was investigated. The cytotoxicity of lycorine against T. vaginalis was studied from 2.5 to 1000 μM and several important ultrastructural alterations were observed by electron microscopy. Lycorine arrested the T. vaginalis cell cycle, although no hallmarks of apoptosis, such as apoptotic bodies, were observed. Consequently, the underlying mechanism of action fails to completely fulfill the criteria for apoptosis. However, some similarities to paraptotic cell death were observed.     

The phylogeny of the hominoid primates,as indicated by DNA-DNA hybridization

Summary The living hominoid primates are Man, the chimpanzees, the Gorilla, the Orangutan, and the gibbons. The cercopithecoids (Old World monkeys) are the sister group of the hominoids. The composition of the Hominoidea is not in dispute, but a consensus has not yet been reached concerning the phylogenetic branching pattern and the dating of divergence nodes. We have compared the single-copy nuclear DNA sequences of the hominoid genera using DNA-DNA hybridization to produce a complete matrix of delta T₅₀H values. The data show that the branching sequence of the lineages, from oldest to most recent, was: Old World monkeys, gibbons, Orangutan, Gorilla, chimpanzees, and Man. The calibration of the delta T₅₀H scale in absolute time needs further refinement, but the ranges of our estimates of the datings of the divergence nodes are: Cercopithecoidea, 27–33 million years ago (MYA); gibbons, 18–22 MYA; Orangutan, 13–16 MYA; Gorilla, 8–10 MYA; and chimpanzees-Man, 6.3–7.7 MYA.     

Cell division of Giardia intestinalis: assembly and disassembly of the adhesive disc, and the cytokinesis

Trophozoites of Giardia are equipped with a special organelle of attachment, essential for parasite survival and pathogenicity, the ventral disc. Although its basic structure is well established, its reorganization and assembly during cell replication is poorly understood. We addressed some of these problems with aid of conventional, confocal and electron microscopy. We found that dividing Giardia alternates attached and free swimming phases in accordance with functional competence of the parent or newly assembled discs. The division started in attached cells by detachment of the disc microtubules from basal bodies. Shortening and eventual loss of the giardin microribbons, and unfolding of the microtubular layer resulting in collapse of the disc chamber and parasite detachment underlined gradual disassembly of the parent disc skeleton. Two daughter discs assembled on the dorsal side of the attached cell, with their ventral sides exposed on the parent cell surface and their microtubular skeletons growing in counter-clockwise direction. A depression between the assembling discs marked the cleavage plane. The splitting continued during the free-swimming phase with ventral-ventral axial symmetry in a plane of the daughter discs. Finally, the daughter cells with fully developed discs but still connected tail to tail by a cytoplasmic bridge, attached to a substrate and terminated the division by a process resembling adhesion-dependent cytokinesis. The mode of assembly of the daughter discs and plane of the division is compatible with maintenance of the left-right asymmetry of the Giardia cytoskeleton in progeny, which cannot be satisfactorily explained by alternative models proposed so far.     

Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.     

Assembly of iron-sulfur clusters mediated by cysteine desulfurases,IscS, CsdB and CSD,from Escherichia coli

Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine. The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters. Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD. We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro. Since IscU, an iron-sulfur protein of E. coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU. By surface plasmon resonance analysis, we found that only IscS interacts with IscU. We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.     

Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells

Iron-sulfur (Fe-S) clusters are cofactors found in many proteins that have important redox, catalytic or regulatory functions. In mammalian cells, almost all known Fe-S proteins are found in the mitochondria, but at least one is found in the cytosol. Here we report cloning of the human homologs to IscU and NifU, iron-binding proteins that play a critical role in Fe-S cluster assembly in bacteria. In human cells, alternative splicing of a common pre-mRNA results in synthesis of two proteins that differ at the N-terminus and localize either to the cytosol (IscU1) or to the mitochondria (IscU2). Biochemical analyses demonstrate that IscU proteins specifically associate with IscS, a cysteine desulfurase that is proposed to sequester inorganic sulfur for Fe-S cluster assembly. Protein complexes containing IscU and IscS can be found in the mitochondria as well as in the cytosol, implying that Fe-S cluster assembly takes place in multiple subcellular compartments in mammalian cells. The possible roles of the IscU proteins in mammalian cells and the potential implications of compartmentalization of Fe-S cluster assembly are discussed.     

Biochemical characterization of iron-sulfur cluster assembly in the scaffold IscU of Escherichia coli

Iron-sulfur cluster is one of the most common prosthetic groups, and it functions in numerous biological processes. However, little is currently known about the mechanisms of iron-sulfur cluster biosynthesis. In this study, we cloned and purified iron-sulfur cluster assembly proteins from Escherichia coli and assembled the cluster in vitro. The results showed that the assembly of iron-sulfur cluster is completed in about 20 min. Although iron or sulfur binds with IscU equivalently, 2-fold amount of iron or cysteine compared with that of IscU is better for the cluster formation, while high concentrations of IscS (IscS/IscU > 1: 10) do not facilitate the cluster formation. Environmental pH plays an important role in iron-sulfur cluster assembly; the cluster was well assembled at pH 7.6–8.0, but was inhibited at pH less than 7.4. On supply of a catalytic amount of IscS (1/50 of IscU) and excess of other substrates, with increasing each of IscU, iron, or cysteine concentration, the iron-sulfur cluster assembly process developed from first order reaction, mixed order reaction to zero order reaction, and up to 64% of apo-IscU was converted to the [2Fe-2S] cluster-bound IscU under the optimal laboratory conditions.