Lidya B. Sánchez

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.

Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis.

Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced. GPI of the parabasalid Trichomonas vaginalis was also sequenced. The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T. vaginalis, a Type II amitochondriate protist. These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica. G. intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs. The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes. The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes. The observation that the two diplomonads and T. vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids. The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria.

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.

Purification and characterization of the acetate forming enzyme, acetyl-CoA synthetase (ADP-forming) from the amitochondriate protist, Giardia lamblia

Giardia lamblia, an amitochondriate eukaryote, contains acetyl‐CoA synthetase (ADP‐forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes. The enzyme has been purified about 350‐fold. The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate. The reverse reaction could not be detected. Succinyl‐CoA, propionyl‐CoA and dADP were utilized with lower efficiency. The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed acetyl‐CoA synthetase (AMP‐forming). The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G. lamblia metabolism.

Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships.

Sequences of putative fructose-1,6-bisphospate aldolases (FBA) in five amitochondriate unicellular eukaryotes, the diplomonads Giardia intestinalis (published earlier) and Spironucleus barkhanus, the pelobiont Mastigamoeba balamuthi,the entamoebid Entamoeba histolytica, and the parabasalid Trichomonas vaginalis all belong to Class II of FBAs and are highly similar to each other (>48% amino acid identity). The five protist sequences, however, do not form a monophyletic group. Diplomonad FBAs share a most recent common ancestor, while FBAs of the three other protist species are part of a lineage that also includes sequences from a few eubacteria (Clostridium difficile, Treponema pallidum, Chlorobium tepidum). Both clades are part of the Type B of Class II aldolases, a complex that contains at least three additional lineages (subgroups) of enzymes. Type B enzymes are distant from Type A Class II aldolases, which consists of a number of bacterial and fungal enzymes and also contains the cytosolic FBA of Euglena gracilis. Class II aldolases are not homologous to Class I enzymes, to which animal and plant enzymes belong. The results indicate that amitochondriate protists acquired their FBAs from separate and different sources, involving lateral gene transfer from eubacteria, than did all other eukaryotes studied so far and underscore the complex composition of the glycolytic machinery in unicellular eukaryotes.

Cloning and sequencing of an acetyl-CoA synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.

A Giardia lamblia gene, Glacs, was cloned, sequenced and expressed in Escheria Coli. This gene codes for a 726 residue long acetyl-CoA synthetase (ADP-forming). This enzyme is responsible for the formation of acetate, a metabolic endproduct of G. lamblia. It is known from only two Type I amitochondriate eukaryotes, G. lamblia and Entamoeba histolytica and from the archaebacterium, Pyrococcus furiosus. With Glacs as query, homologous unidentified open reading frames were detected in the complete genomes of only a few archaebacteria and eubacteria. These form a new protein family present in all three domains of life, which probably plays a central role in the acyl-CoA metabolism but is of restricted taxonomic distribution.

Archaebacterial Relationships of the Phosphoenolpyruvate Carboxykinase Gene Reveal Mosaicism of Giardia intestinalis Core Metabolism

Abstract A gene encoding a putative GTP-specific phosphoenolpyruvate carboxykinase has been cloned and sequenced from the type I amitochondriate protist Giardia intestinalis. The deduced amino acid sequence is related most closely to homologs from hyperthermophilic archaebacteria and only more distantly to homologs from Eubacteria and Metazoa. Most enzymes of Giardia core metabolism, however, are related more closely to eubacterial and metazoan homologs. An archaebacterial relationship has been noted previously for the unusual acetyl-CoA synthetase (ADP-forming) of this organism. The results suggest that phosphoenolpyruvate carboxykinase and acetyl-CoA synthetase have been acquired from different sources than most enzymes of Giardia core metabolism.

NAD(P)H:menadione oxidoreductase of the amitochondriate eukaryote Giardia lamblia: a simpler homologue of the vertebrate enzyme

The amitochondriate eukaryote Giardia lamblia contains an NAD(P)H:menadione oxidoreductase (EC 1.6.99.2) (glQR) that catalyses the two-electron transfer oxidation of NAD(P)H with a quinone as acceptor. The gene encoding this protein in G. lamblia was expressed in Escherichia coli. The purified recombinant protein had an NAD(P)H oxidoreductase activity, with NADPH being a more efficient electron donor than NADH. Menadione, naphthoquinone and several artificial electron acceptors served as substrate for the enzyme. glQR shows high amino acid similarity to its homologues in vertebrates and also to a series of hypothetical proteins from bacteria. Although glQR is considerably smaller than the mammalian enzymes, three-dimensional modelling shows similar arrangement of the secondary structural elements. Most amino acid residues of the mammalian enzymes that participate in substrate binding or catalysis are conserved. Conservation of these features and the similarity in substrate specificity and in susceptibility to inhibitors establish glQR as an authentic member of this protein family.

SEQUENCE OF A MALIC ENZYME GENE OF GIARDIA LAMBLIA

The nucleotide sequence and predicted amino acid sequence of malate dehydrogenase (decarboxylating) or malic enzyme (EC 1.1.1.40) of the amitochondriate protist Giardia lamblia were determined. The overall amino acid identity with malic enzyme sequences from other eukaryotes was between 34 and 39%. Functional domains previously defined in other malic enzymes, the malate-, the ADP- and the NAD(P)-binding domains, were present also in the G. lamblia sequence. In phylogenetic reconstructions, the G. lamblia sequence is part of the eukaryotic clade, but its relative position versus the other early branches of the eukaryotic tree (Trichomonas vaginalis hydrogenosome and plant mitochondria) cannot be firmly established. The results indicate, however, a long, independent evolutionary past of this enzyme.

Comparative analysis of the ribosomal components of the hydrogenosome-containing protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.