Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PP_i)/inorganic phosphate (P_i) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PP_i formation but not in the direction of acetyl phosphate/P_i formation suggests that the physiological direction of the reaction is toward acetate/PP_i production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PP_i-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.     

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO₂ to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PP_i)-type PEPCK (PP_i-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PP_i-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PP_i-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PP_i-PEPCK activity. The primary structure of PP_i-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PP_i-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PP_i-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.     

Utilization of PPi as an Energy Source by a Clostridium sp

The growth of an anaerobic, spore-forming rod we have isolated from the cockroach gut after enrichment on media containing PP_i was stimulated by the presence of PP_i. The doubling time decreased and cell yield increased proportionately to PP_i concentrations of up to 0.35%. A similar stimulation of the growth of Desulfotomaculum sp. by PP_i has been reported. The PP_i-stimulated Clostridium sp. fermented a number of sugars with the production of hydrogen, acetate, and butyrate, with smaller amounts of ethanol and butanol being produced from some substrates. The fermentation products were not qualitatively changed by the presence of PP_i, but significantly more hydrogen was produced. The organism contained several of the enzymes previously reported from Entamoeba sp. and Propionibacterium sp., in which PP_i serves as a source of a high-energy bond in place of ATP. These include significant amounts of pyruvate-phosphate dikinase and phosphoenolpyruvate carboxytransphosphorylase. The activities of many of the catabolic enzymes of the organism, as well as of its phosphatases and pyrophosphatase, were similar whether it was grown in the presence or absence of PP_i. The organism did not accumulate intracellular polyphosphate granules but stored large amounts of glycogen.     

Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PP_i-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PP_i were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PP_i displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.     

Crystal structures of acetate kinases from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans

Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamily and catalyze the reversible phosphorylation of acetate, with ADP/ATP the most common phosphoryl acceptor/donor. While prokaryotic ACKs have been the subject of extensive biochemical and structural characterization, there is a comparative paucity of information on eukaryotic ACKs, and prior to this report, no structure of an ACK of eukaryotic origin was available. We determined the structures of ACKs from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans. Each active site is located at an interdomain interface, and the acetate and phosphate binding pockets display sequence and structural conservation with their prokaryotic counterparts. Interestingly, the E. histolytica ACK has previously been shown to be pyrophosphate (PP_i)-dependent, and is the first ACK demonstrated to have this property. Examination of its structure demonstrates how subtle amino acid substitutions within the active site have converted cosubstrate specificity from ATP to PP_i while retaining a similar backbone conformation. Differences in the angle between domains surrounding the active site suggest that interdomain movement may accompany catalysis. Taken together, these structures are consistent with the eukaryotic ACKs following a similar reaction mechanism as is proposed for the prokaryotic homologs.     

Uniqueness of Entamoeba sulfur metabolism: sulfolipid metabolism that plays pleiotropic roles in the parasitic life cycle

Sulfur metabolism is ubiquitous and terminally synthesizes various biomolecules that are crucial for organisms, such as sulfur‐containing amino acids and co‐factors, sulfolipids and sulfated saccharides. Entamoeba histolytica, a protozoan parasite responsible for amoebiasis, possesses the unique sulfur metabolism features of atypical localization and its terminal product being limited to sulfolipids. Here, we present an overall scheme of E. histolytica sulfur metabolism by relating all sulfotransferases and sulfatases to their substrates and products. Furthermore, a novel sulfur metabolite, fatty alcohol disulfates, was identified and shown to play an important role in trophozoite proliferation. Cholesteryl sulfate, another synthesized sulfolipid, was previously demonstrated to play an important role in encystation, a differentiation process from proliferative trophozoite to dormant cyst. Entamoeba survives by alternating between these two distinct forms; therefore, Entamoeba sulfur metabolism contributes to the parasitic life cycle via its terminal products. Interestingly, this unique feature of sulfur metabolism is not conserved in the nonparasitic close relative of Entamoeba, Mastigamoeba, because lateral gene transfer‐mediated acquisition of sulfatases and sulfotransferases, critical enzymes conferring this feature, has only occurred in the Entamoeba lineage. Hence, our findings suggest that sulfolipid metabolism has a causal relationship with parasitism.     

Acetate kinase (pyrophosphate). A fourth pyrophosphate-dependent kinase from Entamoeba histolytica.

An enzyme from Entamoeba histolytica catalyzes the formation of acetyl phosphate and orthophosphate from acetate and inorganic pyrophosphate (PP_i), but it displays much greater activity in the direction of acetate formation. It has been purified 40-fold and separated from interfering enzyme activities by chromatography. Its reaction products have been quantitatively established. ATP cannot replace PP_i as phosphoryl donor in the direction of acetyl phosphate formation nor will any common nucleoside diphosphate replace orthophosphate as phosphoryl acceptor in the direction of acetate formation. The trivial name proposed for the new enzyme is acetate kinase (PP_i).     

Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3′-Phosphoadenosine 5′-Phosphosulfate and ATP

Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3′-phosphoadenosine 5′-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

PPi-Dependent Phosphofructokinase from Thermoproteus tenax,an Archaeal Descendant of an Ancient Line in Phosphofructokinase Evolution

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PP_i-dependent PFK (PP_i-PFK; EC 2.7.1.90), which uses PP_i instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PP_i-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PP_i-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PP_i-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PP_i-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PP_i as a cosubstrate.As first discovered in Entamoeba histolytica (27), in some members of all three domains of life (Bacteria, Eucarya, and Archaea), the first committing step of glycolysis, the phosphorylation of fructose 6-phosphate (Fru 6-P), is catalyzed not by common ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11) but by an enzyme which uses PP_i as a phosphoryl donor (PP_i-PFK; EC 2.7.1.90) (22–34). The only archaeal PP_i-PFK described so far is the enzyme of Thermoproteus tenax, a hyperthermophilic, anaerobic archaeon which is able to grow chemolithotrophically with CO₂, H₂, and S⁰, as well as chemo-organothrophically in the presence of S⁰ and carbohydrates (11, 41). As shown by enzymatic and in vivo studies (pulse-labeling experiments), T. tenax metabolizes glucose mainly via a variation of the Embden-Meyerhof-Parnas pathway distinguished by the reversible PP_i-PFK reaction (34, 35).In contrast to the virtually irreversible reaction catalyzed by the ATP-PFK, the phosphorylation by PP_i is reversible. Thus, for thermodynamic reasons, the PP_i-PFK should be able to replace the enzymes of both the forward (ATP-PFK) and reverse (fructose-bisphosphatase [FBPase]) reactions. Consistent with the presumed bivalent function of the PP_i-dependent enzyme, in prokaryotes and parasitic protists which possess PP_i-PFK, little, if any, ATP-PFK or FBPase activity is present. Strikingly, the PP_i-PFKs of these organisms proved to be nonallosteric, suggesting that the control of the carbon flux through the pathway is no longer exerted by the PFK in these organisms. A considerably different situation has been described for higher plants and the green alga Euglena gracilis, showing comparable ATP-PFK, FBPase, and PP_i-PFK activities and allosteric regulation of their PP_i-dependent enzyme by fructose 2,6-bisphosphate (12, 22). However, in most cases it is not obvious which physiological role PP_i-PFK performs: reversible catalysis of glycolysis/gluconeogenesis, increase of the energy yield of glycolysis under certain conditions in which the energy charge is low, or ATP-conservation in obligately fermentative organisms (22).Closely related to questions concerning the biological function of PP_i-PFKs is the matter of their evolutionary origin: are these enzymes the result of a secondary adaptation from ATP-PFKs, or do they represent an original phenotype, as suggested by their specificity for PP_i, which is thought to be an ancient source of metabolic energy (9, 18, 19, 26). Indicated by sequence similarity (3, 4), all known PP_i- and ATP-PFKs are homologous and therefore originated from a common ancestral root. From more recent studies of Streptomyces coelicolor PFK (4), the previous assumption of a single event which separated PP_i- and ATP-PFKs had to be revised in favor of a multiple differentiation, leaving open, however, the question of the primary or secondary origin of PP_i-PFK.Understanding of PFK evolution has been impaired by a lack of knowledge concerning archaeal PFK, although the existence of ATP-PFK (31), PP_i-PFK (34), and also ADP-dependent PFK (16, 31) in Archaea has been described. To address the evolution of PFK, we describe the PP_i-PFK from T. tenax and compare its sequence and structure to those of known bacterial and eucaryal PFK enzymes.     

Entamoeba histolytica infections in wild and semi-wild orangutans in Sumatra and Kalimantan

Key to the success of orangutan conservation management practices is the prevention of the introduction of infectious diseases to the remaining populations. Previous reports of Entamoeba spp. positive orangutans are of concern as Entamoeba spp. infection has been linked to morbidity and mortality in primates. It remains to be determined if the Entamoeba species infecting orangutans is the pathogenic Entamoeba histolytica. Orangutan fecal samples have been collected from orangutans from sites in Sumatra (Bukit Lawang, Ketambe, and Suaq, 241 samples from 64 individuals), and two sites in Kalimantan (Sebangau and Tuanan, 129 samples from 39 individuals). All samples were from wild orangutans except for a proportion from Sumatra which were from semi-wild (108 samples, 10 individuals). E. histolytica-specific nested PCR assays were carried out on the fecal samples. A total of 36 samples from 17 individuals tested positive for E. histolytica. When compared with published sequences using NCBI BLAST the E. histolytica positive samples showed a 98–99% concordance. The majority (76%, n = 36) of the positive isolates came from semi-wild orangutans in Bukit Lawang. This study supports the growing body of evidence that contact with humans is an important risk factor for infection of wild primates with E. histolytica.     

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

In vitro construction of the tufB-lacZ fusion: analysis of the regulatory mechanism of tufB promoter

Summary Genetic studies suggest that the so-called phosphorus-family of enzymes inN. crassa are controlled by a complex system of regulatory genes which are responsive to the level of phosphorus in the growth medium. The intracellular metabolite(s) that interact with this system to signal changes in the external phosphorus concentration has not been identified. In this study the pools of acid-soluble, phosphorus-containing, compounds are measured in wild-type and phosphorus-family enzyme regulatory mutant strains ofN. crassa before and during phosphorus starvation.Prolonged phosphorus starvation of wild-typeN. crassa failed to alter significantly the pre-starvation level of intracellular orthophosphate, suggesting that intracellular P_i would be a poor effector signal for the control of the phosphorus family enzymes. However, inorganic pyrophosphate (PP_i) decreased 15-fold, and tri- and tetrapolyphosphate (PPP_i and PPPP_i) increased 3- to 5-fold within 15 minutes after transfer of the wild-type strain to phosphorus-free medium. Phosphate starvation of seven different regulatory gene mutant strains resulted in a rapid decrease in the PP_i pool similar to that which occurred in the wild-type. However, only two of these seven strains showed increased PPP_i and PPPP_i pools following phosphate starvation. Additional experiments demonstrated that PP_i pools, but not PPP_i and PPPP_i pools, were unaffected by several starvation regimens other than phosphorus starvation. Metabolic studies employing H₃ ³²PO₄ showed that the pool of PP_i was labeled to steady-state levels after two minutes of continuous labeling of a phosphate-sufficient culture. Furthermore, long-term steady-state labeling showed that the intracellular PP_i pool was directly responsive to the decrease in the extracellular P_i concentration of the medium resulting from cell growth. Growth on phosphoethanolamine, a phosphorus source that allows a modest degree of derepression even in growing cells, resulted in lower levels of PP_i than were seen in phosphate-grown cells. These observations suggest that PP_i may be involved in the mechanism responsible for the control of phosphorus-family enzyme regulatory gene product activity.     

Dramatic Increase in Glycerol Biosynthesis upon Oxidative Stress in the Anaerobic Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica, a microaerophilic enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. Trophozoites of E. histolytica are exposed to a variety of reactive oxygen and nitrogen species during infection. Since E. histolytica lacks key components of canonical eukaryotic anti-oxidative defense systems, such as catalase and glutathione system, alternative not-yet-identified anti-oxidative defense strategies have been postulated to be operating in E. histolytica. In the present study, we investigated global metabolic responses in E. histolytica in response to H₂O₂- and paraquat-mediated oxidative stress by measuring charged metabolites on capillary electrophoresis and time-of-flight mass spectrometry. We found that oxidative stress caused drastic modulation of metabolites involved in glycolysis, chitin biosynthesis, and nucleotide and amino acid metabolism. Oxidative stress resulted in the inhibition of glycolysis as a result of inactivation of several key enzymes, leading to the redirection of metabolic flux towards glycerol production, chitin biosynthesis, and the non-oxidative branch of the pentose phosphate pathway. As a result of the repression of glycolysis as evidenced by the accumulation of glycolytic intermediates upstream of pyruvate, and reduced ethanol production, the levels of nucleoside triphosphates were decreased. We also showed for the first time the presence of functional glycerol biosynthetic pathway in E. histolytica as demonstrated by the increased production of glycerol 3-phosphate and glycerol upon oxidative stress. We proposed the significance of the glycerol biosynthetic pathway as a metabolic anti-oxidative defense system in E. histolytica.     

PPiase-Activated ATP-Dependent H+ Transport at the Tonoplast of Mesophyll Cells of the CAM Plant Kalanchoë daigremontiana*

The co-ordinated action of the two proton-transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PP_iase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PP_i-dependent H⁺ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H⁺ pumps were non-additive in establishing the steady-state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP-dependent H⁺ transport were stimulated enormously if ATP was added a few minutes after pre-energization of the vesicles with PP_i. This stimulation was observed only when the PP_iase was active. A similar effect was not found for PP_i-dependent H⁺ transport after pre-energization with ATP. Hence, a PP_iase-activated ATP-dependent H⁺ transport can be distinguished from the basic ATP- and the basic PP_i-dependent H⁺ transport. In parallel a PP_i-dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PP_iase. PP_iase-activated ATP-dependent H⁺ transport depends on the presence of permeant anions. It shows properties of both H⁺ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP-dependent H⁺ transport activity, the nitrate stimulation and the KF inhibition of the PP_i-dependent H⁺ transport activity. Only MgPP_i and MgATP were effective as the respective substrates. The PP_iase-activated ATP-dependent H⁺ transport had a half life of about 5–9 minutes. It is concluded that the PP_iase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed.     

<Emphasis Type="Italic">Entamoeba histolytica</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> infections in captive macaques (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>) in the Philippines

Entamoeba histolytica is a protozoan parasite that infects man and animals. This parasite has a global distribution and the disease it causes is usually characterized by diarrhea. In order to detect the parasite, it is necessary to differentiate it from Entamoeba dispar. E. dispar appears morphologically similar to E. histolytica but does not cause disease and tissue invasion. This study reports on the prevalence of E. histolytica and E. dispar among captive macaques in a primate facility in the Philippines. PCR was used to correctly identify both Entamoeba species. Indirect fluorescent antibody test (IFAT) was also performed to determine the seroprevalence of amebiasis in the captive macaques. Based on PCR targeting of the peroxiredoxin gene, of the 96 stool samples collected, 23 (24%) contained E. histolytica while 32 (33%) contained E. dispar. IFAT revealed 26 (27%) serum samples positive for antibodies against E. histolytica. Sequence analysis of the 18S rRNA gene showed that the 23 E. histolytica isolates were identical to human E. histolytica isolates deposited in the GenBank and not Entamoeba nuttalli as found in macaques in other recent reports. The Philippines is a major exporter of monkeys for biomedical research purposes, so screening animals before transporting them to other locations lessens the risk of spreading zoonoses to a wider area. This is the first report of the molecular detection of E. histolytica and E. dispar among macaques in the Philippines. This study complements the limited information available on the animal hosts of E. histolytica in the Philippines.     

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP_i-dependent phosphofructokinase (TvPP_i-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP_i-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.     

Thermotoga maritima phosphofructokinases: expression and characterization of two unique enzymes

A pyrophosphate-dependent phosphofructokinase (PP_i-PFK) and an ATP-dependent phosphofructokinase (ATP-PFK) from Thermotoga maritima have been cloned and characterized. The PP_i-PFK is unique in that the K_m and V_max values indicate that polyphosphate is the preferred substrate over pyrophosphate; the enzyme in reality is a polyphosphate-dependent PFK. The ATP-PFK was not significantly affected by common allosteric effectors (e.g., phosphoenolpyruvate) but was strongly inhibited by PP_i and polyphosphate. The results suggest that the control of the Embden-Meyerhof pathway in this organism is likely to be modulated by pyrophosphate and/or polyphosphate.     

Eukaryotic checkpoints are absent in the cell division cycle ofEntamoeba histolytica

Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite,Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered inE. histolytica.     

Pathogenic and Non-pathogenic Entamoeba: Pore Formation and Hemolytic Activity1

Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the-capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E. histolytica irrespective of the virulence of the particular strain, but low in non-pathogenic E. histolytica-like amebas of human origin as well as in E. invadens, which is pathogenic for reptiles, and in E. moshkovskii isolated from sewage. We conclude that the capacities to insert pores and to lyse are not sufficient for virulence although they may be necessary. The subcellular distribution of the hemolytic activity of E. histolytica and its sensitivity to a variety of inhibitors and activators differ from those of other known amebic cytotoxic activities including pore formation. Therefore, there may be an additional constituent of E. histolytica involved in the cytotoxicity of the parasite.