Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.     

Trichomonas vaginalis: identification of a triacylglycerol acylhydrolase

This work describes the identification of a triacylglycerol lipase named TVLip directly onto blood-LB-agar plates by hemolytic screening of a Trichomonas vaginalis cDNA expression library. Sharing significant similarity in the primary sequence with other lipases, the theoretical 3D structure of the TVLip was resolved. The structure reveals the predictive conserved characteristics of other lipases from EC3.1.1.3 group, although presenting one amino acid change in the catalytic triad Ser-His-Asp. Finally, analysis of Northern blot indicates that the expression of the TVLip gene is up-regulated by iron.     

Purification and characterization of a membrane-associated phospholipase A2 from rat spleen. Its comparison with a cytosolic phospholipase A2 S-1

A membrane-associated phospholipase A2 was purified from rat spleen. The phospholipase A2 was solubilized from the 108,000 x g pellet fraction with 0.3% lithium dodecyl sulfate and then purified to homogeneity by successive DEAE-Cellulofine AM, octyl-Sepharose, Cellulofine GCL 300-m, S-Sepharose, and Bio-Gel P-30 chromatographies in the presence of 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate. The apparent Mr of the enzyme, estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, was about 13,600. The purified enzyme had a pH optimum in the range of pH 8.0-9.5 and required the presence of Ca2+ (4 mM) for its maximal activity. The enzyme preferentially hydrolyzed the 2-acyl ester bonds of phosphatidylglycerol in the presence and absence of sodium cholate or sodium deoxycholate. Unlike the phospholipase A2 of rat spleen supernatant, no immunocross-reactivity was observed between the purified enzyme and anti-rat pancreatic phospholipase A2 antibody. The N-terminal amino acid sequence of the enzyme was determined and found to be homologous to that of viperid and crotalid venom phospholipases A2. The results in this and the preceding report (Tojo, H., Ono, T., Kuramitsu, S., Kagamiyama, H., and Okamoto, M. (1988) J. Biol. Chem. 263, 5724-5731) demonstrate that rat spleen contains two genetically distinct phospholipase A2 isoenzymes.     

Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.     

Hydrolysis of membrane-associated phosphoglycerides by mitochondrial phospholipase A2

Conversion of membrane-bound substrates by membrane-associated enzymes can proceed in principle via intramembrane and intermembrane action. By using rat-liver mitochondria containing labeled phosphatidylethanolamine and inactivated phospholipase A2 as substrate source, and mitochondria containing unlabeled substrate and active enzyme, it is shown that hydrolysis of phosphatidylethanolamine by mitochondrial phospholipase A2 proceeds nearly entirely via intramembrane enzyme action. A study of the characteristics of this mode of enzyme action showed that all mitochondrial phosphoglycerides were hydrolyzed. Plots of approximate initial velocities of hydrolysis against the remaining amounts of each individual phospholipid, indicated that phosphatidylethanolamine was hydrolyzed fastest, with a rate about twice that for phosphatidylcholine and about 10-fold that for cardiolipin. The initial rates remained nearly constant in the initial phase of the hydrolysis, suggesting that the enzyme is surrounded by excess substrate.     

Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.     

The identification of a potent, water soluble inhibitor of lipoprotein-associated phospholipase A2

Modification of the pyrimidone 5-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, lipophilic inhibitors of lipoprotein-associated phospholipase A2, has given inhibitors of nanomolar potency and improved physicochemical properties. Compound 23 was identified as a potent, highly water soluble. CNS penetrant inhibitor suitable for intravenous administration.     

A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ～ 160 Mb) has been sequenced and is composed of ～65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.     

Comparison of phospholipase activity with direct and indirect lytic effects of animal venoms upon human red cells.

1. The venoms of 22 species of arthropods, saurians, elapids and crotalids were studied concerning the phospholipase activity and the presence of a direct and an indirect lytic effect upon human red cells. 2. The venoms from the spiders Latrodectus and "tarantula", and the venoms from the scorpions of the genus Centruroides are not haemolytic and do not have phospholipase activity. 3. Only the venoms of Apis mellifera and Naja naja siamensis have shown direct lytic effect. 4. All other venoms studied have an indirect haemolytic effect associated to a phospholipase activity, but there is indication that other agents might be implicated in the haemolytic processes.     

Purification and some properties of membrane-associated phospholipase A2 of human spleen

Membrane-associated phospholipase A2 was purified to homogeneity from human spleen. The enzyme was solubilized from the particulate fraction by the addition of KBr, and purified by reverse-phase high-performance liquid chromatography. The estimated molecular weight of the enzyme was 14,000. The enzyme had a pH optimum around 9.5, required the presence of Ca2+ for its activity, and hydrolyzed phosphatidylethanolamine more efficiently than phosphatidylcholine.     

A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

Transposons are sequence elements widely distributed among genomes of all three kingdoms of life,providing genomic changes and playing significant roles in genome evolution.Trichomonas vaginalis is an excellent model system for transposon study since its genome(～160 Mb) has been sequenced and is composed of～65%transposons and other repetitive elements.In this study,we primarily report the identification of Kolobok-type transposons(termed tvBac) in T.vaginalis and the results of transposase sequence analy...     

Aspartate:2-oxoglutarate aminotransferase from Trichomonas vaginalis: comparison with pig heart cytoplasmic enzyme

The aspartate:2-oxoglutarate aminotransferase from the protozoon Trichomonas vaginalis exists as a mixture of sub-forms of identical Mr and amino acid composition, and of similar catalytic properties. The amino acid composition closely resembles that of aspartate aminotransferase from prokaryotic and vertebrate sources. Some molecular and catalytic properties of the T. vaginalis aspartate aminotransferase are compared with those of the cytoplasmic pig heart enzyme. A major difference is in the ability of the trichomonal enzyme to transaminate aromatic amino acids and 2-oxo acids. A range of inhibitors have been used to compare the active-site regions of the T. vaginalis and cytoplasmic pig heart aspartate aminotransferases.     

Trichomonas vaginalis: in vitro attachment and internalization of HIV-1 and HIV-1-infected lymphocytes

Sexually transmitted diseases (STDs) caused by bacteria and protozoa play an important role in the epidemiology of human immunodeficiency virus (HIV-1) infection. Human trichomoniasis, produced by the protozoan parasite Trichomonas vaginalis, is one of the most common STDs, and is a cause of mucosal lesions in the urogenital tract, which may increase the risk for HIV infection. However, there are no reports concerning the outcome of in vitro interactions between HIV particles and trichomonads. Therefore, we incubated T. vaginalis with three subtypes of HIV-1 (A, B, and D), as well as with HIV-1-infected lymphocytes, and analyzed the interactions with immunofluorescence microscopy and transmission electron microscopy. Our results demonstrated that HIV-1 particles attach and are incorporated into T. vaginalis through endocytic vesicles and are degraded within cytoplasmic vacuoles in approximately 48 h. There was no ultrastructural evidence of HIV-1 replication in trichomonads. These results demonstrated that trichomonads may internalize and harbor HIV-1 particles for short periods of time. In addition, under in vitro conditions, T. vaginalis ingests and digests HIV-1-infected lymphocytes.     

Quercetin modulates activities of Taiwan cobra phospholipase A2 via its effects on membrane structure and membrane-bound mode of phospholipase A2

The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A2 (PLA2) activities. Sphingomyelin inhibited PLA2 enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA2 differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA2 with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA2 interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA2.     

Beta-succinyl-coenzyme A synthetase from Trichomonas vaginalis is a soluble hydrogenosomal protein with an amino-terminal sequence that resembles mitochondrial presequences.

We describe studies directed toward understanding the biogenesis and origin of the hydrogenosome, an unusual organelle found exclusively in certain anaerobic eukaryotes that lack mitochondria. Hydrogenosomes are involved in fermentative carbohydrate metabolism and are proposed to have arisen through conversion of mitochondria or via endosymbiosis with an anaerobic bacterium. We cloned a gene encoding the beta subunit of the hydrogenosomal protein succinyl-coenzyme A synthetase (beta-SCS) and isolated the protein from Trichomonas vaginalis. The T. vaginalis beta-SCS gene encodes a protein with a calculated molecular mass of 43,980 Da that has 43% amino acid identity (65% similarity) with beta-SCS from Escherichia coli. The trichomonad protein partitions into the soluble fraction of hydrogenosomes treated with sodium carbonate at high pH, consistent with a matrix localization within the organelle. The protein is encoded by a multigene family composed of at least three members. Amino-terminal sequencing of beta-SCS purified from T. vaginalis hydrogenosomes shows that the mature protein lacks the first nine amino acids encoded in the gene. This apparent amino-terminal leader sequence is strikingly similar to that of another hydrogenosomal protein and to mitochondrial presequences.     

Modification of phospholipase C and phospholipase A2 activities during poliovirus infection

The infection of HeLa cells by poliovirus leads to profound alterations in the activities of both phospholipase C and the A23187-stimulated phospholipase A2. As early as the third hour after poliovirus infection, the activity of phospholipase C is enhanced, as measured by the increase in inositol triphosphate (IP3) in the cells. By the fifth hour post-infection there is a 5-fold increase in IP3 in the infected cells. Therefore, the synthesis of the bulk of poliovirus proteins and poliovirus genomes takes place in cells containing a high and sustained increase in IP3. This augmentation in IP3 is dependent on the multiplicity of infection used. Poliovirus gene expression is required to induce the increase in phospholipase C activity, since the presence of cycloheximide or guanidine blocked it. In contrast to the activation of phospholipase C induced by poliovirus, there is a drastic blockade of the A23187-induced phospholipase A2 activity, measured as the release of [3H]arachidonic acid to the medium. This action on phospholipase A2 is dependent on poliovirus gene expression because it was prevented by cycloheximide or 3-methylquercetin. To our knowledge this is the first report analyzing these two activities in animal virus-infected cells. The findings described may help to explain the profound modifications of both membrane permeability and lipid metabolism undergone by poliovirus-infected cells.     

Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2

Recently, we demonstrated that ceramide kinase, and its product, ceramide 1-phosphate (Cer-1-P), were mediators of arachidonic acid released in cells in response to interleukin-1beta and calcium ionophore (Pettus, B. J., Bielawska, A., Spiegel, S., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2003) J. Biol. Chem. 278, 38206-38213). In this study, we demonstrate that down-regulation of cytosolic phospholipase A(2) (cPLA(2)) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA(2) is the key phospholipase A(2) downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 microm) induced the translocation of full-length cPLA(2) from the cytosol to the Golgi apparatus/perinuclear regions, which are known sites of translocation in response to agonists. Cer-1-P also induced the translocation of the CaLB/C2 domain of cPLA(2) in the same manner, suggesting that this domain is responsive to Cer-1-P either directly or indirectly. In vitro studies were then conducted to distinguish these two possibilities. In vitro binding studies disclosed that Cer-1-P interacts directly with full-length cPLA(2) and with the CaLB domain in a calcium- and lipid-specific manner with a K(Ca) of 1.54 microm. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA(2) enzymatic activity as well as lowering the EC(50) of calcium for the enzyme from 191 to 31 nm. This study identifies Cer-1-P as an anionic lipid that translocates and directly activates cPLA(2), demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.     

1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.