Pyruvate produced by Brugia spp. via glycolysis is essential for maintaining the mutualistic association between the parasite and its endosymbiont, Wolbachia.

Human parasitic nematodes are the causative agents of lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness), diseases that are endemic to more than 80 countries and that consistently rank in the top ten for the highest number of years lived with disability. These filarial nematodes have evolved an obligate mutualistic association with an intracellular bacterium, Wolbachia, a symbiont that is essential for the successful development, reproduction, and survival of adult filarial worms. Elimination of the bacteria causes adult worms to die, making Wolbachia a primary target for developing new interventional tools to combat filariases. To further explore Wolbachia as a promising indirect macrofilaricidal drug target, the essential cellular processes that define the symbiotic Wolbachia-host interactions need to be identified. Genomic analyses revealed that while filarial nematodes encode all the enzymes necessary for glycolysis, Wolbachia does not encode the genes for three glycolytic enzymes: hexokinase, 6-phosphofructokinase, and pyruvate kinase. These enzymes are necessary for converting glucose into pyruvate. Wolbachia, however, has the full complement of genes required for gluconeogenesis starting with pyruvate, and for energy metabolism via the tricarboxylic acid cycle. Therefore, we hypothesized that Wolbachia might depend on host glycolysis to maintain a mutualistic association with their parasitic host. We did conditional experiments in vitro that confirmed that glycolysis and its end-product, pyruvate, sustain this symbiotic relationship. Analysis of alternative sources of pyruvate within the worm indicated that the filarial lactate dehydrogenase could also regulate the local intracellular concentration of pyruvate in proximity to Wolbachia and thus help control bacterial growth via molecular interactions with the bacteria. Lastly, we have shown that the parasite's pyruvate kinase, the enzyme that performs the last step in glycolysis, could be a potential novel anti-filarial drug target. Establishing that glycolysis is an essential component of symbiosis in filarial worms could have a broader impact on research focused on other intracellular bacteria-host interactions where the role of glycolysis in supporting intracellular survival of bacteria has been reported.

Intravascular filarial parasites elaborate cyclooxygenase-derived eicosanoids.

The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Since prostanoids formed from arachidonic acid can modulate interactions among platelets, leukocytes, and endothelial cells, we examined whether intravascular nematode parasites can elaborate prostanoids. Microfilariae of Brugia malayi utilize exogenous and endogenous arachidonic acid to generate and release two predominant prostanoids, prostacyclin and prostaglandin E2. Filarial metabolism of host fatty acids to form these vasodilatory, antiaggregatory, and immunomodulatory eicosanoids provides a means by which these helminthic parasites may influence host immune and other cellular responses.

Physicochemical characteristics of non-electrolytes and their uptake by Brugia pahangi and Dipetalonema viteae.

The uptake of a diverse set of 14C-labelled non-electrolytes by Brugia pahangi and Dipetalonema viteae was measured relative to the free diffusion of tritiated water. Inulin was used as a non-absorbable surface marker to account for non-electrolyte adherent to the surface of the parasite which had not crossed the cuticle. B. pahangi and D. viteae took up the non-electrolytes to a similar degree; a comparison of tissue uptake indices gave a correlation coefficient of 0.99. Worm uptake could not be described by non-electrolyte octanol/aqueous partition coefficients alone. However, greater success was achieved using further descriptors and pattern recognition techniques for data analysis. The whole molecule descriptors log P, molar refraction, melting point, dipole moment and CNDO total energy were obtained from computer chemistry and the literature. Using a linear learning machine to relate uptake to these 5 physicochemical descriptors it was possible to successfully classify non-electrolytes as high or low uptake. Multivariate regression analysis of uptake versus these 5 parameters gave a correlation coefficient of 0.77. However, this was not statistically significant and therefore could not be used for quantitative predictions of substance uptake by worms. This illustrates the value of 'pattern recognition' techniques such as the linear learning machine. Using such 'pattern recognition' methods on a chemically related set of compounds it is anticipated that predictions of uptake can be achieved and improved upon. Such predictions could then be used in drug design.

N-acetylation of serotonin, octopamine and dopamine by adult Brugia pahangi.

The metabolism of biogenic amines by the filarial worm, Brugia pahangi, was investigated by incubating cut worms with radio-labelled amine substrates. Two-dimensional thin-layer chromatography and analysis on two high-performance liquid chromatography systems showed that [14C]5-hydroxytryptamine was metabolised to a less polar compound that was identified as N-acetyl 5-hydroxytryptamine. N-Acetyloctopamine and N-acetyldopamine were also formed when cut B. pahangi were incubated with [14C]octopamine and [3H]dopamine, respectively. N-Acetyltransferase activity towards 5-hydroxytryptamine was readily detected in nematode homogenates. This enzyme was localised in a 50,000 x g supernatant and required the addition of the co-substrate, acetyl CoA, for activity. No evidence was obtained for the involvement of monoamine oxidases in the metabolism of 5-HT in these filarial worms.

Chitin synthesis and sheath morphogenesis in Brugia malayi microfilariae.

Brugia malayi microfilariae and gravid adult females were examined to determine whether chitin (poly beta(1----4)-linked N-acetylglucosamine) is a structural component of the microfilarial sheath. Two lectins which are specific for beta(1----4)-linked oligomers of N-acetylglucosamine bind to the sheaths of living microfilariae. Diflubenzuron, a potent inhibitor of chitin synthesis in insects and crustaceans, causes gravid female worms to shed progeny microfilariae with truncated sheaths. A chitin-like fraction (hot alkali-insoluble and chitinase-sensitive) can be isolated from gravid female (but not male) worms. This fraction can be metabolically labelled with radioactive glucosamine, but such labelling is inhibited by diflubenzuron. These data suggest that chitin synthesis is critical to microfilarial sheath morphogenesis in this parasitic nematode.

Turnover of the surface proteins of adult and third and fourth stage larval Brugia pahangi.

The turnover of surface proteins in adults, fourth-stage and third-stage larvae of Brugia pahangi was measured using [125I]iodosulfanilic acid. Groups of worms (n = 10 adult, 20 L4, 50 L3) were labelled and surgically implanted into the peritoneal cavity of naive jirds. The amount of radioactivity remaining on worms recovered over a 7-8 day period was determined. Adult females showed no significant loss of label during a 7 day period. The recovery of fourth-stage larvae was low but the counts per minute remaining on each group of larvae recovered over an 8 day period, encompassing the major part of the instar, did not fall below the limits of the standard deviation of the time 0 groups, indicating that no significant loss of surface label had occurred. Third-stage larvae showed a significant loss of 125I-labelled proteins prior to the third moult, although it was not confirmed that these proteins occur on the worm surface. Electrophoresis and autoradiography of labelled homogenates of adult, fourth and third stage larvae suggested that [125I]iodosulfanilic acid labels polypeptides of different molecular weights on each life cycle stage of B. pahangi.

Synthesis of ubiquinone 9 by adult Brugia pahangi and Dirofilaria immitis: evidence against its involvement in the oxidation of 5-methyltetrahydrofolate.

Among various ubiquinone (Q) isoprenologues tested, only Q7 was more efficient than menadione in promoting the oxidation of 5-methyltetrahydrofolate (CH3FH4) by 5,10-methylenetetrahydrofolate reductase isolated from adult Brugia pahangi, whereas Q10 was the best cofactor in the same reaction catalysed by the analogous enzyme from adult Dirofilaria immitis. Menoctone (3-[1-cyclohexyloctyl] -2-hydroxy-1,4-naphthoquinone) was a strong competitive inhibitor of both these ubiquinone isoprenologues in the respective reactions. When incubated in the presence of D,L-[14C]-mevalonate, adult B. pahangi and D. immitis synthesized radiolabelled Q9 only, in addition to other isoprenoid derivatives in the neutral lipid fraction. In view of the inability of Q9 to promote the oxidation of CH3FH4 by 5,10-methylenetetrahydrofolate reductase from B. pahangi, it seems unlikely that this filaria uses Q9 as a cofactor in this reaction. Conceivably, D. immitis could use Q9 as a cofactor in its enzymatic oxidation of CH3FH4, since in this circumstance, it was a better cofactor than menadione.

Thymidine kinase activity and thymidine salvage in adult Brugia pahangi and Dirofilaria immitis.

Cytosolic thymidine kinase (EC 2.7.1.75), the initial enzyme in the thymidine salvage pathway, was detected in crude homogenates of adult female Brugia pahangi and Dirofilaria immitis, with respective specific activities of 100 and 460 nmol/h/mg protein. Partially purified filarial thymidine kinases were found to have molecular weights of approximately 180 000, to be most active in the presence of Mg2+ and ATP, to have a sharp pH optimum (pH 7.0) and to be heat-labile in the absence of added thymidine. For both, the respective Km values for thymidine and ATP were 60 muM and 1.6 mM, and 5-iodo-2'-deoxyuridine was as good a substrate as thymidine. A distinguishing property was the 3-fold higher sensitivity of the B. pahangi enzyme to feedback inhibition by thymidine 5'-triphosphate. Adult female B. pahangi took up and incorporated [methyl-3 H] thymidine into DNA when they were exposed to this radiolabeled deoxynucleoside in vivo, but the thymidine salvage pathway in these worms was essentially nonfunctional in vitro.

Rapid uptake and esterification of arachidonic acid and other fatty acids by microfilariae of Brugia malayi.

We have examined the differential incorporation and esterification of exogenous fatty acids by microfilariae of the human filarial parasite Brugia malayi. Microfilariae incubated with 2 nM [3H]arachidonic acid over 1 h rapidly took up this fatty acid. Palmitic, oleic and linoleic acids were also incorporated by parasites. In contrast to these other fatty acids, little incorporated arachidonic acid remained as free fatty acid within microfilariae. Arachidonate was rapidly esterified into phospholipids, with 66% of incorporated arachidonate esterified into phospholipids at 1 min. Esterification of other fatty acids into phospholipids was quantitatively lesser and occurred into phosphatidylcholine and phosphatidylethanolamine. Arachidonate was preferentially esterified into phosphatidylinositol, which constituted only 10% of the total parasite phospholipid pool, and into phosphatidylcholine. By 1 min these two phospholipid classes, respectively, comprised 53% and 43% of [3H]arachidonyl-phospholipids. Neither the microfilarial incorporation of arachidonate nor its esterification into parasite phospholipids could be saturated by noncytotoxic concentrations of up to 600 microM. Microfilariae, which in vivo are exposed to arachidonate in blood, can rapidly, avidly and with high capacity incorporate exogenous arachidonate and esterify it preferentially into specific classes of phospholipids, including phosphatidylinositol. Like many mammalian cells, these phylogenetically distinct metazoan parasites possess efficient means for utilizing host-derived arachidonic acid.

An NMR study on the effect of glucose availability on carbohydrate metabolism in Dipetalonema viteae and Brugia pahangi.

Adult Brugia pahangi and Dipetalonema viteae utilise a percentage of absorbed glucose (ca. 15%) in the formation of the disaccharide trehalose [8]. This paper reports an investigation, employing 13C-NMR techniques, of the utilisation of trehalose by these nematodes and also the effect of glucose availability on metabolic product composition. The metabolism of [1-13C]trehalose in D. viteae differed dramatically from that of [1-13C]glucose under normal experimental conditions. A succinate/lactate ratio of 0.73 was obtained from the metabolism of [1-13C]trehalose compared with 0.05 from [1-13C]glucose at an initial concentration of ca. 5 mM. Similar, but less consistent, results were obtained from B. pahangi adults. Macrofilariae of D. viteae were fed variable, low levels of glucose at hourly intervals for 8 h, and a significant relationship (P less than 0.001) between the glucose addition rate and the ratio of succinate to lactate production was obtained. The lower the amount of glucose added each hour, the higher was the observed succinate to lactate ratio. The percentage yield of succinate increased greatly as the amount of added glucose was diminished. Parallel experiments performed on B. pahangi macrofilariae indicated that B. pahangi did not increase their succinate output so greatly with reduced glucose availability. It is clear that in the absence of available external glucose, B. pahangi and D. viteae draw on their internal trehalose reserves as a source of carbohydrate for energy generation.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation of the glucose metabolism of Brugia pahangi and Dipetalonema viteae by nuclear magnetic resonance spectroscopy.

This study followed the metabolism of [13C]glucose under anaerobic and aerobic conditions in the adult filarial nematodes Brugia pahangi and Dipetalonema viteae using non-invasive 13C nuclear magnetic resonance techniques. Adult B. pahangi and D. viteae showed a rapid uptake of labelled glucose which remained linear over at least 4 h. Both species of worm removed significantly more glucose from the medium under aerobic conditions than under anaerobic conditions. The principal product of metabolism, under both anaerobic and aerobic conditions, was lactate, which accounted for 62-71% of the original [13C]glucose. Examination of the maintenance medium following worm incubation revealed a further excretory product which was identified as succinate. This product accounted for 1-2% of labelled glucose in adult B. pahangi and 2-5% in adult D. viteae. The presence of succinate as an excretory product suggests that a partial reversed tricarboxylic acid cycle is active in these filarial nematodes. A further peak was identified in the worm homogenate and identified as trehalose. The disaccharide was not an excretory product and occurred only within the worm. The peak accounted for 13-14% of the 13C-labelled glucose in B. pahangi and 15-16% in D. viteae. Trehalose has not been previously recorded in either of these nematodes and is likely to have a storage function.

Investigation of ecdysteroid excretion by adult Dirofilaria immitis and Brugia pahangi.

The excretion of ecdysteroids by the filarial nematode species, Dirofilaria immitis and Brugia pahangi, was examined both in vitro, by the analysis of culture medium, and in vivo, through analysis of serum samples from experimentally infected hosts. There was no evidence of ecdysteroid excretion by intact parasites of either species in vitro. Free ecdysteroids were detected in the serum of ferrets and dogs infected with D. immitis, but concentrations would be at or below the limit of detection in sub-millilitre serum samples. The detection of ecdysteroids in the serum of potential hosts is unlikely to be of value in the diagnosis of filarial infections due to a combination of low titre in the presence of current infection and measurable titre in its absence. Ecdysteroids of dietary origin may contribute to the latter.

Involvement of tetrahydrofolate cofactors in de novo purine ribonucleotide synthesis by adult Brugia pahangi and Dirofilaria immitis.

Adult Brugia pahangi in vitro, unlike mouse leukemia L1210 cells, converted 5-methyltetrahydrofolate (CH3FH4) directly to 5,10-methylenetetrahydrofolate and thence to other FH4 cofactors. The excreted CO2 that was derived from CH3FH4 was due to the presence within the filariae of 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) which catalyzes the deformylation of 10-formyl-tetrahydrofolate. Adult B. pahangi and Dirofilaria immitis, incubated in a purine-free medium containing [5-14C]CH3FH4 as the only form of folate, synthesized purine ribonucleotides radiolabeled at positions 2 and 8 of the purine ring. Presumably, 10-formyl[14C]FH4 donated Carbon 2 during the synthesis de novo of the purine ring and 5,10-methenyl[14C]FH4 donated Carbon 8.

Polyamine metabolism in filarial worms.

The human and animal filarial parasites Onchocerca volvulus, Dirofilaria immitis, Brugia patei and Litomosoides carinii contained low levels of putrescine but much higher levels of spermidine and spermine as estimated by ion-pair high pressure liquid chromatography; N-acetylated polyamines were present only in minute amounts. Enzyme activities of ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), respectively, were not detectable. Experiments carried out with O. volvulus and D. immitis demonstrated the uptake and bioconversion of labeled polyamines. There is evidence for the existence of a complete reverse pathway generating putrescine from spermidine and spermine, respectively, in both worms. N-Acetylating enzyme activities were detected in 100,000 X g preparations of homogenates from D. immitis which were capable to acetylate putrescine, spermidine and spermine. Long term incubation of the worms in the presence of labeled polyamines resulted in the excretion of putrescine and N-acetylputrescine.

Evidence for the occurrence of respiratory electron transport in adult Brugia pahangi and Dipetalonema viteae.

Mixed-sex adult stages of Brugia pahangi and Dipetalonema viteae, in the absence of exogenous substrate, consumed oxygen at rates of 4.18 +/- 0.38 and 2.12 +/- 0.20 ngatoms O2 min-1 mg-1 dry wt. respectively. When calculated on a unit dry weight basis the endogenous O2 consumption rates (E-QO2) of mature adult male macrofilariae of B. pahangi and D. viteae were significantly greater than those of mature females, although the E-QO2 calculated per individual worm was essentially similar irrespective of sex. When assayed as separate unisexual groups, the oxygen uptake of male and female macrofilariae of both species was inhibited by classical inhibitors of respiratory electron transport (RET), and showed classical substrate bypass phenomena in response to succinate and ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine with respect to the RET inhibitors rotenone (inhibitor of complex I) and antimycin A (inhibitor of complex III). Since male worms elicited similar responses to the classical RET inhibitors as did mixed-sex and/or adult female populations, the possibility that developmental stages contained within the female filariids were contributing in any significant manner to the overall responses observed with the RET inhibitors can be discounted. Such responses as observed with live-intact macrofilariae are normally elicited only by mitochondrial preparations and suggest that the cuticles of both species are permeable to rotenone, succinate, antimycin A, N,N,N',N'-tetramethyl-p-phenylenediamine, azide and cyanide. The uncoupler 2,4-dinitrophenol stimulated the endogenous rate of oxygen consumption (E-QO2) of intact B. pahangi at 33-160 microM, indicating the probable occurrence of RET-coupled oxidative phosphorylation. Higher concentrations of 2,4-dinitrophenol proved inhibitory. Respiratory studies on subcellular fractions substantiated the responses elicited by the intact parasites, suggesting the presence of antimycin A-sensitive and -insensitive RET pathways capable of utilising alpha-glycerophosphate, succinate, and malate as substrates. Both B. pahangi and D. viteae macrofilariae therefore probably possess branched RET-pathways bifurcating on the substrate side of RET-complex III. The rates of substrate oxidation in terms of QO2 mg-1 mitochondrial protein compare well with those observed with other nematode parasites.

Diethylcarbamazine inhibits endothelial and microfilarial prostanoid metabolism in vitro.

Diethylcarbamazine (DEC) rapidly lowers the number of microfilariae in the peripheral circulation. The mechanism of action is unknown, but may involve alterations of arachidonic acid metabolism in vascular tissues. We studied the effects of DEC on arachidonic acid metabolism by bovine pulmonary arterial endothelium monolayers, human platelets and Brugia malayi microfilariae. DEC at a concentration of 2.5 microM, a level achieved in vivo, rapidly decreased prostacyclin, prostaglandin E2 and thromboxane B2 release from endothelial monolayers by 78% (P less than 0.001), 57% (P = 0.05), and 75% (P less than 0.05), respectively. High-pressure liquid chromatography of extracts of endothelial monolayers incubated with DEC showed similar inhibition of these cyclooxygenase pathway products, but exposure to the drug did not result in formation of new eicosanoids. DEC did not inhibit endothelial phospholipase A2-dependent release of arachidonate from membrane stores, whereas prostaglandin H2 synthase activity (cyclooxygenae, EC 1.14.99.1) was reduced to a degree similar to that effected by acetylsalicylic acid. Microfilarial but not platelet synthesis of cyclooxygenase products was also reduced by DEC. These data suggest that the mechanism by which DEC lowers the level of microfilariae in the circulation may in part involve its effects on host endothelial and parasite eicosanoid production.

Lectin binding to larval stages of Brugia patei.

Surface properties of microfilariae (mf) and infective larvae of Brugia patei were investigated to compare them to previous studies with the other brugian species. Of all the lectins tested, only wheat germ agglutinin (WGA) binds to the sheath surface of mf indicating the presence of N-acetyl-D-glucosamine as a major surface carbohydrate. However, cuticle of infective larvae failed to show binding of these lectins. Enzyme treatment of mf with N-acetyl-D-glucosaminidase and L-fucosidase has exposed D-mannose, N-acetyl-D-galactosamine and L-fucose on the sheath surface. The binding of lectins to intact mf and to enzyme-treated mf appeared to be specific as pretreatment with specific inhibitory sugars completely abolished the binding activity. This is the first study conducted with this filarial parasite and it established that B. patei is similar to other species of Brugia but differs from Wuchereria in its surface-lectin binding properties.

Respiration in the filarial nematode Brugia pahangi.

Glucose-supported O2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30-40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO2 by B. pahangi in the presence of D-glucose was stimulated by O2; the stimulation of CO2; the stimulation of CO2 production was sensitive to antimycin A. The O2 dependencies of respiration showed that the apparent O2 affinity for B. pahangi was diminished in the presence of antimycin A; O2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O2 concentrations. H2O2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H2O2 production suggest two sites of H2O2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O2 dependencies of H2O2 production and respiration may indicate that H2O2 production is involved in O2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O2.- radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H2O2 production and O2.- production by 30, 20 and 25%, respectively, whereas higher concentrations (greater than 2.5 mM) inhibited respiration by 75% and H2O2 and O2.- production by up to 85%.

Release of prostaglandin E2 by microfilariae of Wuchereria bancrofti and Brugia malayi.

To elucidate the local release of immunomodulatory prostaglandins by intravascular filarial parasites, the formation of prostaglandin E2 (PGE2) was examined in individual microfilariae of Wuchereria bancrofti and Brugia malayi. Following incubation of living microfilariae immobilized in an agar matrix, prostaglandins released by the parasites were fixed by carbodiimide and localized by indirect immunofluorescence. Prostaglandin E2 was specifically detected around the entire surface of microfilariae with anti-PGE2 antiserum, but not with control nonimmune or PGE2 affinity-immunoadsorbed antiserum. These results provide direct evidence that individual microfilariae of W. bancrofti as well as B. malayi release prostaglandins into their microenvironment. The release of PGE2 by these intravascular parasites may modulate host leukocyte responses, and thereby contribute to the immune defects observed in infected humans with peripheral microfilaremia.

Cuticular proteins of Brugia filarial parasites.

Surface-labelling techniques have been used to delineate a number of constituent molecules of the cuticle in adult stage Brugia malayi and Brugia pahangi. These molecules can be separated by virtue of their physical properties, and localised either by sequential solubilisation of intact cuticles or immunoelectron microscopy with relevant antisera. The major structural components of the cuticular matrix consist of a set of collagenous proteins of diverse molecular weight ranging from 36 to 160 kDa, cross-linked by disulphide bonds and confined to the basal and inner cortical layers. Each stage of the parasite has a distinctive set of between 12 to 25 collagenous proteins whose synthesis is regulated temporally with respect to moulting. As in other nematodes, the outer cortex and epicuticle is composed of a cross-linked insoluble proteinaceous structure. Two non-structural and water-soluble proteins are also resolved by Iodogen-mediated labelling; a 15 kDa peptide which shows no evidence of glycosylation, and a major 29 kDa glycoprotein, which carries at least two N-linked oligosaccharide chains and which we have termed Gp29. The former protein can be detected in L3, L4 and adult B. malayi by surface labelling, whereas Gp29 appears to be restricted to L4 and adult worms. The possible significance of cuticular proteins as targets of immunity or causative agents of pathology is discussed.