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.

Wolbachia lipoprotein stimulates innate and adaptive immunity through Toll-like receptors 2 and 6 to induce disease manifestations of filariasis.

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed 3-11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirB6 were consistently predicted, and B. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-alpha and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp38 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4+ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88-/- mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive interferon-gamma-dependent CD4+ T cell polarization and antibody switching.

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.

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.

Effect of bis(benzyl)polyamine derivatives on polyamine transport and survival of Brugia pahangi.

A potent filaricidal effect of bis(benzyl)polyamine derivatives is reported; the addition of 1 microM MDL 27695 to Brugia pahangi maintained in vitro killed the worms within 1 week. Using the labelled derivative, MDL 27391 uptake was demonstrated and evidence was provided for an uptake system that is independent of and clearly distinguishable from those for polyamines. The Km value for the uptake of MDL 27391 was determined to be 2 microM, and that for putrescine, spermidine, and spermine was 4.9, 1.7, and 4.8 microM, respectively. The uptake of MDL 27391 was not affected by polyamines. In contrast, bis(benzyl)polyamines were shown to be strong inhibitors of both the putrescine and the spermidine/spermine uptake system. As shown for MDL 27391, bis(benzyl)polyamines are not metabolized after uptake by Brugia worms; therefore, it is expected that the filaricidal effect of the drug depends on its interaction with potential polyamine-binding sites.

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.

Brugia malayi and Brugia pahangi: transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti.

Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L2, and L3 larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L3 larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L3's recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microfilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microfilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microfilariae to develop to the L3 stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microfilariae also failed to develop to L3 in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 ng/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microfilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Comparison of the cuticular structure of parasitic nematodes recognized by immunocytochemical and lectin binding studies.

The cuticle structure of some nematode species was studied by immunogold and lectin-gold techniques. The gold labelling made it possible to distinguish the cuticle layers by the distribution and/or the density of the marker. On the other hand, no labelling pattern was found which led to a clear grouping of the layers into larger 'zones', since there were no subunits consisting of more than one layer which reacted in a characteristic way as compared to the rest of the cuticle. The outer surface of the epicuticle of parasitic adult worms turned out to be highly inert; it did not react with any of the antibodies or lectins tested. The cuticle of parasitic nematodes seems to function as a protection against the host's defense mechanisms rather than as an interaction site. An immunogenic component on the surface was only found in infective larvae. All antibodies and lectins showed a preferential binding to the electron dense layers and fibrous structures (HPL/GalNAc, WGA/GlcNAc) or to the amorphous ground-substance (Con A/Glc, RCA I/Gal).

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.

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.

Comparison of glycolysis and glutaminolysis in Onchocerca volvulus and Brugia pahangi by 13C nuclear magnetic resonance spectroscopy.

Comparison of glycolysis in Brugia pahangi and Onchocerca volvulus by 13C nuclear magnetic resonance (NMR) spectroscopy showed that the former organism is predominantly a lactate fermenter and the latter resembles more closely the metabolism of a mixed acid fermenter producing lactate, succinate, acetate, ethanol, formate and carbon dioxide. Both organisms synthesize glycogen as a storage carbohydrate. Glutaminolysis in both organisms proceeds by the delta-amino-butyrate shunt to produce succinate which is then further metabolized to acetate and carbon dioxide as end-products.

Studies on the biosynthesis and fate of ecdysteroids in filarial nematodes.

Experiments were performed to investigate whether adult Dirofilaria immitis and Brugia pahangi were capable of synthesising ecdysteroids from cholesterol or various intermediates from the biosynthetic pathway functioning in insects. Metabolites of radioactively-labelled cholesterol and 5 beta-ketodiol (2,22,25-trideoxy-ecdysone) were detected in the filarial nematodes, but there was no radioactivity corresponding to ecdysteroid. Uptake of tritiated 2-deoxyecdysone was poor and metabolism was not observed. [3H]Ecdysone was absorbed sparingly by adult D. immitis, but was metabolised efficiently to several less polar products. There was no evidence of C-20 hydroxylation capability.

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.

The further application of MTT-formazan colorimetry to studies on filarial worm viability.

Experiments have confirmed that MTT-formazan colorimetry in its simplest form (incubation of intact worms with MTT and direct visualisation of any formazan formed) can be readily applied to several species of filariae including Onchocerca volvulus. Data is presented which will assist the development of quantitative MTT reduction viability tests for a selection of the smaller filarial species. Assays of pieces of Onchocerca gutturosa and O. volvulus females have led us to tentatively conclude that the tips of filariae, particularly the anterior ends, may well be metabolically the most active part of the worm. Selective sampling of these regions for Onchocerca might therefore be a useful indicator for the viability of the parasite. An example of how MTT-formazan colorimetry has been applied to yield additional data to support motility observations on the in vitro survival of male O. gutturosa is also given. The in vitro timecourse of worm death caused by 10 microM CGP 20376 on Acanthocheilonema viteae females has been examined by MTT reduction and compared with 6 other non-subjective parameters. The results suggests that the parameters examined could be divided into two groups according to the time taken for CGP 20376 to cause 50% inhibition (t50) of the parameter. Fast response parameters had t50's between 1 and 6 h (motility indices, 14CO2 evolution, adenine uptake and leucine uptake), they are more sensitive measures of viability and indicate possible worm damage which may or may not be reversible. Slow response parameters had t50's between 34 and 48.5 h (lactate output, MTT reduction and adenine leakage), and are probably linked with severe degenerative changes and are indicative of worm death.(ABSTRACT TRUNCATED AT 250 WORDS)

Brugia malayi: microfilarial polyunsaturated fatty acid composition and synthesis.

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2 omega 6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4 omega 6), as well as the 18:3 omega 6, 20:2 omega 6, and 20:3 omega 6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [14C]linoleic acid incorporated by microfilariae was converted to [14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.

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%.

pH-dependent uptake and macrofilaricidal effects of chloroquine on adult filarial parasites in vitro.

Uptake and macrofilaricidal effects of chloroquine (CQ) and other aminoquinolines were found to be highly pH dependent in Brugia pahangi, Acanthocheilonema viteae, Onchocerca volvulus, and Dirofilaria immitis. Using [3H]CQ, it was found that all of the parasites took up more drug under alkaline conditions (RPMI 1640 at pH 8.4) than in neutral (pH 7.4) or acidic (pH 6.8) media. Differences were seen in the amount of drug taken up among the filariae studied. B. pahangi and A. viteae took up 7 times more chloroquine per milligram of tissue than did O. volvulus, and 30 times more than D. immitis during a 60-min incubation period at pH 8.4. Sensitivity to the aminoquinolines also increased with increasing media pH, and was measured using parasite motility as an indicator of drug efficacy. Potency of chloroquine against B. pahangi increased 100-fold at pH 8.4 compared to pH 7.4. A. viteae and O. volvulus showed similar sensitivity to chloroquine compared to B. pahangi; D. immitis was less sensitive. While uptake of chloroquine was linear from pH 6.8 to 8.4, B. pahangi was unaffected by 32 microM of the drug below pH 7.6; at any pH above this, motility of this parasite was completely inhibited. Calculations of the internal pH of this parasite indicated that it shifted upwards significantly with changes in media pH. It was concluded that these shifts in internal pH may render parasites more sensitive to the effects of chloroquine.

Leakage of incorporated radiolabelled adenine--a marker for drug-induced damage of macrofilariae in vitro.

An objective in vitro assay has been developed for quantifying drug-induced damage in Dipetalonema viteae macrofilariae. The method involves radiolabelling the worms ATP pool by incubating macrofilariae with [U-14C]-adenine. As determined by HPLC 72% of the incorporated label was in ATP, 15% in ADP and about 4% in each of NAD and AMP. Macrofilariae labelled with [U-14C]-adenine show a linear efflux of [14C]-label amounting to 21.3% of the total incorporation (mainly as uncharged catabolites) over a time course of 120 h in vitro incubation. When prelabelled worms were exposed to compounds exerting macrofilaricidal effects in vitro a marked stimulation in the leakage of [14C]-label from the worms was noted. The [14C]-label leakage appears to be linked with membrane (or cuticle) damage and the reduction of macrofilarial ATP levels. Determination of the amount of [14C]-label remaining in drug-treated worms relative to appropriate control provides a simple, sensitive and quantitative measure of drug-induced damage in macrofilariae (including Onchocerca). The method has been used to describe the macrofilaricidal activity of a wide range of antifilarial standards, membrane disruptive agents, respiratory inhibitors, fasciolicides and anti-cestode compounds.