Lr Walker

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta.

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30h and adult worms for 4-6h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30h. An initial external ammonia concentration of 600 microM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.

Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: a critical junction between aerobic and anaerobic metabolism.

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L(3)) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L(3)s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L(3) and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.

The tricarboxylic acid cycle in L3 Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃ Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD+ and NADP+ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃ T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD+ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD+] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists withinL₃ T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle inL₃ T. circumcincta are probably similar to those in the tissues of their host species.

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta

Phosphofructokinase (PFK-1) activity was examined in L(3) and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4±0.2 to 1.7±0.2 in L(3)s and 2.0±0.3 to 2.4±0.4 in adults) and the K(½) for fructose 6 phosphate (from 0.35±0.02 to 0.75±0.05mM in L(3)s and 0.40±0.03 to 0.65±0.05mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.

The initial kinetics of NH3/NH4(+) efflux from L3 Teladorsagia circumcincta.

The initial rate of NH(3)/NH(4)(+) accumulation in a medium containing L(3) Teladorsagia circumcincta was 0.18-0.6 pmol h(-1) larva(-1), which increased linearly with larval density. However it appeared that the larva-generated external concentration of NH(3)/NH(4)(+) did not exceed about 130 μM. The rate of NH(3)/NH(4)(+) accumulation increased with temperature between 4 °C and 37 °C, declined with increasing pH or increasing external NH(3)/NH(4)(+) concentration and was not significantly affected by the concentration of the phosphate buffer or by exsheathing the larvae. We infer from these data that the efflux of NH(3)/NH(4)(+) is a diffusive process and that the secreted or excreted NH(3)/NH(4)(+) is generated enzymatically rather than dissociating from the surface of the nematode. The enzymatic source of the NH(3)/NH(4)(+) is yet to be identified. Since the concentration of NH(3)/NH(4)(+) in the rumen and abomasum is higher than 130 μM, it is unlikely that T. circumcincta contributes to it, but NH(3)/NH(4)(+) may be accumulated from the rumen fluid by the nematode.

The glyoxylate cycle in Ostertagia (Teladorsagia) circumcincta

Ostertagia (Teladorsagia) circumcincta is a nematode parasite which infects the abomasum of sheep and goats. The utilisation of substrates for energy production in O. circumcincta is largely unknown. This parasite has been shown to consume oxygen, and have a full glycolytic and TCA cycle in both L3 and adult stages, although the metabolism of carbohydrates in adults appears to be more geared towards an anaerobic pathway. As well as glycogen, O. circumcincta also contains high levels of stored lipid in both L3 and adult stages, which could also be utilised to provide energy. However, the relative importance of glycogen and lipid for energy production is unknown: in the L3, in which nutrient intake is thought to be restricted by the presence of the sheath; or the adult, in which lipid metabolism may be restricted by oxygen supply. The key enzymes of the glyoxylate cycle, malate synthase and isocitrate would allow this parasite to convert lipid to glucose for metabolism via glycolysis. The enzyme phosphoenolpyruvate carboxykinase (PEPCK) has also been detected in both L3 and adult O. circumcincta. Although this enzyme is typically associated with anaerobic metabolism in nematode parasites, it is also central to gluconeogenesis. As the O. circumcincta has a full TCA cycle and consumes oxygen, gluconeogenesis may be the key function of PEPCK in this parasite species, particularly in L3s. The activity of the glyoxylate cycle along relative levels of glycogen and lipid in L3 and adults of different ages will be discussed.

Teladorsagia circumcincta: survival of adults in vitro is enhanced by the presence of a mammalian cell line.

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO(2), 5% O(2) 85% N(2), 65% humidity at 37 degrees C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO(2) or O(2), or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.

Effect of excretory/secretory products of abomasal parasites on epithelial tight junctions

The presence of abomasal parasites is thought to be associated with an increase in the permeability of the gastric epithelium. Epithelial permeability is regulated by junctional complexes between adjacent cells. The most apical component of this junctional complex is the tight junction which functions as a paracellular diffusion barrier. Any disruption of tight junctions results in impaired barrier function and an associated increase in epithelial permeability. To investigate the effect of abomasal parasites on the integrity and barrier function of epithelia, Caco-2 cell monolayers were exposed to the excretory/ secretory products (ES) of adult Ostertagia (Teladorsagia) circumcincta and Haemonchus contortus. Changes in epithelial barrier function were monitored by measuring transepithelial electrical resistance (TEER) and tight junction integrity was visualised using immunofluorescence localisation of the tight junction-associated proteins, occludin and zonula occludens-1 (ZO-1), by confocal microsopy. Under control conditions, occludin and ZO-1 were localised to a continuous pericellular ring around individual cells when viewed from the apical surface. In cells exposed to ES for 24 h, staining of this pericellular ring was diminished in intensity and corresponded with an increase in the presence of punctuate, intracellular staining. Exposure to ES was also shown to interfere with tight junction integrity, which was detected as a decrease in TEER from 678 ± 10 Ωcm2 (control) to 526 ± 8 Ωcm2 (ES-treated) (n = 12) in 6 h. These alterations in TEER, along with intracellular changes in occludin and ZO-1 distribution, suggest that parasite ES disrupts tight junctions, leading to an increase in epithelial permeability which may be of importance in the pathology of abomasal parasitism.Mucins play important roles in host-pathogen interactions, influencing host resistance and establishment of infection, as pathogen recognition sites and as a source of nutrients. They are highly glycosylated molecules and changes in monosaccharide composition during parasitism have been reported in pigs, mice and rats. There are no data on sheep gastrointestinal (GI) mucin modifications after infection with nematodes. Experiments were designed to examine the effects of parasites on GI monosaccharide component of mucins of sheep: (1) non-infected; (2) sheep infected with 10 000 Haemonchus contortus and slaughtered 21 days post infection (p.i.); (3) sheep infected with 15 000 Ostertagia circumcincta and slaughtered 28 days p.i. Mucus was scraped off the surface of the abomasal fundus. Gel filtration and CsCl density gradient centrifugation were used to purify the mucins. Mucins were hydrolysed in 2M HCl to release monosaccharides that were quantified with a HPAEC CarboPac-PA20 column. Four monosaccharides that were detected in mucin glycoproteins were fucose, glucosamine, galactosamine and galactose. In uninfected animals, the predominant of glucosamine and galactosamine (24.6 and 21.9 respectively). Fucose (12.6) approached a significant decrease in H. contortus infected (6.3) and O. circumcincta infected sheep (8.3). Galactosamine was lower in infected animals than in worm-free sheep. There was no difference in the proportion of galactose between uninfected and H. contortus infected animals (40.2) whereas it increased in those O. circumcincta infected (62.9). The study showed that parasitism caused changes in the ratio of hexoses and hexosamines in gastrointestinal mucins of sheep.Vacuolated parietal cells have been observed in tissue sections from parasitised sheep, and in vitro preparations of Haemonchus contortus excretory/ secretory (ES) products are able to induce vacuolation in HeLa and AGS cells. The mechanisms and active components have not yet been identified, although different components of ES products are being tested as possible candidates. Prostaglandins play a role in many biological processes, including host-parasite interactions, and are synthesised by both host and a number of parasites, including nematodes. Prostaglandins have been reported to be involved in vacuole formation in protozoa, but it is not certain whether prostaglandins are capable of vacuolating mammalian cells or playing a role in the pathophysiology of nematode parasitism. The aim of this study was to separate lipid components of adult H. contortus ES products and assess their possible role in vacuolation of HeLa cells. Lipids in ES products were separated by thin layer chromatography (TLC) with phosphomolybdic acid staining and commercial prostaglandin standards (PGA2, PGB2, PGD2, PGE2 and PGF2α) used as a reference. ES product lipids were additionally identified by SDSPAG E with Sudan Black staining. The prostaglandin standards were also tested on both HeLa and AGS cells cultivated on glass cover slips and the formation of vacuoles microscopically examined using Neutral Red. Lipids, but not prostaglandins, were detected by TLC and these spots were excised and tested for vacuolating activity in HeLa cells. Several lipid bands were also detected by SDS-PAG E. None of the prostaglandin standards or ES product lipids were able to induce vacuolation. These results show that it is very unlikely that the lipids in H. contortus ES products are responsible for vacuole formation in HeLa cells.