Search

Expression of cholines terases and muscarinic acetylcholine receptors in the jejunal mucosa has been investigated during infection of rats with the nematode parasite Nippostrongylus brasiliensis. Selective expression of m3 receptors was observed on epithelial cells from uninfected rats and animals 7 days postinfection, and saturation binding with [(3)H]quinuclidinyl benzilate indicated that receptor expression on cell membranes was unaltered by infection. Butyrylcholinesterase was highly expressed in mucosal epithelia, but acetylcholinesterase was present at low levels in uninfected animals. In contrast, discrete foci of intense acetylcholinesterase activity were observed on the basement membrane of intestinal epithelial cells in animals infected with N. brasiliensis. This was demonstrated to be due to upregulation of expression of endogenous enzyme, which peaked at Day 10 postinfection and subsequently declined to preinfection levels. It is suggested that this occurs in response to hyper-activation of the enteric nervous system as a result of infection, and may benefit the host by limiting excessive fluid secretion due to cholinergic stimulation., (Copyright 2000 Academic Press.)

We have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic (G1na) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts of N. brasiliensis and report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (G4a) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 microM for acetylthiocholine was determined. It was highly sensitive to the AChE-specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 microM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50 of 46.5 +/- 6.1 microM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.

We have examined the susceptibility of cuticular membrane lipids of Brugia malayi to oxidants generated in vitro. Live parasites as well as extracted cuticular lipids were treated with hydrogen peroxide and hypochlorous acid and the extent of lipid peroxidation was quantified. The cuticular membranes of B. malayi were found to be resistant to lipid peroxidation at hydrogen peroxide concentrations which were lethal to the organism. This resistance was partly due to the inherently low unsaturation indices of the fatty acyl residues, but complete protection was afforded by lipid-soluble antioxidants present in the neutral lipid fraction of the parasites. We have identified alpha-tocopherol as a major antioxidant present in both adult and microfilarial B. malayi. In addition, we report that although hypochlorous acid chemically modifies isolated parasite lipids, the latter do not appear to be the primary substrate for the oxidant in live worms. The data are discussed in terms of the susceptibility of B. malayi to products of the respiratory burst from activated myeloid cells.

A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from a Dirofilaria immitis adult female cDNA expression library by a combination of polymerase chain reaction amplification with primers designed from the Brugia pahangi glutathione peroxidase gene sequence and hybridization screening of D. immitis cDNA libraries. The Di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for selenocysteine in the active site. The cDNA clone was expressed in Escherichia coli and Spodoptera frugiperda Sf9 insect cells, and the resulting recombinant proteins were purified for antibody production and assessment of enzymatic properties, respectively. An antiserum generated against the E. coli-expressed protein detected a protein of 29 kDa in D. immitis via immunoblotting. This protein is expressed in adult worms (both sexes) and fourth stage larvae generated via 6 days of in vitro culture, but was undetectable in microfilariae, and third stage larvae obtained either directly from mosquitoes or following 2 days of culture. The Di29-encoded recombinant protein was secreted from Sf9 insect cells and displayed low-level glutathione peroxidase activity against a range of hydroperoxide substrates, including hydrogen peroxide.

We have obtained and examined several fracture planes close to the surface of the cuticle of adult Brugia malayi by freeze-fracture electron microscopy. We observed three exocytoplasmic and two protoplasmic fracture faces exhibiting the periodic annulations characteristic of the outer layers of the cuticle. The outermost exocytoplasmic fracture face we observed contained small randomly distributed particles and appears to originate from within the epicuticle. The second exocytoplasmic face appeared as a featureless surface representing a nonmembranous proteinaceous structural arrangement adjacent to the epicuticle. The innermost fracture plane was represented by an exocytoplasmic face with a morphology dominated by large particles regularly appearing as organised linear arrays. This fracture plane seems to originate from a membrane-like organisation directly below the annulated proteinaceous layer. The two annulated protoplasmic faces were exposed by two discrete planes of fracture and were complementary to the exocytoplasmic planes. The above observations are considered in terms of the overall structural organisation of the cuticle of adult B. malayi.

We have determined the levels of superoxide dismutase (SOD) in different stages of the lymphatic filarial nematode parasite of man, Brugia malayi. Adult male worm extracts showed the highest levels of enzyme activity at 34.5 U mg-1, and there was no significant difference in the overall levels of SOD in extracts of adult female worms and microfilariae (27.1 and 26.7 U mg-1, respectively). SOD activity was detected in the culture medium of parasites maintained in vitro, with particularly high levels of specific activity in media in which males and females were maintained (357 and 339 U mg-1, respectively), indicative of active secretion. In all cases, this was accounted for predominantly by CuZn SOD, assessed by potassium cyanide inhibition. Northern blots with cDNA probes specific for cytoplasmic and extracellular CuZn SODs indicated that levels of mRNA for the cytoplasmic form were similar between adults and microfilariae, whereas expression of the extracellular form was 10x higher in adult worms. Western blots with an antibody to recombinant CuZn SOD demonstrated that higher levels of the extracellular protein were present in adult male worms, whereas the cytoplasmic form was present in roughly equivalent amounts in males, females, and microfilaria. Iodination and immunoprecipitation experiments indicated that the extracellular enzyme was accessible to surface labeling of both male and female adult worms, but not microfilaria. Immuno-electron microscopy showed that CuZn SOD was localised predominantly in the hypodermis of adult parasites, with an asymmetric distribution in the intercordal regions suggestive of compartmentalisation into several distinct syncytia. No labeling was evident in the cuticle, and thus the accessibility of the extracellular enzyme to extrinsic iodination in adult worms remains unclear. No binding of antibody was demonstrable in the glandular region of the oesophagus or the uterus of females, presumed to be major sites of synthesis for secreted proteins. Dense labeling was observed in the seminal fluid surrounding spermatazoa in the vas deferens of male parasites. These data also suggest that, as observed in mammals, nematode spermatazoa are particularly susceptible to oxidative damage and are protected during storage by secreted anti-oxidant enzymes.

The sensitivity of microfilariae and adult Brugia malayi to hydrogen peroxide (H2O2) was determined in vitro, and parasite viability assessed by incorporation of 2-deoxy-D-[1-3H]glucose. Both stages were surprisingly resistant to peroxide stress. Microfilariae tolerated the direct addition of H2O2 to medium in which they were incubated at concentrations up to 50 microM, whereas adult worms survived the addition of 100 microM H2O2 and showed slightly impaired viability at 150 microM H2O2. Higher concentrations were lethal in both cases. This observation of differential susceptibility was reproducible when parasites were subjected to continuous generation of H2O2 via glucose/glucose oxidase. Microfilariae remained viable over a 4-hr period when challenged with concentrations which generated 20 microM H2O2 in the absence of parasites. Adults survived higher concentrations of glucose oxidase, which generated 200 microM H2O2 over the same time period. Under these conditions the parasites effectively countered the rate of peroxide generation by metabolising the product. Protein carbonyl formation was detectable at sublethal concentrations of glucose/glucose oxidase, but malonaldehyde formation was only detectable coincident with parasite death. The rate of H2O2 consumption by parasites was determined and showed that adult worms metabolised it at a rate 23x faster than microfilariae, expressed as activity per wet weight. Assessment of enzyme activities in parasite extracts demonstrated that H2O2 metabolism was effected principally by catalase activity, which was elevated in adult worms relative to microfilariae. Cytochrome c peroxidase activity was also detected and was roughly equivalent in both stages. Glutathione peroxidase and NADH/NADPH-dependent consumption of H2O2 were absent, and the rate of nonenzymic reduction of H2O2 coupled to glutathione oxidation did not contribute significantly to metabolism. Glutathione reductase activity and total glutathione content were equivalent in adults and microfilariae. This study illustrates that Brugia malayi are much more resistant to H2O2 than other filarial species examined to date and can effectively metabolise levels in excess of those potentially generated by activated leucocytes.

The cuticle of filarial nematode parasites contains distinct and separable sets of soluble and structural proteins. Surface-labeling techniques have previously identified a soluble protein complex in adult stage Brugia which ranges in molecular weight from 15 to 200 kDa. Using an antiserum directed to the 15-kDa basal subunit of this complex, we show here that this complex is synthesized and processed from a single, very large precursor protein with a molecular weight of approximately 400 kDa. Molecular cloning, sequencing, and Southern analysis indicates that the protein is encoded by a single gene composed predominantly of approximately 20 tandemly repeated segments of 396 bp. The two complete copies of these repeated segments in our cDNA sequence are identical. Each subunit of 132 amino acids bears a consensus site for N-linked glycosylation, and glycosidase treatment indicates that this corresponds to an oligosaccharide side chain of 2 kDa. The protein displays no significant homology to sequences lodged in databases corresponding to molecules of known function. Nevertheless, a significant similarity (19/41 residues) is observed with the N-terminal sequence of a protein termed ABA-1, an allergen from Ascaris.