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-1-3Hglucose. 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 μM, whereas adult worms survived the addition of 100 μM H2O2 and showed slightly impaired viability at 150 μM 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 μM H2O2 in the absence of parasites. Adults survived higher concentrations of glucose oxidase, which generated 200 μM 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 23× 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.
We recently reported on a non‐neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties ...of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met).
A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10‐fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the ‘peripheral site’ inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase‐specific inhibitor iso‐OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.
The major soluble cuticular glycoprotein of lymphatic filariae, gp29, has been expressed in the Salmonella typhimurium aroA aroD live vaccine strain BRD509. Two distinct constructs were generated: a) ...pgp29, in which gp29 was expressed directly via the inducible promoter nirB, or b) pTetC-gp29, in which it was expressed as a C-terminal fusion to the non-toxic immunogenic fragment C of tetanus toxin, again under the control of nirB. In both cases, plasmid stability in vivo was demonstrated by recovery of recombinant bacteria from livers and spleens of mice immunized via the intravenous route. Negligible gp29-specific antibodies were detected in animals immunized with bacteria expressing the fragment C fusion protein, but bacteria expressing the non-fused protein resulted in gp29-specific antibody production in a proportion of animals immunized. Notably, a number of BALB/c and B10.D2/n (i.e. mice of the H-2d haplotype) responded, in contrast to previously documented nonresponsiveness during infection, or immunization with parasite extracts. Presentation of gp29 by live attenuated S. typhimurium resulted in a broad spectrum of antigen-specific IgG isotypes.
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