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Title: A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

Author
item NISBET, A - The Moredun Group
item Zarlenga, Dante
item KNOX, D - The Moredun Group
item MEIKLE, L - The Moredun Group
item WILDBLOOD, L - The Moredun Group
item MATTHEWS, J - The Moredun Group

Submitted to: Parasite Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2010
Publication Date: 4/10/2011
Citation: Nisbet, A.J., Zarlenga, D.S., Knox, D.P., Meikle, L.I., Wildblood, L.A., Matthews, J.B. 2011. A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses? Parasite Immunology. 33:236-243.

Interpretive Summary: Teladorsagia circumcincta, which is very similar to the cattle brown stomach worm Ostertagia ostertagi, damages the abomasum during larval development, and during the tissue feeding adult stage. Teladorsagia is among the most pathogenic of this parasite group found in sheep and goats. Little is known of how this and similar worms escape the strong immune responses normally exhibited by the host. Herein, we identified an ATP diphosphohydrolase secreted by the parasite which is capable of degrading key host nucleotides required for cell signaling and initiating an inflammatory response. We further demonstrate for the first time a relationship between the protein and immune responses in the host. Controlling the local concentration of nucleotides therefore confers an advantage to nematode establishment and survival. Given that the parasite secretes this apyrase through the feeding end of the worm, such a molecule warrants targeting for vaccine development because interrupting this process could drastically affect the ability of the worm to metabolize nutrients and/or modulate the local immune responses. Inasmuch as the crystal structure of the human homologue is known, this could help in the design of compounds to specifically attenuate the function of this enzyme in Teladorsagia as well as other related GI nematodes.

Technical Abstract: A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth stage larval (L4) cDNA library. The closest orthologue of this gene is Oos-apy-1, a Ca2+-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, with the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third stage larvae (L3). The associated protein, Tci-APY-1, was detected by immunoblotting in soluble extracts of L4 nematodes and was also present in excretory/secretory products derived from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in Escherichia coli as an active enzyme with a nucleoside triphosphate substrate preference as follows: dATP>>dTTP>dGTP>>dCTP. Apyrase activity of the recombinant protein was found to be divalent cation-dependent, with no hydrolysis observed in the presence of Mg2+, but activation in the presence of Ca2+. Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in the abomasal mucus of trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection.