Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: USING GENOMICS TO DEFINE AND CONTROL PARASITIC INFECTIONS IN CATTLE Title: A calcium-activated nucleotidase secreted from Ostertagia ostertagi fourth stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods

Authors
item Zarlenga, Dante
item Gasbarre, L -
item Nisbet, A -
item Garrett, Wesley

Submitted to: Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 20, 2010
Publication Date: September 1, 2010
Citation: Zarlenga, D.S., Gasbarre, L.C., Nisbet, A.J., Garrett, W.M. 2010. A calcium-activated nucleotidase secreted from Ostertagia ostertagi fourth stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods. Parasitology. 138:333-243.

Interpretive Summary: Ostertagia ostertagi, often called the brown stomach worm of cattle, damages the abomasum during larval development, and during the tissue feeding adult stage. Ostertagia is among the most pathogenic of this parasite group in the world. 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. 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 Ostertagia as well as many other related GI nematodes.

Technical Abstract: Apyrases (ATP diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyze extracellular ATP and ADP to orthophosphate and AMP. Most apyrases have been structurally linked to the heat shock70/sugar kinase/actin superfamily which is associated with motility, adhesion, secretion, regulation of hemostasis, non-synaptic information transfer and platelet formation. A second class of newly-described, calcium-dependent, salivary apyrases known to counteract blood-clotting, has been identified in several hematophagous arthropods. Herein, we have identified an apyrase gene (OoAp) encoding a protein that structurally conforms to the calcium-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library with hyperimmune bovine serum. The full-length sequence was subcloned and a biochemically-functional protein (rOoAP-64) was expressed in bacteria. Hydrolysis of various dNTPs showed that the rOoAP-64 is Ca2+-dependent with greatest activity on ATP, ADP, UTP and UDP, coinciding with the range of nucleotides involved in purinergic signaling across the P2 receptor complex. Negligible activity was observed in the presence of Mg2+ and hydrolysis did not occur on CTP or AMP. Host antibodies to rOoAp-64 appeared as early as 14 days post infection (dpi) and increased through 30 dpi. Immunohistochemical and Western blot analyses demonstrated that the native OoAP is produced or collects within the glandular bulb of the esophagus at the esophageal/intestinal junction, and is developmentally-regulated appearing predominantly in the L4. A putative signal sequence at the N-terminus is consistent with the native OoAP protein being secreted at the cellular level during the more rapid feeding stage of development. Predicated upon the range of substrate activity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides secreted by locally-damaged tissues in the gastric glands in an effort to modulate host immune intervention.

Last Modified: 10/21/2014