Progress 12/15/15 to 12/14/17
Outputs
Target Audience:Target audience for this project are public, beef industry, ranchers, outdoor workers, agriculture workforce, and researchers from variety of disciplines including veterinary sciences, vector biology, allergy and immunity, and food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One doctoraland three undergraduate students were trained. All students had an opportunaity to present their rseearch findings at the scientific meetings. Students were trained in bioinformatics, reverse genetics, protein analysis, enzymetic assays, and handling of laboratory animals. All the students were provided an opportunity to conduct research, analyze results, copile data, and present their research at the scientific conferences. How have the results been disseminated to communities of interest?Presentations at the scientific conferences: Commins SP, Karim S.- Develoment of a novel murane model of alpha-gal meat allergy. 2017 American Academy of allergy, Asthma, & Immunology annual meeting, March 3-6, Atlanta, GA. Cox, C., Crispell, G., Lange, J., and Karim, S. - Molecular Characterization of a-D-galactosidase in the Lone-Star Tick, Amblyomma americanum. Poster presentation at the Mississippi INBRE Symposium 2017, July 27, 2017 in Jackson, Mississippi. Lange, J., Crispell, G., Cox, C., and Karim, S. - Investigating the Functional Role of b-1-4-Galactosyltransferase in the Lone-Star Tick, Amblyomma americanum. Poster presentation at the Mississippi INBRE Symposium 2017, July 27, 2017 in Jackson, Mississippi. Gary Crispell and Shahid Karim - Characterization of Putative Tick Salivary Antigens Responsible for Red Meat Allergy. Oral presentation at the 81st Annual meeting of Mississippi Academy of Sciences, February 23-47, 2017 in Hattiesburg, Mississippi. Crispell, G. Science Cafe' at Cook Library: A Carnivore's Nightmare. Invited speaker at the University of Southern Mississippi's Science Café at Cook Library April 25, 2016 in Hattiesburg, Mississippi. Crispell, G. Tick Saliva Induces Human Hypersensitivity to a-Galactose in Red Meat Allergy. Oral presentation at the Center for Basic Biomedical Sciences Meeting March 2, 2016 at the University of Southern Mississippi in Hattiesburg, MS. Crispell, G., Balamurugan, K., Karim, S. Uncovering the Molecular Action of Tick Saliva in the Induction of a-Galactose Hypersensitivity in Red Meat Allergy. Oral presentation in the division of Cellular, Molecular, and Developmental Biology at the 80th annual meeting of the Mississippi Academy of Sciences February 18-19, 2016 in Hattiesburg, MS. 2nd place award. Crispell, G., Balamurugan, K., Karim, S. Elucidating the Molecular Action of Tick Saliva in the Induction of a-Galactose Hypersensitivity in Red Meat Allergy. Graduate Poster Competition: Medical, Urban, and Veterinary Entomology (MUVE) - Ticks and Mosquitoes at the 63rd Annual Meeting of the Entomological Society of America November 16-18, 2015 in Minneapolis, Minnesota. Crispell, G., Karim, S. Elucidating the Molecular Action of Tick Saliva in the Induction of a-Galactose Hypersensitivity in Red Meat Allergy. Student Three-minute Presentation Competition at the 63rd Annual Meeting of the Entomological Society of America November 16, 2015 in Minneapolis, Minnesota. Crispell, G. - The Role of Tick Saliva in α-Galactose Hypersensitivity (Red-Meat Allergy). Three-minute Thesis Program at the University of Southern Mississippi November 4, 2015 in Hattiesburg, Mississippi. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall goal was to identify tick salivary allergens responsible for inducing red meat allergy. To identify tick proteins, unfed adult lone-star tick (Amblyomma americanum) and the gulf-coast tick (Amblyomma maculatum) were blood feed on sheep. Tick tissues (salivary glands and midguts) were dissected from unfed, 1, 3, 5, 7, and 9 days post infestation on the sheep. Additionally, saliva was also collected from partially blood-fed ticks. The salivary tissues proteins were solubilized and fractionated on 4-20% SDS-PAGE. One gel was stained with Coommassie brilliant blue, and second gel was trabsfered onto nitrocellulose membrane, and blotted with alpha-gal monoclonal antibody. Results of immunoblot showed immune-reacrtivity of alpha-gal antibody in saliva andblood-fed salivary glands samples. The alpha-gal antibody did not react to any salivary protein in A. maculalatum saliva, supportingthe presence of galactosylated antigens only in the A. americanum saliva and salivary glands. Interestingly,lack of immuno-reactivity of alpha-gal antibodyin unfed salivary glands suggests that the blood feeding induced expression of immunogenic glycoprotein 9tick allergen). This experiment provided the basis to further identify the immune-reactive allergens by using peptide fingerprinting through Mass spoectrometery (LC-MS/MS) approach. Immuno-reactive protein bands were exised from stained gel followed by in gel trypsin digestion for LC-MS/MS analysis. Our search revealed homology to several tick glycoproteins. Additionally, a magnetic pull-down assay was conducted to further identify glycosylated tick proteins responsible for induced red meat allergy. The magnetic pull-down assays was able to capture the alpha-gal bearing epitope from A. americanum salivary tissues, and LC-MS/MS analysis reveals several glycoproteins and ~900 tick peptides. Furthermore, we utilized the alpha-gal antibody to localize the alpha-gal in partially fed salivary gland secretory vesicle using confocal microscopy. To further confirm the source of alpha-gal in tick salivary glands, peptide-N-glycosidae F (PNGase F) was used for the deglycosylation of tick salivary gland and saliva glycoproteins. Immuno-blot analysis of deglycosylated tick salivary glands showed successful cleageb of the sugar from the tick glycoproten cross-reacted with alpha-gal antibody. These results confirmed that the tick allergens criss-reacting with alpha-gal antibody are indeed glycosylated.N-linked glycan profiling of Amblyomma americanum and Amblyomma maculatum revealed interesting results concerning the glycosylation of tick proteins. The unfed salivary glands of both tick species lacked the presence of detectable alpha-gal. Interestingly, the partially blood fed salivary glands and saliva from A. americanum had detected quantities of alpha-gal, whereas A. maculatum lacked the alpha-gal. The overall abundance of alpha-gal glycoforms in A. americanum was more than 1.12% of the total glycans detected, but saliva contained ~0.15% alpha-galactosyl glycoforms. This information supported our earlier immuno-blot analysis. Currently,thefunctionalrole of beta-1,4-Galactosyltransferase (enzyme required to produce theprecursor glycan to alpha-galactose), and alpha-D-Galactosidase (enzyme required to cleave alpha-linked galactose from glycan chains) is being investigated using RNA interference approach.
Publications
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