Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Richard F. Ross
A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by using purified swine tracheal cilia which are the natural targets for the mycoplasma. M. hyopneumoniae bound specifically to solubilized cilia immobilized onto microtiter plates. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited binding of the mycoplasmas to cilia. Heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesins on the surface of mycoplasmas, whereas laminin blocked the receptors in cilia. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas; whereas, treatment of cilia with sodium metaperiodate decreased the binding. In the second study, the natural glycolipid receptors in cilia for Mycoplasma hyopneumoniae adherence were analyzed by using a thin-layer chromatography (TLC) overlay assay. M. hyopneumoniae bound specifically to sulfatide, globoside, ganglioside GM3, and three glycolipids (La, Lb, and Lc) extracted from swine tracheal cilia. La, Lb, and Lc were determined to be sulfated glycolipids. Sensitive and dose-dependent binding of M. hyopneumoniae was also detected to La, Lb, and Lc immobilized onto microtiter plates. In the third study, the adhesins of M. hyopneumoniae were identified and characterized. A monoclonal antibody (Mab F2G5) predominantly recognized a 97 KDa (P97) protein of M. hyopneumoniae and inhibited the adherence of the mycoplasma to cilia. Immunolabelling demonstrated that the proteins recognized by Mab F2G5 were located at the surface of the mycoplasmas, predominantly on a surface fuzzy layer. Antibody affinity chromatography-purified P97 was able to bind to cilia and blocked the adherence of intact M. hyopneumoniae cells to cilia. Surface proteolysis of M. hyopneumoniae with trypsin selectively digested P97 and decreased the adherence activity of the mycoplasma. The predominant proteins detected by Mab F2G5 were different in size among various strains, and multiple proteins were detected with Mab F2G5 in a single strain, indicating that the antigens bearing the epitope for Mab F2G5 undergo intraspecies and intrastrain size variations. In conclusion, adherence of M. hyopneumoniae to cilia was mainly mediated by ligand-receptor interactions. Three sulfated glycolipids (La, Lb, and Lc) in cilia were the major native receptors for M. hyopneumoniae. A major adhesin of M. hyopneumoniae was determined to be P97; evidence was obtained that M. hyopneumoniae adhesins undergo size variations, which may be a pathogenic mechanism utilized by M. hyopneumoniae to evade the porcine immune system.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Zhang, Qijing, "Molecular mechanisms of Mycoplasma hyopneumoniae adherence to swine respiratory epithelial cells" (1994). Retrospective Theses and Dissertations. 10528.