Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.
Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.
Hsu
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Department of Microbiology, Immunology, and Preventive Medicine, Veterinary Medical Research Institute, Iowa State University, Ames 50011, USA.
Artiushin
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Minion
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D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D001483:Base Sequence; D002923:Cilia / Q000378:metabolism*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006493:Heparin / Q000494:pharmacology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D009693:Nucleic Acid Hybridization; D011134:Polysaccharides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013552:Swine; D014774:Virulence
D018829:Adhesins, Bacterial; C092236:P97 protein, Mycoplasma; D011134:Polysaccharides; D011994:Recombinant Proteins; D006493:Heparin; C007789:fucoidan