CLONING AND FUNCTIONAL-ANALYSIS OF THE P97 SWINE CILIUM ADHESIN GENE OF MYCOPLASMA-HYOPNEUMONIAE

Colonization of the swine respiratory tract by Mycoplasma hyopneumonia e is accomplished by specific binding to the cilia of the mucosal epit helial cells, Previous studies have implicated a 97-kDa outer membrane -associated protein, P97, that appeared to mediate this interaction. I n order to further define the role of P97 in adherence to porcine cili a, the structural gene was cloned and sequenced, and the recombinant p roducts were analyzed, Monoclonal antibodies were used to identify rec ombinant clones in a genomic library expressed in an opal suppressor h ost because of alternate codon usage by mycoplasmas. The gene coding f or P97 was then identified by Tn1000 mutagenesis of recombinant clones , DNA sequence analysis revealed an open reading frame coding for a 12 4.9-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 occ urred in M. hyopneumoniae. Both recombinant P97 protein expressed in a n Escherichia coli opal suppressor host and M. hyopneumoniae bound spe cifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involv ed in binding to swine cilia in vivo.