IDENTIFICATION OF THE CILIUM BINDING EPITOPE OF THE MYCOPLASMA-HYOPNEUMONIAE P97 ADHESIN

Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membra ne. This interaction involves an adhesin called P97; the cilium bindin g activity of this protein was localized to the carboxy terminus, whic h included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C, Minion, J, Bacteriol. 179:1317-1323, 1997), To further delineate t he molecular mechanisms of M. hyopneumoniae interactions with ciliated epithelium, me used a bank of transposon inserts in the cloned P97 ge ne to identify the site for cilium binding by testing the truncated ge ne products in an in vitro microtiter plate adherence assay, These stu dies showed that the cilium binding site was located in the AAKPV(E) r epeat sequence of P97, referred to as the R1 repeat. For functional bi nding, at least seven AAKPV(E) repeats were required. The adherence-bl ocking monoclonal antibody F1B6 also recognized this region hut requir ed fewer AAKPV(E) repeats for recognition. We then constructed R1 regi on-lacZ gene fusions and used the resulting R1 repeat-P-galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cili um binding. A comparison of the R1 regions of M.. hyopneumoniae strain s displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strai ns. Thus, we concluded that other proteins, in addition to P97, must b e involved in cilium adherence, possibly in combination with P97.