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Infection and Immunity, October 1998, p. 4762-4766, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of the Cilium Binding Epitope of the Mycoplasma hyopneumoniae P97 Adhesin

Tsungda Hsu, and F. Chris Minion*

Department of Microbiology, Immunology and Preventive Medicine, Veterinary Medical Research Institute, Iowa State University, Ames, Iowa 50011

Received 28 May 1998/Returned for modification 24 June 1998/Accepted 8 July 1998

Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. This interaction involves an adhesin called P97; the cilium binding activity of this protein was localized to the carboxy terminus, which included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C. Minion, J. Bacteriol. 179:1317-1323, 1997). To further delineate the molecular mechanisms of M. hyopneumoniae interactions with ciliated epithelium, we used a bank of transposon inserts in the cloned P97 gene to identify the site for cilium binding by testing the truncated gene products in an in vitro microtiter plate adherence assay. These studies showed that the cilium binding site was located in the AAKPV(E) repeat sequence of P97, referred to as the R1 repeat. For functional binding, at least seven AAKPV(E) repeats were required. The adherence-blocking monoclonal antibody F1B6 also recognized this region but required fewer AAKPV(E) repeats for recognition. We then constructed R1 region-lacZ gene fusions and used the resulting R1 repeat-beta -galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cilium binding. A comparison of the R1 regions of M. hyopneumoniae strains displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strains. Thus, we concluded that other proteins, in addition to P97, must be involved in cilium adherence, possibly in combination with P97.

* Corresponding author. Mailing address: Iowa State University, Veterinary Medical Research Institute, 1802 Elwood Dr., Ames, IA 50011. Phone: (515) 294-6347. Fax: (515) 294-1401. E-mail: fcminion{at}iastate.edu.

Infection and Immunity, October 1998, p. 4762-4766, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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