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Infection and Immunity, October 1998, p. 4762-4766, Vol. 66, No. 10
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-
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Identification of the Cilium Binding Epitope of the
Mycoplasma hyopneumoniae P97 Adhesin
-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.
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