The type 3 fimbrial adhesin (MrkD) of Klebsiella pneumoniae mediates binding of the bacteria to specific types of collagen. The receptors for this adhesin molecule are located in the basement membrane region of tissues, and denuded epithelial surfaces are likely to present binding sites. Since K. pneumoniae is a frequent cause of respiratory infections of patients with epithelial trauma, it is possible that type 3 fimbriate bacteria colonize the airways in these individuals due to the presence of the MrkD adhesin. In this proposal we describe experiments we describe experiments to determine the role of the MrkD adhesin in facilitating colonization, in vivo, of the respiratory tract. A murine model of respiratory infection will be used and the animals will be treated with dilute HCI or hexamethylphosphoramide to cause desquamation of the mucosa to mimic similar histological lesions seen in compromised individuals. An mrkD-positive isolate of K. pneumoniae, and derivatives of this strain that do not express the MrkD adhesin, will be used to investigate the role of the adhesin in colonization. Molecular analysis of variant MrkD adhesin proteins will be performed in order to determine the binding domains within these molecules. Chimeric adhesins possessing defined regions of two different molecules exhibiting receptor binding specificities will be constructed. Binding to specific collagen types will be investigated by standard techniques. Site-directed mutagenesis will be used to further delineate the collagen-binding sites of the adhesin. Also, collagen subunits and fragments will be examined in order to localize the receptor sites on whole collagen molecules. This material will be used in several binding assays using purified fimbrial protein as well as fimbriate bacteria. The identification of receptor sites will allow further investigations into the development of synthetic analogs that could inhibit bacterial colonization of susceptible individuals.
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