The long-term goal of this project is to understand the role of host macromolecules in bacterial colonization of the mucosal surfaces of the oropharynx. We will focus principally on the role salivary molecules play in the modulation of adherence of type 1 fimbriated gram negative bacteria frequent agents of nosocomial pneumonia) to mucosal epithelial cells. We will purify type 1 fimbrial-binding glycoproteins (FBGs) from saliva by routine chromatographic procedures. We will also test sample of known salivary glycoproteins obtained from other investigators in the field. The FBGs will be characterized by amino acid analysis, two- dimensional SDS polyacrylamide gel electrophoresis, peptide mapping, and/or immunological crossreactivity. Oligosaccharides will be characterized by chemical and lectin-binding assays and, where warranted, by mass spectrometry and nuclear magnetic resonance spectrometry. The specific antibody probes generated will be used to assay for the concentration of the various molecules in saliva. Purified FBGs will be radiolabeled and used to determine the specificity, affinity and number of binding sites on type 1 fimbriated E. coli and FN. Appropriate controls will include E. coli mutants which lack or overproduce the 29 kDa mannose-binding adhesin and other species which express type 1 adhesins. Attention will be given to the influence of pH, ionic strength and divalent cations. Competitive inhibition studies using FN will be used to further characterize these interactions. Radiolabeled FBGs will also be used in binding studies to determine the specificity, affinity and number of binding sites on epithelial cells. Attention will be given to the influence of pH, ionic strength, divalent cations and to the effects of saliva and FN on this interaction. Since the interaction of FBGs with type 1 fimbriae may be mediated through the oligosaccharide portion of the glycoprotein, we will study the effects of glycosidases on the interaction of E. coli with the purified FBGs and determine whether exposure of saliva to these glycosidases exposes additional FBGs. We will use an animal model to study the hypothesis that the increased gram negative colonization following trauma is due, at least in part, to changes in salivary or buccal cell-associated molecules. Samples of epithelial cells and saliva will be assayed for: 1) changes in E. coli (or other gram negative bacteria) adherence using standard adherence assays, 2) changes in levels of FBGs, Fn or Fn fragments on buccal cells and in saliva using immuno-fluorescence, Western blot analysis and quantitative immunological assays, and 3) changes in the levels of FBG- or Fn- degrading activity using will add significantly to the information needed to understand important aspects of bacterial pathogenesis in the oropharyngeal cavity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE007218-07
Application #
3220797
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Project Start
1985-04-01
Project End
1993-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
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Dale, J B; Baird, R W; Courtney, H S et al. (1994) Passive protection of mice against group A streptococcal pharyngeal infection by lipoteichoic acid. J Infect Dis 169:319-23
Watanabe-Ohnishi, R; Aelion, J; LeGros, L et al. (1994) Characterization of unique human TCR V beta specificities for a family of streptococcal superantigens represented by rheumatogenic serotypes of M protein. J Immunol 152:2066-73
Courtney, H S; Li, Y; Dale, J B et al. (1994) Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci. Infect Immun 62:3937-46
Courtney, H S; Bronze, M S; Dale, J B et al. (1994) Analysis of the role of M24 protein in group A streptococcal adhesion and colonization by use of omega-interposon mutagenesis. Infect Immun 62:4868-73
Ofek, I; Kabha, K; Athamna, A et al. (1993) Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a. Infect Immun 61:4208-16
Hasty, D L; Ofek, I; Courtney, H S et al. (1992) Multiple adhesins of streptococci. Infect Immun 60:2147-52
Sokurenko, E V; Courtney, H S; Abraham, S N et al. (1992) Functional heterogeneity of type 1 fimbriae of Escherichia coli. Infect Immun 60:4709-19
Courtney, H S; Hasty, D L; Dale, J B et al. (1992) A 28-kilodalton fibronectin-binding protein of group A streptococci. Curr Microbiol 25:245-50
Abraham, S N; Land, M; Ponniah, S et al. (1992) Glycerol-induced unraveling of the tight helical conformation of Escherichia coli type 1 fimbriae. J Bacteriol 174:5145-8

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