Mucins of the respiratory tract appear to serve a defensive role against bacterial infection by trapping bacteria and removing them from contact with epithelial surfaces. However in certain diseases e.g. cystic fibrosis (CF), the mucin becomes persistently colonized by Pseudomonas aeruginosa and the mucin-bacterial association appears to serve a pathogenetic role rather than a defensive one. While there may be many factors contributing to this association, a very important one must be a special ability of P. aeruginosa to adhere tightly to mucins and to persist in them. Another important attribute would be some versatility in evading the host's immune system. This proposal seeks to understand the basis of the first attribute, adherence to mucins, and to provide some rationale for the development of therapeutic modalities aimed at preventing or palliating the state of chronic colonization.
The specific aims are (i) to study and compare the adherence of P. aeruginosa strains to normal and CF mucin by mathematical modelling, (ii) to examine whether elevated Ca++ concentrations play a role in adherence, (iii) to ascertain the chemical nature of the receptor(s) for this organism and, (iv) to examine the role of bacterial structures such as pili and the mucoid covering, as adhesins for mucin. Tracheobrochial mucins will be isolated from sputum or tracheal secretions of CF and nonCF patients by combination of methods involving centrifugation, precipitation and column chromatography. Adherence of P. aeruginosa to the mucins will be tested in a microtiter plate assay which has been worked out in this laboratory. The affinity of mucoid and nonmucoid strains for these mucins will be compared mathematically by Langmuir isotherm and Michaelis-Menten analyses to detect differences between strains and between mucins. The effect of calcium concentration on affinity will also be examined. The receptor(s) involved in this interaction will be ascertained by using sugars, oligosaccharides and lectins in blocking experiments with whole bacteria and by use of established methods of oligosaccharide degradation such as enzyme treatment, metaperiodate and acid treatment of mucins. Finally the adhesins responsible for adherence will be ascertained by preparing the possible adhesins such as pili and the mucoid exopolysaccharide and testing them in competitive assays against whole bacteria. Antibodies against the putative adhesins will also be tested for their ability to inhibit adherence of whole bacteria. In total these studies could suggest whether there are differences between CF respiratory mucin and normal mucin.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Modified Research Career Development Award (K04)
Project #
5K04HL001479-03
Application #
3073807
Study Section
Bacteriology and Mycology Subcommittee 1 (BM)
Project Start
1984-07-01
Project End
1989-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Kwiatkowski, P; Puc, J; Rotbart-Fiedor, M et al. (1997) Effects of pefloxacin on rat pancreatic islet cell function after 24-hour cold preservation. Transplant Proc 29:1984-5
Konopka, L M; Cooper, R; Crayton, J W (1996) Serotonin-induced increases in platelet cytosolic calcium concentration in depressed, schizophrenic, and substance abuse patients. Biol Psychiatry 39:708-13
Cooper, R S; Rotimi, C N (1995) Absence of black-white differences in sodium and calcium in platelets. Am J Hypertens 8:558-64
Cooper, R S; Cheng, H Y; Rotimi, C (1995) Basal and stimulated platelet calcium and sodium in hypertensive versus normotensive black people. J Hum Hypertens 9:747-52
Ramphal, R; Carnoy, C; Fievre, S et al. (1991) Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1 (Gal beta 1-3GlcNAc) or type 2 (Gal beta 1-4GlcNAc) disaccharide units. Infect Immun 59:700-4
Ramphal, R; Houdret, N; Koo, L et al. (1989) Differences in adhesion of Pseudomonas aeruginosa to mucin glycopeptides from sputa of patients with cystic fibrosis and chronic bronchitis. Infect Immun 57:3066-71
Houdret, N; Ramphal, R; Scharfman, A et al. (1989) Evidence for the in vivo degradation of human respiratory mucins during Pseudomonas aeruginosa infection. Biochim Biophys Acta 992:96-105
Ramphal, R; Lhermitte, M; Filliat, M et al. (1988) The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum. J Antimicrob Chemother 22:483-90
Vishwanath, S; Ramphal, R; Guay, C M et al. (1988) Respiratory-mucin inhibition of the opsonophagocytic killing of Pseudomonas aeruginosa. Infect Immun 56:2218-22
Ramphal, R; Guay, C; Pier, G B (1987) Pseudomonas aeruginosa adhesins for tracheobronchial mucin. Infect Immun 55:600-3

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