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.
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