title: Addition of a bacterial alginate lyase to purulent CF sputum in vitro can result in the disruption of alginate and modification of sputum viscoelasticity.
abstract: Alginate is a large molecular weight exopolysaccharide present in the purulent airway secretions of cystic fibrosis (CF) patients. This polymer, produced by some of the opportunistic pathogens associated with the recurrent lung infections characteristic of CF, has been suggested to effect an increase in the viscoelastic properties of purulent CF airway secretions. We have investigated the use of an enzyme targeted at this exopolysaccharide, an alginate lyase obtained from a bacterial source, to disrupt its polymeric nature and effect a change in the rheological properties of CF sputum in vitro. Expectorated sputum samples obtained from hospitalized CF patients were found to contain 80-200 micrograms alginate per ml sputum with no measurable endogenous alginate lyase activity. Treatment with exogenous alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa resulted in the disruption of alginate and a decrease in sputum viscoelasticity in a small percentage of the samples tested. Similar treatment of these samples with recombinant human deoxyribonuclease I to cleave DNA present in purulent sputum and the use of alginate extracted from sputum as an alginate lyase assay substrate suggested that the inability of the exogenous alginate lyase to disrupt sputum alginate was not due to substrate inaccessibility or an unresponsive substrate. Concentrations of Ca2+ and Zn2+ in alginate lyase-resistant sputum samples, determined by metal ion analysis, were found to inhibit enzyme activity in studies using seaweed alginate as a substrate. High concentrations of Ca2+ and Zn2+ in sputum samples initially resistant to lyase activity could be reduced significantly in some samples by dialysis and these same samples acquired sensitivity to the lyase. Other sputum samples did not show reduced concentrations of Ca2+ and Zn2+ following dialysis and these samples remained lyase-insensitive. Together, these results suggest that bacterial alginate present within purulent CF sputum may be quite stable, that endogenous alginate lyase activities appear to be limited and that the in vitro addition of exogenous alginate lyase can lead to the disruption of alginate and a change in the viscoelastic properties of some purulent CF sputum samples.
authors.affiliation: Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Mrsny
authors.forename: B A
authors.initials: BA
authors.lastname: Lazazzera
authors.forename: A L
authors.initials: AL
authors.lastname: Daugherty
authors.forename: N L
authors.initials: NL
authors.lastname: Schiller
authors.forename: T W
authors.initials: TW
authors.lastname: Patapoff
mesh_terms: D000464:Alginates / Q000737:chemistry*; D003550:Cystic Fibrosis / Q000378:metabolism*; D004548:Elasticity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008670:Metals / Q000032:analysis; D011133:Polysaccharide-Lyases / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013183:Sputum / Q000737:chemistry / Q000187:drug effects*; D014783:Viscosity
chemical_list: D000464:Alginates; D008670:Metals; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase