ADDITION OF A BACTERIAL ALGINATE LYASE TO PURULENT CF SPUTUM IN-VITROCAN RESULT IN THE DISRUPTION OF ALGINATE AND MODIFICATION OF SPUTUM VISCOELASTICITY

Alginate is a large molecular weight exopolysaccharide present in the purulent airway secretions of cystic fibrosis (CF) patients. This poly mer, produced by some of the opportunistic pathogens associated with t he recurrent lung infections characteristic of CF, has been suggested to effect an increase in the viscoelastic properties of purulent CF ai rway secretions. We have investigated the use of an enzyme targeted at this exopolysaccharide, an alginate lyase obtained from a bacterial s ource, to disrupt its polymeric nature and effect a change in the theo logical properties of CF sputum in vitro. Expectorated sputum samples obtained from hospitalized CF patients were found to contain 80-200 mu g alginate per ml sputum with no measurable endogenous alginate lyase activity. Treatment with exogenous alginate lyase prepared from a muc oid strain of Pseudomonas aeruginosa resulted in the disruption of alg inate and a decrease in sputum viscoelasticity in a small percentage o f the samples tested, Similar treatment of these samples with recombin ant human deoxyribonuclease I to cleave DNA present in purulent sputum and the use of alginate extracted from sputum as an alginate lyase as say substrate suggested that the inability of the exogenous alginate l yase to disrupt sputum alginate was not due to substrate inaccessibili ty or an unresponsive substrate. Concentrations of Ca2+ and Zn2+ in al ginate lyase-resistant sputum samples, determined by metal ion analysi s, were found to inhibit enzyme activity in studies using seaweed algi nate as a substrate. High concentrations of Ca2+ and Zn2+ in sputum sa mples initially resistant to lyase activity could be reduced significa ntly in some samples by dialysis and these same samples acquired sensi tivity to the lyase, Other sputum samples did not show reduced concent rations of Ca2+ and Zn2+ following dialysis and these samples remained lyase-insensitive. Together, these results suggest that bacterial alg inate present within purulent CF sputum may be quite stable, that endo genous alginate lyase activities appear to he 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 purule nt CF sputum samples.