Chronic pulmonary infection with Pseudomonas aeruginosa (P. aeruginosa) results in a variety of clinical complications and progressive loss of lung function in cystic fibrosis (CF) patients. In particular, the mucoid P. aeruginosa strains typically found in the CF lung dominate the microbial flora, contribute to obstruction of the airway, and cause tissue damage as a result of a hyperactive inflammatory immune response. The persistent nature of these infections is due in large part to the bacteria's ability to form biofilms that protect it from the human immune system and various antibiotics. In the case of mucoid P. aeruginosa, the biofilm matrix is composed predominantly of alginate, a polysaccharide copolymer of mannuronic and guluronic acids. Motivated by the success of PulmozymeŽ as a treatment for extracellular DNA, it has been proposed that inhaled alginate lyase enzymes capable of degrading the exopolysaccharide alginate would ease clearance of airway obstructions, render the infecting P. aeruginosa more susceptible to the immune response and antibiotics, and result in an overall improvement in lung function. While a variety of alginate lyases have shown therapeutic potential in in vitro experiments, the bacterial origin of these enzymes predisposes them towards excessive immunogenicity in higher organisms. The putative immunogenicity of the enzymes in the already inflamed CF lung constitutes a barrier to utilizing this class of biotherapeutics in a clinical setting. Chemical modification of therapeutic proteins with polyethylene glycol (PEG) is one common approach to modulating immunogenicity. However, conventional strategies for conjugating PEG to the therapeutic candidate Sphingomonas sp. A1-III alginate lyase (A1-III) have been shown to negatively impact catalytic activity. Therefore, we genetically engineered the A1-III enzyme to facilitate site-specific conjugation of PEG with the expectation that selective labeling at positions distal to the active site would decrease immunogenicity while maintaining high levels of activity. Seven different mutant constructs of A1-III have been designed in which each contains a single non-native cysteine substitution that allows controlled conjugation of PEG by means of a maleimide-thiol reaction. We demonstrate that at least one of these site-specific PEG conjugates exhibits solution phase kinetics equal to the native enzyme, but is significantly less immunoreactive towards antiserum raised against the native protein. These results indicate that the modified protein may have practical therapeutic potential, a possibility that is currently being explored by both in vivo immunoreactivity studies and advanced in vitro biofilm disruption assays.

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