(47e) Genetically Engineered Alginate Lyase for the Treatment of Bacterial Biofilms

Alginate lyases, enzymes that degrade the alginate copolymer of guluronic and mannuronic acid, have often been proposed as a therapeutic candidate for the disruption of alginate-rich biofilms associated with Cystic Fibrosis (CF) related mucoid Pseudomonas aeruginosa (P. aeruginosa) infections. It is hypothesized that disruption of these protective bacterial biofilms would allow better antibiotic access to the infection and increase overall lung function. One therapeutic candidate in particular, Sphingomonas sp. A1-III alginate lyase (A1-III), has already shown significant potential for the degradation of bacterial alginate, but is unfortunately predisposed towards excessive immunogenicity as a result of its bacterial origin. To address this problem, we site-specifically engineered a panel of A1-III enzymes through a varied single cysteine substitution that allowed for controlled, orthogonal, maleimide-thiol conjugation of polyethylene glycol (PEG). This rational approach produced a 40-90% decrease in antigenicity for the A1-III mutants when compared to the wild type via enzyme-linked immunosorbent assays (ELISA). The most active enzyme, A53Ch-PEG, displayed a 48% decrease in antigenicity and a 44% decreased in vivo immunogenicity when evaluated in a rabbit model. Further antibody binding studies using a naïve library of human single chain variable fragments (scFv) displayed on yeast produced a 90% reduction in human scFv binding to A53Ch-PEG when compared to the wild type enzyme, suggesting that blocking of the antigenic epitopes translates to the human immune system. Additionally, A53Ch-PEG was found to possess Michaelis-Menten kinetics equivalent to the wild type enzyme when assayed in the presence of soluble brown seaweed alginate and displayed an 80% increase in specific activity over the wild type when assayed against alginate purified from the mucoid P. aeruginosa clinical isolate FRD1. A similar increase in functionality was observed in a series of biofilm disruption studies in which A53Ch-PEG managed to remove 94% of a mucoid clinical isolate biofilm grown in the wells of a 96-well plate, while the wild type enzyme only managed a 75% reduction. These results, along with the various immunogenicity studies, suggest that genetically and chemically modified A1-III alginate lyases posses therapeutically relevant, enhanced alginate degrading activity while concurrently possessing a decreased potential for producing an immune response in patients.