More than 2 decades of study support the hypothesis that alginate lyases are promising therapeutic candidates for treating mucoid Pseudomonas aeruginosa infections. In particular, the enzymes' ability to degrade alginate, a key component of mucoid biofilm matrix, has been the presumed mechanism by which they disrupt biofilms and enhance antibiotic efficacy. The systematic studies reported here show that, in an in vitro model, alginate lyase dispersion of P. aeruginosa biofilms and enzyme synergy with tobramycin are completely decoupled from catalytic activity. In fact, equivalent antibiofilm effects can be achieved with bovine serum albumin or simple amino acids. These results provide new insights into potential mechanisms of alginate lyase therapeutic activity, and they should motivate a careful reexamination of the fundamental assumptions underlying interest in enzymatic biofilm dispersion. P remature mortality among cystic fibrosis (CF) patients is typically associated with chronic airway infection by the opportunistic pathogen Pseudomonas aeruginosa (1). Upon colonization of the airway, P. aeruginosa causes a range of clinical complications, including stimulation of a hyperinflammatory response, obstruction of the pulmonary tract, and progressive loss of lung function (1-3). The bacterium's persistence within the CF lung is thought to result in large part from a biofilm mode of growth (2, 4), which can subvert both the host immune response and antibacterial therapies (5). In contrast to environmental niches, in the CF lung, P. aeruginosa generally transitions to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate (6). This copolymer of (1,4)-linked ␤-D-mannuronic acid and ␣-L-guluronic acid alters biofilm architecture and function (7) and thereby compounds P. aeruginosa's persistence in the chronically inflamed airway (5). Indeed, alginate is one of the most extensively studied P. aeruginosa virulence factors (8).Given alginate's contribution to mucoid biofilm structure, its function in bacterial virulence, and its role in the persistent nature of lung infections, it has long been considered an attractive target for interventional therapies (6). In particular, biocatalytic degradation of mucoid P. aeruginosa biofilms using alginate lyase enzymes (EC 4.2.2.3) has been the subject of more than 20 years of research. Alginate lyase treatment has been shown to reduce viscosity in cultures of clinical isolates and in CF sputum (9, 10); it strips biofilms from abiotic surfaces (11, 12), it enhances phagocytosis and killing of P. aeruginosa by human immune cells (13)(14)(15), and it improves the efficacy of various antipseudomonal antibiotics (14,(16)(17)(18)(19). In aggregate, these reports make a rather convincing case for the use of inhaled alginate lyases for treating chronic P. aeruginosa infections of the CF lung, although no clinical trials have been conducted to date.In an effort to further bolster the rationale for therapeutic alginate lyases, we set out to conduct systemati...

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted towards the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high throughput screen was implemented to functionally interrogate combinatorial libraries of charge engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing.

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

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Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50 – 80 kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in E. coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli’s ease of culture, short doubling time, and facile genetics to yield upwards of 30 mg/L of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC chromatography, yielding as much as 21 mg/L of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications.