Alginate Lyase Exhibits Catalysis-Independent Biofilm Dispersion and Antibiotic Synergy

Abstract

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...

Innovative Strategies for Combating Biofilm-Based Infections

“…Dispersin B also sensitizes S. epidermidis biofi lm cells to the action of antimicrobials (Izano et al 2008 ;Donelli et al 2007 ) and is active in vivo, lowering the rate of catheter colonization by S. aureus in combination with triclosan in a rabbit model of infection (Darouiche et al 2009 ). Alginate lyase degrades the polysaccharide alginate and enhances the microbicidal activity of aminoglycosides against P. aeruginosa biofi lms in vitro (Alkawash et al 2006 ;Alipour et al 2009 ;Lamppa and Griswold 2013 ). Alginate lyase has demonstrated in vivo effi cacy, enhancing the clearance of mucoid P. aeruginosa when coadministered with amikacin in a rabbit model of endocarditis (Bayer et al 1992 ).…”

What's on the Outside Matters: The Role of the Extracellular Polymeric Substance of Gram-negative Biofilms in Evading Host Immunity and as a Target for Therapeutic Intervention

“…Given the abundance of alginate in biofilms produced by many mucoid Pa isolates, the use of alginate lyase to disperse biofilms has been investigated (75). A recent study (76) reinforced the conclusions about the biofilm-disruptive properties of this approach as well as its synergistic interaction with antibiotics. However, they present data that question the mechanisms that underlie alginate lyase enzyme-based therapies.…”

Modeling Biofilms: From Genes to Communities

“…Detachment and dispersal of planktonic cells from biofilms could also have a genetic basis. It has been suggested that increased expression of the alginate lyase in the mucoid strain of P. aeruginosa led to alginate degradation and increased cell detachment [27,28]. Antibiotic therapy in patients colonized with P. aeruginosa often gives a measure of relief from symptoms, but fails to cure the basic ongoing infection [29,30].…”