Applying a polysaccharide lyase from Stenotrophomonas maltophilia to disrupt alginate exopolysaccharide produced by Pseudomonas aeruginosa clinical isolates

Pseudomonas aeruginosa is considered one of the most challenging, drug-resistant, opportunistic pathogens partly due to its ability to synthesize robust biofilms. Biofilm is a mixture of extracellular polymeric substances (EPS) that encapsulates microbial cells, leading to immune evasion, antibiotic resistance, and thus higher risk of infection. In the cystic fibrosis lung environment, P. aeruginosa undergoes a mucoid transition, defined by overproduction of the exopolysaccharide alginate. Alginate encapsulation results in bacterial resistance to antibiotics and the host immune system. Given its role in airway inflammation and chronic infection, alginate is an obvious target to improve treatment for P. aeruginosa infection. Previously, we demonstrated polysaccharide lyase Smlt1473 from Stenotrophomonas maltophilia strain k279a can catalyze the degradation of multiple polyuronides in vitro, including D-mannuronic acid (poly-ManA). Poly-ManA is a major constituent of P. aeruginosa alginate, suggesting that Smlt1473 could have potential application against multidrug-resistant P. aeruginosa and perhaps other microbes with related biofilm composition. In this study, we demonstrate that Smlt1473 can inhibit and degrade alginate from P. aeruginosa. Additionally, we show that tested P. aeruginosa strains are dominant in acetylated alginate and that all but one have similar M-to-G ratios. These results indicate that variation in enzyme efficacy among the isolates is not primarily due to differences in total EPS or alginate chemical composition. Overall, these results demonstrate Smlt1473 can inhibit and degrade P. aeruginosa alginate and suggest that other factors including rate of EPS production, alginate sequence/chain length, or non-EPS components may explain differences in enzyme efficacy.

Importance: Pseudomonas aeruginosa is a major opportunistic human pathogen in part due to its ability to synthesize biofilms that confer antibiotic resistance. Biofilm is a mixture of polysaccharides, DNA, and proteins that encapsulate cells, protecting them from antibiotics, disinfectants, and other cleaning agents. Due to its ability to increase antibiotic and immune resistance, the exopolysaccharide alginate plays a large role in airway inflammation and chronic P. aeruginosa infection. As a result, colonization with P. aeruginosa is the leading cause of morbidity and mortality in CF patients. Thus, it is an obvious target to improve the treatment regimen for P. aeruginosa infection. In this study, we demonstrate that polysaccharide lyase, Smlt1473, inhibits alginate secretion and degrades established alginate from a variety of mucoid P. aeruginosa clinical isolates. Additionally, Smlt1473 differs from other alginate lyases in that it is active against acetylated alginate, which is secreted during chronic lung infection. These results suggest that Smlt1473 may be useful in treating infections associated with alginate-producing P. aeruginosa, as well as have the potential to reduce P. aeruginosa EPS in non-clinical settings.