Molecular modeling and redesign of alginate lyase from Pseudomonas aeruginosa for accelerating CRPA biofilm degradation

Abstract

Administration of an efficient alginate lyase (AlgL) or AlgL mutant may be a promising therapeutic strategy for treatment of cystic fibrosis patients with Pseudomonas aeruginosa infections. Nevertheless, the catalytic activity of wild-type AlgL is not sufficiently high. It is highly desired to design and discover an AlgL mutant with significantly improved catalytic efficiency against alginate substrates. For the purpose of identifying an AlgL mutant with significantly improved catalytic activity, in this study, we first constructed and validated a structural model of AlgL interacting with substrate, providing a better understanding of the interactions between AlgL and its substrate. Based on the modeling insights, further enzyme redesign and experimental testing led to discovery of AlgL mutants, including the K197D/K321A mutant, with significantly improved catalytic activities against alginate and acetylated alginate in ciprofloxacin-resistant P. aeruginosa (CRPA) biofilms. Further anti-biofilm activity assays have confirmed that the K197D/K321A mutant with piperacillin/tazobactam is indeed effective in degrading the CRPA biofilms. Co-administration of the potent mutant AlgL and an antibiotic (such as a nebulizer) could be effective for therapeutic treatment of CRPA-infected patients with cystic fibrosis. Proteins 2016; 84:1875-1887. © 2016 Wiley Periodicals, Inc.

Advanced strategies for combating bacterial biofilms

“…Many studies stated that treatment with the liquid crystals containing alginate lyase (AlgL) and gentamicin resulted in a greater 2‐log reduction in mucoid P. aeruginosa (clinical isolate) biofilms (Thorn, Prestidge, Boyd, & Thomas, ). Furthermore, a combination of AlgL and other antibiotics, such as Piperacillin/Tazobactam, Tobramycin, and Clarithromycin, enhanced the disruption of Ciprofloxacin‐resistant P. aeruginosa (CRPA) biofilms and other bacterial biofilms significantly (Bugli, Palmieri et al, , Cho, Huang et al, , Jang, Piao et al, , Lamppa & Griswold, ).…”

Dispersal and inhibition of biofilms associated with infections

“…The efficacy of the combination of a mutant alginate lyase and piperacillin-tazobactam was compared to piperacillin-tazobactam alone and piperacillin-tazobactam with the wild-type enzyme against ciprofloxacin-resistant P. aeruginosa (CRPA) biofilms. The combination therapy with the mutant enzyme reduced biofilm formation and a lower dose of piperacillin-tazobactam was required to degrade the CRPA biofilm (Cho et al 2016).…”

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum

“…Genetically engineered MC5Es from A. vinelandii altered the epimerization pattern of alginate [20,21]. Mutants of ALs from alginate-producing bacteria exhibited improved activity in degrading P. aeruginosa biofilms associated with cystic fibrosis [22,23]. Consequently, MC5Es and ALs hold promise for tailoring alginate for a wide range of applications.…”