Characterization of different alginate lyases for dissolving Pseudomonas aeruginosa biofilms

Abstract

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II’ (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.

Structure Characteristics, Biochemical Properties, and Pharmaceutical Applications of Alginate Lyases

“…Núria et al observed the contradiction between different reports, and they tested five different alginate lyases in order to find the secrets behind the contradiction. According to their results, only enzymes with polyM/G activity are effective in dissolving biofilms and have a synergistic effect with ciprofloxacin antibiotic, while strict polyM or polyG specific alginate lyases show fewer activities and do not have the synergistic effect [92], which verifies that substrate specificity acts as a key factor in pharmaceutical applications, as described above. Here, this paper summarizes some online studies posted in recent years.…”

“…As a protective barrier, the thick biofilm is composed of several components [91]. Alginates are the major component of P. aeruginosa biofilm, which is responsible for surface adhesion and stabilization of biofilm to resist antibiotics [92,93]. Moreover, alginates can interrupt the phagocytosis of macrophages and neutrophils, limit the lymphocyte function, and induce inflammatory reactions to aggravate the lung infec-tion [91].…”

Advancements in Biodegradable Active Films for Food Packaging: Effects of Nano/Microcapsule Incorporation

“…Alginate is the salt of alginic acid, a bio-based polymer consisting of D-mannuronic and L-guluronic monomers extracted from brown marine algae (brown seaweeds) and some bacteria, such as Pseudomonas aeruginosa [ 93 , 94 ]. Alginate is nontoxic, non-antigenic, biocompatible, and biodegradable, and it has the capacity to form a hydrogel when applied in encapsulation.…”

Separation and quantification of 2-keto-3-deoxy-gluconate (KDG) a major metabolite in pectin and alginate degradation pathways

“…CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 3, 2020. ; https://doi.org/10.1101/2020.07.24.220400 doi: bioRxiv preprint 4 [19]. KdgF was identified as the missing link in the microbial metabolism of uronate sugars from pectin and alginate|20] (Figure 1).…”