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.
Highlights
Biofilms are complex and dynamic structures formed by different pathogens that cause chronic persistent and recurrent infections
Multiple studies have reported that the biofilm inhibitory concentration (BIC) of antibiotics against P. aeruginosa biofilm cultures is lowered when combined with alginate lyase activity[18,19,20,21,47], it was shown that synergy between tobramycin and the A1-III and AlgL enzymes is completely decoupled from their catalytic activity[47]
An upper band of approximately 40 kDa was enriched in fraction B5-B4 and a lower band of about 30 kDa was enriched in fractions B3 to B1, both being consistent across two different samples fractionated the same way
Summary
Biofilms are complex and dynamic structures formed by different pathogens that cause chronic persistent and recurrent infections. Multiple studies have reported that the biofilm inhibitory concentration (BIC) of antibiotics against P. aeruginosa biofilm cultures is lowered when combined with alginate lyase activity[18,19,20,21,47], it was shown that synergy between tobramycin and the A1-III and AlgL enzymes is completely decoupled from their catalytic activity[47]. This raises the question of whether it is their enzyme activity that is important for dissolving biofilms. A careful reexamination of the alginate lyases responsible for the enzymatic activities present in these extracts could clarify whether these proteins can really be used to dissolve P. aeruginosa biofilms and reduce the required dose of antibiotics in CF patients
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