Study of CAZymes from >i<Thermothelomyces thermophilus>/i< M77 as antibiofilm agents: an oxidative and hydrolytic approach

Abstract

This work presents the biochemical characterization of relevant CAZymes from Thermothelomyces thermophilus M77, which includes a cellobiose dehydrogenase (TthCDHIIa), an endoglucanase (TthCel7B), and cellobiohydrolases (TthCel7A and TthCel6A). Furthermore, it explores their application as antimicrobial and antibiofilm agents.In the first part of this study, it was demonstrated that TthCDHIIa is thermostable in different ionic solutions and is capable of oxidizing multiple mono and oligosaccharide substrates and to continuously produce H2O2. Kinetics measurements depict the enzyme catalytic characteristics consistent with an Ascomycota class II CDH. Our structural analyses show that TthCDHIIa substrate binding pocket is spacious enough to accommodate larger cello and xylooligosaccharides. We also reveal that TthCDHIIa supplemented with cellobiose reduces the viability of Staphylococcus. aureus ATCC 25923 up to 32% in a planktonic growth model and inhibits its biofilm growth on 62.5%. Furthermore, TthCDHIIa eradicates preformed S. aureus biofilms via H2O2 oxidative degradation of the biofilm matrix, making these bacteria considerably more susceptible to gentamicin and tetracycline. In the second part of this study, the investigated cellulases exhibited a preference for acidic conditions and high temperatures in the hydrolysis of substrates. Additionally, we described the functionality of the carbohydratebinding module. The structural characteristics of cellobiohydrolases and endoglucanases, such as the loop arrangement, aligned with the type of recognized substrate. The optimization of the mixture of TthCel7A, TthCel7B, and TthCel6A using a Simplex-lattice design model revealed that for a higher degradation of Gluconoacetobacter hansenii BC, a 49.3% TthCel7A and 50.7% TthCel6A enzymatic load is required. To achieve optimal degradation of a pathogenic model and a clinical Escherichia coli biofilm, binary mixtures comprising 56.5% TthCel7B + 43.5% TthCel6A and 59.6% TthCel7A + 40.4% TthCel7B were found to be effective, respectively. This optimization resulted in a reduction in the quantity of enzymes required for biofilm eradication, with EC50 values of 0.086 μM and 0.63 μM for the hydrolysis of clinical and pathogenic E. coli, respectively. Confocal laser scanning microscopy demonstrated changes in protein and carbohydrate content under cellulase treatment. Notably, TthCel7B played a key role as a potent eradication agent, particularly since extracted cellulose from E. coli biofilms is amorphous. These findings provide valuable insights for the prospective use of endoglucanase enzymes in the treatment of biofilm-associated diseases. In summary, T. thermophilus emerges as a promising source of antibiofilm enzymes.