Staphylococcus aureus microbial biofilms degradation using cellobiose dehydrogenase from Thermothelomyces thermophilus M77

Samaniego, Lorgio Victor Bautista; Higasi, Paula Miwa Rabêlo; Capetti, Caio Cesar de Mello; Cortez, Anelyse Abreu; Pratavieira, Sebastião; Pellegrini, Vanessa de Oliveira Arnoldi; Dabul, Andrei Nicoli Gebieluca; Segato, Fernando; Polikarpov, Igor

doi:10.1016/j.ijbiomac.2023.125822

Cited by 1 publication

(21 citation statements)

References 72 publications

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“…So, for all the exposed above, in this study we proposed as a new antibiofilm candidate, a CDH enzyme form Thermothelomyces thermophilus (TthCDHIIa) 46 . T. thermophilus is a filamentous fungus that grows in soils and high temperature compost (35-48°C) and has an excellent lignocellulose degrading capability 47 .…”

Section: Searching For New Antibiofilm Agentsmentioning

confidence: 99%

“…The enzyme is completely inactivated after 30 min at 70 °C (Figure 6c). 46 An initial screening of substrate preference using the DCPIP test was conducted, and it identified disaccharides like lactose and cellobiose, in combination with cellooligosaccharides, as some of the most favorable substrates (Figure 6d). Also, we evaluated the effect of different ions on TthCDHIIa enzymatic activity and thermal stability.…”

Section: Production and Characterization Of Tthcdhiiamentioning

confidence: 99%

“…In general, there was a slight boosting effect in the presence of divalent cations, such as Co +2 (114%), Ca +2 (109%), Zn +2 (107%), Ni +2 (106%) and Mg +2 (104%) at 10 mM concentration (Figure 7a). Divalent cations like Ca +2 show an ion bridge effect, that stabilizes and bring together both CDH domains 46 improving the IET and substrate accommodation 57 . Conversely, Mn +2 , Cu +2 and Li +1 ions decreased the enzymatic activity by an average of 30%, while Fe +3 can inhibit more than 90% of the capacity of CDH to reduce DCPIP (Figure 7a).…”

Section: Production and Characterization Of Tthcdhiiamentioning

confidence: 99%

“…After 24 h of enzymatic reaction, a residual production of approximately 20 µM H2O2 could be quantified. The monitored fluorescence and the peak temperature in Δϕ/Δt curve were used to extract the melting temperature (Tm) Source: Adapted from SAMANIEGO et al 46 In other studies, a group of class I (basidiomycetes) CDHs from Phlebia lindtneri (PlCDH), Phanerodontia (Phanerochaete) chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) revealed variable capacities to produce hydrogen peroxide 41 . In 24h reactions and using cellobiose as a substrate, the maximum production of H2O2 by CuCDH reached about 150 µM, both PchCDH and PsCDH produced 80 µM H2O2, whereas PlCDH was only capable to deliver about 1 µM H2O2 41 .…”

Section: Hydrogen Peroxide Productionmentioning

confidence: 99%

“…The capacity of TthCDHIIa to distinguish between lactose and cellobiose can be calculated by the ratio of catalytic efficiencies [kcat/KM Cellobiose]/[kcat/KM Lactose], giving a factor of 3.3, revealing relatively low substrate discrimination in comparison to the more selective class I CDHs (basidiomycetes), but within the range observed for class II CDHs 1 . Source: Adapted from SAMANIEGO et al 46 Despite their bigger molecular size, the cellooligosaccharides are recognized and oxidized by the enzyme (Table 2). Apparently, the enzymatic efficiency stays approximately the same and even grows a little as the chain length of substrate increases .…”

Section: Substrate Preference and Steady-state Kinetics Of Tthcdhiiamentioning

confidence: 99%

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

Samaniego

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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.

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Section: Searching For New Antibiofilm Agentsmentioning

confidence: 99%

Section: Production and Characterization Of Tthcdhiiamentioning

confidence: 99%

Section: Production and Characterization Of Tthcdhiiamentioning

confidence: 99%

Section: Hydrogen Peroxide Productionmentioning

confidence: 99%

Section: Substrate Preference and Steady-state Kinetics Of Tthcdhiiamentioning

confidence: 99%

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

Samaniego

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show abstract

Staphylococcus aureus microbial biofilms degradation using cellobiose dehydrogenase from Thermothelomyces thermophilus M77

Cited by 1 publication

References 72 publications

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

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

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