DNase increases the efficacy of antimicrobial photodynamic therapy on Candida albicans biofilms.

Antimicrobial Photodynamic Therapy (aPDT) has been proposed as a means to treat Candida infections. However, microorganisms in biofilms are less susceptible to aPDT than planktonic cultures, possibly because the matrix limits the penetration of the photosensitizer. Therefore, the goals here were: (1) to target biofilm matrix components of a fluconazole-susceptible (S) and a fluconazole-resistant (R) C. albicans (Ca) strains using the hydrolytic enzymes ß-glucanase and DNase individually or in combination; (2) to apply the best enzyme protocol in association with aPDT mediated by Photodithazine® (PDZ); (3) to verify under confocal microscope the penetration of PDZ in biofilms pre-treated or not with DNase at different periods of incubation. CaS and CaR 48h-old biofilms were incubated with the hydrolytic enzymes (5 min) and evaluated by cell viability, biomass, and matrix components. DNase showed the best outcomes by significantly reducing extracellular DNA (eDNA) and soluble proteins from the matrix of both strains; and water-soluble polysaccharides from CaR matrix. Subsequently, 48h-old biofilms were incubated with DNase for 5 min, followed by incubation with PDZ for 20 min and exposure to LED light (660 nm, 50 J/cm²). Controls were biofilms treated only with aPDT without DNase, PDZ only, PDZ + DNase, light only, light + DNase, and biofilm without treatment. Pre-treatment with DNase allowed PDZ penetration into deeper biofilm layers, and the aPDT effect was enhanced, showing a significant reduction of the cell viability (p = 0.000) and eDNA amounts (p ≤ 0.047). DNase affected the matrix composition improving the penetration of the photosensitizer, thereby, improving the effectiveness of subsequent aPDT.

Recombinant ß-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth.

In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a ß-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of ß-1,3-1,4-glucanases and presented high protein identity with ß-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with ß-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches' broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao ß-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a ß-1,3-1,4-glucanase, in particular against M. perniciosa.

Potential fungal inhibition by immobilized hydrolytic enzymes from Trichoderma asperellum.

The use of cell wall degrading enzymes from Trichoderma asperellum immobilized on biodegradable support is an alternative for food packaging. In this study, hydrolytic enzymes produced by T. asperellum were tested as a fungal growth inhibitor, in free form or immobilized on a biodegradable film composed of cassava starch and poly(butylene adipate-co-terephtalate) (PBAT). The inhibitory activity was tested against Aspergillus niger , Penicillium sp., and Sclerotinia sclerotiorum , microorganisms that frequently degrade food packaging. The use of chitin as carbon source in liquid medium induced T. asperellun to produce N-acetylglucosaminidase, ß-1,3-glucanase, chitinase, and protease. The presence of T. asperellun cell wall degradating enzymes (T-CWD) immobilized by adsorption or covalent attachment resulted in effective inhibition of fungal growth. The enzymatic activity of T-CWD was stronger on S. sclerotiorum than on the Aspergillus or Penicillum isolates tested. These results suggest that T-CWD can be used in a free or immobilized form to suppress fungi that degrade food packaging.