Synergistic Antifungal Activity of Chito-Oligosaccharides and Commercial Antifungals on Biofilms of Clinical Candida Isolates.

The development of yeast biofilms is a major problem due to their increased antifungal resistance, which leads to persistent infections with severe clinical implications. The high antifungal activity of well-characterized chitosan polymers makes them potential alternatives for treating yeast biofilms. The activity of a chito-oligosaccharide with a depolymerization degree (DPn) of 32 (C32) and a fraction of acetylation (FA) of 0.15 on Candida sp. biofilms was studied. The results showed a concentration-dependent reduction in the number of viable cells present in C. albicans, C. glabrata, and C. guillermondii preformed biofilms in the presence of C32, especially on intermediate and mature biofilms. A significant decrease in the metabolic activity of yeast biofilms treated with C32 was also observed. The antifungals fluconazole (Flu) and miconazole (Mcz) decreased the number of viable cells in preformed early biofilms, but not in the intermediate or mature biofilms. Contrary to Flu or Mcz, C32 also reduced the formation of new biofilms. Interestingly, a synergistic effect on yeast biofilm was observed when C32 and Flu/Mcz were used in combination. C32 has the potential to become an alternative therapeutic agent against Candida biofilms alone or in combination with antifungal drugs and this will reduce the use of antifungals and decrease antifungal resistance.

Genomic and Proteomic Study of Andreprevotia ripae Isolated from an Anthill Reveals an Extensive Repertoire of Chitinolytic Enzymes.

Chitin is an abundant natural polysaccharide that is hard to degrade because of its crystalline nature and because it is embedded in robust co-polymeric materials containing other polysaccharides, proteins, and minerals. Thus, it is of interest to study the enzymatic machineries of specialized microbes found in chitin-rich environments. We describe a genomic and proteomic analysis of Andreprevotia ripae, a chitinolytic Gram-negative bacterium isolated from an anthill. The genome of A. ripae encodes four secreted family GH19 chitinases of which two were detected and upregulated during growth on chitin. In addition, the genome encodes as many as 25 secreted GH18 chitinases, of which 17 were detected and 12 were upregulated during growth on chitin. Finally, the single lytic polysaccharide monooxygenase (LPMO) was strongly upregulated during growth on chitin. Whereas 66% of the 29 secreted chitinases contained two carbohydrate-binding modules (CBMs), this fraction was 93% (13 out of 14) for the upregulated chitinases, suggesting an important role for these CBMs. Next to an unprecedented multiplicity of upregulated chitinases, this study reveals several chitin-induced proteins that contain chitin-binding CBMs but lack a known catalytic function. These proteins are interesting targets for discovery of enzymes used by nature to convert chitin-rich biomass. The MS proteomic data have been deposited in the PRIDE database with accession number PXD025087.

Antifungal activity of well-defined chito-oligosaccharide preparations against medically relevant yeasts.

Due to their antifungal activity, chitosan and its derivatives have potential to be used for treating yeast infections in humans. However, to be considered for use in human medicine, it is necessary to control and know the chemical composition of the compound, which is not always the case for polymeric chitosans. Here, we analyze the antifungal activity of a soluble and well-defined chito-oligosaccharide (CHOS) with an average polymerization degree (DPn) of 32 and fraction of acetylation (FA) of 0.15 (C32) on 52 medically relevant yeast strains. Minimal inhibitory concentrations (MIC) varied widely among yeast species, strains and isolates (from > 5000 to < 9.77 µg mL-1) and inhibition patterns showed a time- and dose-dependencies. The antifungal activity was predominantly fungicidal and was inversely proportional to the pH, being maximal at pH 4.5, the lowest tested pH. Furthermore, antifungal effects of CHOS fractions with varying average molecular weight indicated that those fractions with an intermediate degree of polymerization, i.e. DP 31 and 54, had the strongest inhibitory effects. Confocal imaging showed that C32 adsorbs to the cell surface, with subsequent cell disruption and accumulation of C32 in the cytoplasm. Thus, C32 has potential to be used as a therapy for fungal infections.

Antibiotic saving effect of combination therapy through synergistic interactions between well-characterized chito-oligosaccharides and commercial antifungals against medically relevant yeasts.

Combination therapies can be a help to overcome resistance to current antifungals in humans. The combined activity of commercial antifungals and soluble and well-defined low molecular weight chitosan with average degrees of polymerization (DPn) of 17-62 (abbreviated C17 -C62) and fraction of acetylation (FA) of 0.15 against medically relevant yeast strains was studied. The minimal inhibitory concentration (MIC) of C32 varied greatly among strains, ranging from > 5000 µg mL-1 (Candida albicans and C. glabrata) to < 4.9 (C. tropicalis). A synergistic effect was observed between C32 and the different antifungals tested for most of the strains. Testing of several CHOS preparations indicated that the highest synergistic effects are obtained for fractions with a DPn in the 30-50 range. Pre-exposure to C32 enhanced the antifungal effect of fluconazole and amphotericin B. A concentration-dependent post-antifungal effect conserved even 24 h after C32 removal was observed. The combination of C32 and commercial antifungals together or as part of a sequential therapy opens new therapeutic perspectives for treating yeast infections in humans.

Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bacillus subtilis (BsCsn46A).

BsCsn46A, a GH family 46 chitosanase from Bacillus subtilis had been previously shown to have potential for bioconversion of chitosan to chito-oligosaccharides (CHOS). However, so far, in-depth analysis of both the mode of action of this enzyme and the composition of its products were lacking. In this study, we have employed size exclusion chromatography, 1H NMR, and mass spectrometry to reveal that BsCsn46A can rapidly cleave chitosans with a wide-variety of acetylation degrees, using a non-processive endo-mode of action. The composition of the product mixtures can be tailored by varying the degree of acetylation of the chitosan and the reaction time. Detailed analysis of product profiles revealed differences compared to other chitosanases. Importantly, BsCsn46A seems to be one of the fastest chitosanases described so far. The detailed analysis of preferred endo-binding modes using H218O showed that a hexameric substrate has three productive binding modes occurring with similar frequencies.