Targeting Non- <i>albicans Candida</i> Strains and Phytopathogenic <i>Fusarium</i> Species with Chitooligosaccharides: Insights into the Antifungal Mechanism

The worldwide emergence of resistance to commonly used licensed antifungal drugs, such as azoles and echinocandins, has raised significant concerns for public health and stimulated the search for new antifungal candidates. Chitooligosaccharides (COSs) are water-soluble, biocompatible compounds that have several potential applications in diverse fields, such as human health (e.g., as antimicrobial agents or immune system modulators) and agriculture (e.g., as plant growth promoters or biopesticides). Several <i>Candida</i> species, such as <i>Candida albicans</i>, affect human health by causing invasive infections such as candidemia. Similarly, <i>Fusarium</i> species are economically important phytopathogenic fungi that lead to significant crop losses in wheat, maize, and various other agricultural plants. This work aimed to evaluate the antimycotic activity of low-molecular-weight COSs against <i>Candida</i> strains and phytopathogenic fungi. The COS mixture was obtained via hydrolysis of commercial chitosan via the bacterial GH46 chitosanase <i>Cv</i>Csn46 from <i>Chromobacterium violaceum</i>. Mass spectrometry (ESI-MS) analysis of the enzymatically prepared COS mixture revealed the predominant presence of the monosaccharide β-d-glucosamine (GlcN) and its oligomers (GlcN_2-4). In silico protein-ligand complexes between a GlcN tetrasaccharide and both the open (apo-form) and closed (substrate-bound form) conformation tridimensional models of <i>Cv</i>Csn46 were generated, providing insights into the possible mechanism responsible for the formation of GlcN and GlcN_2-3 through the hydrolysis of GlcN₄ and other GlcN oligomers. Additionally, FTIR spectroscopy analysis confirmed the characteristic amine and hydroxyl group signals in the enzymatically produced COS samples. Broth microdilution and time-to-kill assays revealed that the COS samples inhibited the growth of <i>Candida krusei</i> (<i>Pichia kudriavzevii</i>), <i>C. parapsilosis</i> and <i>C. tropicalis</i> <i>in vitro</i>, exhibiting both fungistatic (minimum inhibitory concentration range: 78-312 μg/mL) and fungicidal (minimum lethal concentration range: 156-625 μg/mL) activities. Among the filamentous fungi tested, COSs significantly (<i>p</i> < 0.05) inhibited the mycelial growth of <i>F. oxysporum</i> (IC₅₀ = 298 μg/mL) and <i>F. solani</i> (IC₅₀ = 316.5 μg/mL) <i>in vitro</i>. Fluorescence and scanning electron microscopy revealed that treatment with COSs caused cell membrane disruption and permeabilization, increased the production of reactive oxygen species (ROS), and led to morphological alterations such as cell shrinkage and surface deformation, ultimately resulting in the death of susceptible strains of <i>Candida</i> and <i>Fusarium</i>. The effects of ionic strength and pH on the antifungal action of COSs revealed that both ionic and nonionic interactions play a role in their antimicrobial activity. Collectively, the results revealed that COSs act primarily on the cell membrane of susceptible fungi and are promising antifungal drug candidates.