Multifaceted antifungal and antibiofilm activities of Mo-CBP2 against Candida species: Structural disruption, cell membrane, cell wall interaction, and synergistic potential.

Clinically relevant species of Candida are opportunistic pathogens capable of forming complex and resilient biofilms, which contribute to antifungal resistance and poor clinical outcomes. In the search for alternative therapeutic agents, plant-derived proteins with antifungal properties have gained attention. This study investigates the antifungal and antibiofilm activities of Mo-CBP2, a chitin-binding protein isolated from Moringa oleifera seeds, against clinically relevant Candida species. Mo-CBP2 effectively inhibited planktonic cells, biofilm formation, and mature biofilms of C. albicans, C. tropicalis, C. parapsilosis, and C. krusei. Scanning electron microscopy revealed significant morphological alterations, including collapsed cells, porosity, and cytoplasmic leakage. Mechanistic assays suggested that Mo-CBP2 does not act on cell wall biosynthesis, as osmoprotective rescue with sorbitol was ineffective. Additionally, Mo-CBP2 did not inhibit ergosterol biosynthesis but showed partial activity reversal in the presence of exogenous ergosterol, suggesting membrane interaction. The antifungal effect was also attenuated by ascorbic acid, indicating potential involvement of reactive oxygen species (ROS). Furthermore, Mo-CBP2 displayed strong synergism with conventional antifungals, reducing their inhibitory concentrations against biofilms. The protein's lack of cytotoxicity, combined with its multitargeted antifungal profile, supports its potential as a safe and effective bioactive compound. These findings underscore the value of Mo-CBP2 as a candidate for developing novel antifungal strategies, particularly against biofilm-associated infections.