Buagafuran

Candida tropicalis is a WHO-prioritised opportunistic fungal pathogen capable of causing superficial-to-systemic fungal infections and forming biofilms on both biotic and abiotic surfaces, such as tissues and medical implants. The rising incidence of fungal infections and the emergence of drug resistance underscore the urgent need for alternative, natural therapeutic agents such as antifungal macromolecules. AF₄ and AF₅, natural antifungal lipopeptide homologs derived from the Bacillus subtilis RLID strain, were purified to homogeneity from the cell free supernatant using semi-preparative scale HPLC with a C18 column at the final step. Using these lipopeptide fractions AF₄ and AF₅, the anti-biofilm efficacies were evaluated against C. tropicalis ATCC 750 biofilms and their synergistic potential with the standard antifungal fluconazole. Two compounds with amphiphilic nature were highly effective in preventing adhesion and inhibiting the formation of biofilm within 6 h. Additionally, they eradicated 24 h mature biofilms, as were demonstrated through various biofilm quantification techniques, including the crystal violet assay, XTT reduction assay, quantification of EPS, and ATR-FTIR analysis of biochemical composition of treated biofilms. In vitro studies revealed that AF₄ and AF₅ significantly reduced biofilm biomass and metabolic activity of cells embedded within the biofilm. Furthermore, morphological analysis of biofilms stained with Con A and FUN-1 using CLSM and COMSTAT analyses demonstrated a strong antibiofilm effect, including damaged cell surfaces and reduced hyphae structures, indicating effective biofilm disruption. Combinations of fluconazole with AF₅ exhibited improved synergistic anti-biofilm effects. However, AF₄ alone demonstrated strong potential to combat all phases of C. tropicalis biofilms.

Invasive fungal diseases are an important public health concern due to an increase in the at‐risk population and high mortality associated with these infections. Managing invasive fungal infections poses a significant challenge given the limited antifungal options and the emergence of resistance in key fungal pathogens. Through a comprehensive approach, we evaluated the in vitro antifungal activity and the in vivo efficacy of two novel lipopeptides, AF4 and AF5 in murine models of disseminated candidiasis, cryptococcosis, and aspergillosis. Flow cytometry analysis with propidium iodide showed a dose‐dependent increase in the permeability and disruption of fungal cell membranes, underscoring the membrane perturbing effects of AF4 and AF5. These observations were further substantiated by SEM analyses that showed yeast cell and hyphal deformation and leakage of cellular contents. Our in vivo investigations utilizing two doses (5 and 10 mg/kg bodyweight) of each lipopeptide and its comparison with standard antifungal therapies showed lipopeptides significantly improved the odds of mice survival in invasive candidiasis and cryptococcosis models, with a reduction in organ fungal burden by 2 to 3‐log10 order. Additionally, in the disseminated aspergillosis model, treatment with 10 mg/kg of AF4 significantly improved median survival from 4 to 10 days while achieving a notable 1‐log10 order reduction in organ fungal burden. Overall, our study underscores the potent and broad‐spectrum antifungal activity of lipopeptides in mouse infection models, hinting at their promising therapeutic potential in invasive fungal disease.