Intrinsic antifungal activity of curli nanofibers expands the design space for programmable antimicrobial biomaterials

The rise of antifungal resistance and the limited number of available antifungal drug classes have created an urgent need for biomaterials capable of localized, programmable antifungal activity. Microbial extracellular protein nanofibers have emerged as versatile scaffolds for engineering functional biomaterials, yet whether these structural proteins possess intrinsic biological activities remains largely unexplored. Here, we engineered curli nanofibers displaying the antifungal peptide heliomicin and unexpectedly discovered that wild-type CsgA itself exhibits intrinsic antifungal activity against Candida albicans , reducing fungal viability by approximately 2 log units. Genetic fusion of heliomicin enhanced this intrinsic activity to a 3.5-log fungicidal reduction while preserving nanofiber self-assembly, hydrogel formation, mechanical properties, and 3D printability. Mechanistic analyses identified membrane disruption as the primary mode of action and linked the enhanced activity of Hel-CsgA to expansion of the cationic surface of CsgA. Heliomicin-CsgA hydrogels further reduced fungal burden and suppressed hyphal development in an ex vivo porcine skin infection model. These findings demonstrate that microbial extracellular protein nanofibers can encode intrinsic biological functions that can be uncovered and further enhanced through protein engineering, expanding the design space for intrinsically bioactive antimicrobial biomaterials.