A trypsin inhibitor from Poincianella pyramidalis (Tul) L.P Queiroz seeds exhibits antimicrobial and antibiofilm properties against Candida albicans and Candida tropicalis.

Proteaseinhibitors are molecules with antifungal potential. We described the antifungal and antibiofilm activity, mechanisms of action, and toxicity of the Poincianella pyramidalis trypsin inhibitor (PpyTI), a kunitz inhibitor. The minimum inhibitory concentration (MIC) of PpyTI was determined using broth microdilution according to Clinical & Laboratory Standards Institute (CLSI) guidelines. Mechanisms of action were elucidated using sorbitol, ergosterol and Sytox™ Green probe. Amphotericin B and fluconazole were used to investigate the combined effect with the inhibitor. Biofilm formation and eradication were assessed using crystal violet staining, while SYTO9 and propidium iodide were used to evaluate PpyTI's effect on biofilm through fluorescence microscopy. Toxicity was evaluated in vivo in Galleria mellonella larvae. PpyTI showed antifungal activity against various Candida species. Notably, it exhibited fungicidal and antibiofilm activity against Candida tropicalis and Candida albicans, with MICs of 3.28 μM and 6.56 μM, respectively. These effects were associated with damage to cellular membranes, as confirmed by the increase in MIC in medium supplemented with ergosterol and increased Sytox™ Green uptake. PpyTI also showed a synergistic effect when combined with fluconazole against C. albicans, resulting in reduced MIC for both composts. Importantly, PpyTI exhibited no toxicity in G. mellonella larvae. PpyTI presented antifungal activity against Candida species through promising mechanisms of action and exhibited a synergistic effect with standard antifungals. It effectively inhibited biofilm formation, outperforming standard antifungals used in clinical settings, and exhibited no significant toxicity, emphasizing the safety and applicability of the inhibitor.