Antifungal and Therapeutic Evaluation of l- and d‑Enantiomers of a Plant Defensin-Inspired Peptide against Sporothrix schenckii and Sporothrix brasiliensis.

Sporotrichosis is an emerging subcutaneous mycosis and an important public and veterinary health concern in Brazil. The ongoing expansion of feline-associated transmission, particularly involving Sporothrix brasiliensis, has reinforced the need for new antifungal strategies. In this study, we evaluated the antifungal potential of the bioinspired peptide L-CaDef2.1G27‑K44 (L-CDF-GK) and its enantiomer D-CaDef2.1G27‑K44 (D-CDF-GK), derived from a defensin identified in Capsicum annuum fruits. Antifungal activity was assessed through in vitro and in vivo assays, together with the investigation of possible mechanisms of action. The analyses included fungal growth inhibition, cytoplasmic membrane permeabilization, oxidative stress induction, mitochondrial depolarization, and erythrocyte cytotoxicity. In vivo, Galleria mellonella larvae were used to evaluate therapeutic potential by survival analysis, melanization quantification, and hemocyte density determination. Both peptides exhibited significant dose-dependent antifungal activity against Sporothrix schenckii and S. brasiliensis. The L-isoform completely inhibited the growth of both species at 100 μM, whereas the D-isoform inhibited 90% of S. schenckii growth and 70% of S. brasiliensis growth. IC50 analysis and functional assays performed with S. schenckii showed that both peptides induced membrane permeabilization, increased reactive oxygen species production, and loss of mitochondrial membrane potential. In the G. mellonella infection model, lethal dose determination indicated greater virulence of S. brasiliensis. Both peptides showed low toxicity and significantly improved the survival of larvae infected with S. schenckii, with minimal activation of cellular and humoral immune responses. Together, these findings support the potential of CDF-GK-derived peptides as promising candidates for the development of new antifungal strategies against sporotrichosis.