João Moreira de Matos Neto¹, Maria Laína Silva¹, Ubirajara Moreira Paz Júnior¹, Queilane Lemos de S. G. Chaves¹, Laís Helena Sousa Vieira¹, Mariana Ferreira de Oliveira¹, Viviane Otaviana Silva¹, Rossana de Aguiar Cordeiro¹, Cleverson Diniz Teixeira de Freitas¹

Confronting the rise of antimicrobial resistance requires multiple and coordinated strategies to mitigate the thrive of resistant microbial isolates within infected hosts. Antimicrobial peptides (AMPs), bioactive molecules with multiple mechanisms, as well as combined therapy represent favorable approaches that achieve this goal, narrowing the antibiotic-resistance selection window, thereby mitigating the acquisition of resistant traits. Novel antimicrobial molecules, such as AMPs, are commonly associated with classic drugs, combining the attributes of both molecules, enhancing their individual potential and resulting in better outcomes in terms of treatment effectiveness and rapidity. Our evaluation of the synergistic profile of an AMP identified in the floral nectar of ornamental tobacco plants (Ni-I) in combination with three antifungal drugs revealed a strong synergy with fluconazole (FICI = 0.1406) and caspofungin (FICI = 0.1915) but only an additive effect with amphotericin B (FICI = 1.0) against Candida tropicalis. Bioinformatics analysis confirmed that no significant direct interactions among Ni-I and the test compounds or their targets, findings consistent with a non-antagonistic profile. Further analysis revealed that the peptide has a strong affinity for the fungal membrane, adhering to the cell membrane and neutralizing the surface ζ-potential, shifting from -31.5mV to +4.32mV at 4 x MIC, being partially internalized in the process as observed by confocal laser scanning microscopy (CLSM). Our hypothesis regarding the synergism between these molecules relies on the capacity of Ni-I to damage the phospholipid bilayer of yeast during its action, causing a greater influx of exogenous molecules, including the peptide itself and the test drugs, which supports the caspofungin and fluconazole results. The absence of synergy observed with amphotericin B is justified by both antifungals acting on the microbial membrane, without favorable cross-interactions as seen by molecular docking, which may cause a reciprocal interference at their membrane-binding sites. Despite this, it is impossible to obtain a complete picture of the metabolic responses underlying peptide’s solo and joint actions. Thus, further proteomic and transcriptomic studies are key to properly interpreting the results hereby presented. Nevertheless, this study highlights the potential of the use of this nectar peptide in combined therapies to tackle Candida infections, highlighting its capacity to be used in therapeutic approaches to circumvent infections mediated by azole-resistant yeast.

Agradecimentos: O trabalho aqui presente só foi possível graças às seguintes entidades acadêmicas e agências de fomento: PPGBioquímica, PPGMM, CAPES e CNPq.