Background: Cryptococcus neoformans  is a common human fungal pathogen in immunocompromised people, as well as a prevalent cause of meningitis in HIV-infected individuals. With the emergence of clinical fungal resistance and the shortage of antifungal drugs, it is urgent to discover novel antifungal agents. AMP-17, a novel antimicrobial peptide from Musca domestica , has antifungal activity against C. neoformans . However, its antifungal and anti-biofilm activities remain unclear. Thus, this study aimed to evaluate the antifungal activity of AMP-17 against planktonic cells and biofilms of C. neoformans .
Methods: The minimum inhibitory concentration (MIC), the biofilm inhibitory and eradicating concentration (BIC and BEC) were determined by the broth microdilution assay or the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay, respectively. The inhibitory and killing activities of AMP-17 against C. neoformans were investigated through the time-inhibition/killing kinetic curves. The potential antifungal mechanism of AMP-17 was detected by flow cytometry, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The efficiency of AMP-17 against biofilm formation or preformed biofilm was evaluated by crystal violet staining and XTT reduction assays. The morphology of pre-biofilms was tested by optical microscopy (OM) and CLSM.
Results: AMP-17 exhibited in vitro antifungal activity against C. neoformans planktonic cells and biofilms, with MICs of 4∼ 16 μg/ml, BIC₈₀ and BEC₈₀ of 16∼ 32 μg/ml, 64∼ 128 μg/ml, respectively. In addition, the 2× and 4× MIC of AMP-17 exhibited similar inhibition levels compared to the 2× and 4× MIC of the clinical drugs FLC and AMB in C. neoformans growth. Moreover, the time-kill results showed that AMP-17 (8× MIC) did not significantly eliminate colony forming units (CFU) after 6 h of treatment; however, there was 2.9-log reduction in CFU of C. neoformans . Furthermore, increasing of the permeability of the fungal cell membrane was observed with the treatment of AMP-17, since the vast change as fungal leakage and cell membrane disruption. However, the DNA binding assay of AMP-17 indicated that the peptide did not target DNA. Besides, AMP-17 was superior in inhibiting and eradicating biofilms of C. neoformans compared with FLC.
Conclusion: AMP-17 exhibited potential in vitro antifungal activity against the planktonic cells and biofilms of C. neoformans , and it may disrupt fungal cell membranes through multi-target interactions, which provides a promising therapeutic strategy and experimental basis for Cryptococcus -associated infections.
Keywords: antimicrobial peptides, AMP-17, Cryptococcus neoformans , antifungal mechanism, anti-biofilm