In vitro Antifungal Activity of a Novel Antimicrobial Peptide AMP-17 Against Planktonic Cells and Biofilms of Cryptococcus neoformans

Abstract

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

Acknowledgments

This work was supported by the National Natural Science Foundation of China [grant numbers: 81760647, 82060381] and Key Technologies R&D Program for Science and Technology Department of Guizhou Province ([2019]2823).

Author Contributions

All authors contributed toward data analysis, drafting and critically revising the paper, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

Prof. Dr. Guo Guo reports a licensed patent ZL 2016 10428119. 8. The authors report no conflicts of interest in this work.