Infection and Drug Resistance (Jan 2022)
In vitro Antifungal Activity of a Novel Antimicrobial Peptide AMP-17 Against Planktonic Cells and Biofilms of Cryptococcus neoformans
Abstract
Longbing Yang,1 Zhuqing Tian,1 Luoxiong Zhou,1,2 Lijuan Zhu,1 Chaoqin Sun,1 Mingjiao Huang,1 Jian Peng,1,3 Guo Guo1,3,4 1School of Basic Medical Sciences, The Key and Characteristic Laboratory of Modern Pathogen Biology, Guizhou Medical University, Guiyang, 550025, People’s Republic of China; 2School of Public Health, Guizhou Medical University, Guiyang, 550025, People’s Republic of China; 3Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, 550025, People’s Republic of China; 4Translational Medicine Research Center, Guizhou Medical University, Guiyang, 550025, People’s Republic of ChinaCorrespondence: Guo GuoBuilding Wuben, School of Basic Medical Sciences, Guizhou Medical University, College Town, Gui’an New District, Guiyang, 550025, People’s Republic of China, Tel/fax +86 851 882 59268, Email [email protected]: 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
