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

Author

Yang,Longbing, Tian,Zhuqing, Zhou,Luoxiong, Zhu,Lijuan, Sun,Chaoqin, Huang,Mingjiao, Peng,Jian, Guo,Guo, Yang,Longbing, Tian,Zhuqing, Zhou,Luoxiong, Zhu,Lijuan, Sun,Chaoqin, Huang,Mingjiao, Peng,Jian, and Guo,Guo

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 Guo, Building 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 guoguojsc@163.comBackground: 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 kine

Published
2022

14. Antimicrobial peptide AMP-17 exerts anti–Candida albicanseffects through ROS-mediated apoptosis and necrosis

Author

Ma, Huiling, Yang, Longbing, Tian, Zhuqing, Zhu, Lijuan, Peng, Jian, Fu, Ping, Xiu, JiangFan, and Guo, Guo

Abstract

There is a need for new anti–Candida albicans(C. albicans) drugs owing to the emergence of drug resistance in recent years. AMP-17, an antimicrobial peptide from Musca domestica(M. domestica), is known to be an effective inhibitor of many fungal pathogens, including C. albicans. In this study, we investigated the potential mechanism underlying the anti–C. albicanseffects of AMP-17 using flow cytometry, transmission electron microscopy, fluorescent probes, fluorescence microplate reader, and confocal laser microscopy. Transmission electron microscopy showed that, following AMP-17 treatment, the shape of C. albicanscells became irregular, and vacuoles could be seen in the cytoplasm. Furthermore, AMP-17 treatment resulted in an increase in reactive oxygen species (ROS) levels, depolarization of the mitochondrial membrane potential (MMP), and changes in the cell cycle, leading to the apoptosis and necrosis, which ultimately contributed to the death of C. albicanscells.

Published
2023
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15. Antimicrobial Peptide AMP-17 Affects Candida albicans by Disrupting Its Cell Wall and Cell Membrane Integrity

Author

Ma, Huiling, Zhao, Xinyu, Yang, Longbing, Su, Peipei, Fu, Ping, Peng, Jian, Yang, Na, and Guo, Guo

Subjects
membrane dynamics and permeability, C. albicans, Infection and Drug Resistance, cell wall, AMP-17, Original Research
Abstract

Huiling Ma,1,2 Xinyu Zhao,1 Longbing Yang,1 Peipei Su,1 Ping Fu,1,3 Jian Peng,1,3 Na Yang,4 Guo Guo1,3,4 1Key and Characteristic Laboratory of Modern Pathogen Biology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, People’s Republic of China; 2Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Sanquan College of Xinxiang Medical University, Xinxiang 453003, 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; 4School of Food Science, Guizhou Medical University, Guiyang 550025, People’s Republic of ChinaCorrespondence: Guo Guo Tel +86 13765024379Email guoguojsc@163.comBackground: Candida albicans is associated with high mortality among immunocompromised patients. Resistance to and toxic side effects of antifungal drugs require the development of alternative antifungal agents. AMP-17 is a novel antimicrobial peptide derived from Musca domestica that exerts excellent antifungal effects against the Candida species. In this article, we discuss the potential mechanism of AMP-17 against C. albicans from the perspective of affecting the latter’s cell external structure.Methods: Recombinant AMP-17 was prepared by prokaryotic expression system, and its anti-C. albicans activity was detected by microdilution method. Microscopy and scanning electron microscopy were used to examine morphological changes in C. albicans. Cell wall-specific staining method was used to detect the change of cell wall integrity of C. albicans after AMP-17 treatment. AMP-17-induced damage to the C. albicans cell membrane was analyzed by fluorescent probes and glycerol assay kit. The expression of genes related to fungal cell wall and cell-membrane synthesis was detected by qRT-PCR.Results: Morphological observations showed that the growth of C. albicans was significantly inhibited in AMP-17-treated cells; the cells appeared aggregated and dissolved, with severe irregularities in shape. Furthermore, AMP-17 damaged the integrity of C. albicans cell walls. The cell wall integrity rate of AMP-17-treated cells was only 21.7% compared to untreated cells. Moreover, the change of membrane dynamics and permeability suggested that the cell membrane was disrupted by AMP-17 treatment. Genetic analysis showed that after AMP-17 treatment, the cell wall synthesis-related gene FKS2 of C. albicans was up-regulated 3.46-fold, while the cell membrane ergosterol synthesis-related genes ERG1, ERG5, ERG6, and MET6 were down-regulated 5.88-, 17.54-, 13.33-, and 7.14-fold, respectively.Conclusion: AMP-17 treatment disrupted the cell wall integrity and membrane structure of C. albicans and is likely a novel therapeutic option for prevention and control of C. albicans infections.Keywords: AMP-17, C. albicans, cell wall, membrane dynamics and permeability

Published
2020

16. Antimicrobial Peptide AMP-17 Affects Candida albicans by Disrupting Its Cell Wall and Cell Membrane Integrity

Author

Ma,Huiling, Zhao,Xinyu, Yang,Longbing, Su,Peipei, Fu,Ping, Peng,Jian, Yang,Na, Guo,Guo, Ma,Huiling, Zhao,Xinyu, Yang,Longbing, Su,Peipei, Fu,Ping, Peng,Jian, Yang,Na, and Guo,Guo

Abstract

Huiling Ma,1,2 Xinyu Zhao,1 Longbing Yang,1 Peipei Su,1 Ping Fu,1,3 Jian Peng,1,3 Na Yang,4 Guo Guo1,3,4 1Key and Characteristic Laboratory of Modern Pathogen Biology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, People’s Republic of China; 2Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Sanquan College of Xinxiang Medical University, Xinxiang 453003, 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; 4School of Food Science, Guizhou Medical University, Guiyang 550025, People’s Republic of ChinaCorrespondence: Guo Guo Tel +86 13765024379Email guoguojsc@163.comBackground: Candida albicans is associated with high mortality among immunocompromised patients. Resistance to and toxic side effects of antifungal drugs require the development of alternative antifungal agents. AMP-17 is a novel antimicrobial peptide derived from Musca domestica that exerts excellent antifungal effects against the Candida species. In this article, we discuss the potential mechanism of AMP-17 against C. albicans from the perspective of affecting the latter’s cell external structure.Methods: Recombinant AMP-17 was prepared by prokaryotic expression system, and its anti-C. albicans activity was detected by microdilution method. Microscopy and scanning electron microscopy were used to examine morphological changes in C. albicans. Cell wall-specific staining method was used to detect the change of cell wall integrity of C. albicans after AMP-17 treatment. AMP-17-induced damage to the C. albicans cell membrane was analyzed by fluorescent probes and glycerol assay kit. The expression of genes related to fungal cell wall and cell-membrane synthesis was detected by qRT-PCR.Results: Morphological observations showed that the growth of C. albica

Published
2020

18. Unveiling the roles of CaSDH8 in Candida albicans : Implications for virulence and azole resistance.

Author

Huang M, Song D, Zhou L, Jiao Z, Yang L, Yang Y, Peng J, and Guo G

Subjects
Virulence, Animals, Membrane Potential, Mitochondrial drug effects, Reactive Oxygen Species metabolism, Mice, Mitochondria drug effects, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Hyphae growth & development, Hyphae drug effects, Hyphae genetics, Fluconazole pharmacology, Mice, Inbred BALB C, Adenosine Triphosphate metabolism, Female, Gene Deletion, Candida albicans pathogenicity, Candida albicans genetics, Candida albicans drug effects, Candida albicans enzymology, Fungal Proteins genetics, Fungal Proteins metabolism, Antifungal Agents pharmacology, Drug Resistance, Fungal genetics, Azoles pharmacology, Candidiasis microbiology
Abstract

Candida albicans is the most common pathogen in systemic fungal diseases, exhibits a complex pathogenic mechanism, and is increasingly becoming drug tolerant. Therefore, it is particularly important to study the genes associated with virulence and resistance of C. albicans . Here, we identified a gene ( orf19.1588 ) that encodes a conserved mitochondrial protein known as CaSDH8 , upon deletion of CaSdh8 , the deleted strain ( Casdh8Δ/Δ ) experienced impaired growth, hyphal development, and virulence. Casdh8Δ/Δ displayed a reduced capacity to utilize alternative carbon sources, along with detrimental alterations in reactive oxygen species (ROS), mitochondrial membrane potential (MMP) depolarization, and adenosine triphosphate (ATP) levels. Interestingly, Casdh8Δ/Δ demonstrated resistance to azole drugs, and under the influence of fluconazole, the cell membrane permeability and mitochondrial function of Casdh8Δ/Δ were less compromised than those of the wild type, indicating a reduction in the detrimental effects of fluconazole on Casdh8Δ/Δ . These findings highlight the significance of CaSDH8 as a crucial gene for the maintenance of cellular homoeostasis. Our study is the first to document the effects of the CaSDH8 gene on the virulence and azole resistance of C. albicans at both the molecular and animal levels, providing new clues and directions for the antifungal infection and the discovery of antifungal drug targets.

Published
2024
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19. [Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans ].

Author

Yang L, Tian Z, Zhou L, Sun C, Huang M, Tian C, Peng J, and Guo G

Subjects
Humans, Proteomics, Peptides pharmacology, Transcription Factors metabolism, Antifungal Agents pharmacology, Candida albicans, Antimicrobial Peptides
Abstract

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti- Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall ( XOG1 ) and oxidative stress ( SRR1 ) genes upon the action of AMP-17 on C . albicans , suggesting that AMP-17 may exert anti- C . albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C . albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C . albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C . albicans . Drug sensitivity assay showed that the MIC 80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C . albicans SC5314) to 16 μg/mL, while the MIC 80 values against srr1Δ/Δ : : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti -C . albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Published
2023
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