Novel antimicrobial peptide DvAMP serves as a promising antifungal agent against Cryptococcus neoformans.

Cryptococcus neoformans is an important opportunistic human fungal pathogen that causes cryptococcosis in immunocompromised patients. However, the number of drugs for the treatment of cryptococcosis is restricted, and the development of novel antifungal drugs and innovative strategies for the treatment of cryptococcosis is urgently needed. In this study, we validated that DvAMP is a novel antimicrobial peptide with antimicrobial activity and that it was obtained by pre-screening from the UniProt database of more than three million unknown functional sequences based on the quantitative structure-activity relationships (QSARs) protocol (http://www.chemoinfolab.com/antifungal). The peptide exhibited satisfactory biosafety and physicochemical properties, and relatively rapid fungicidal activity against C. neoformans. Meanwhile, DvAMP was able to inhibit the static biofilm of C. neoformans and cause a reduction in the thickness of the capsule. In addition, DvAMP exerts antifungal effects through membrane-mediated mechanisms (membrane permeability and depolarization) and mitochondrial dysfunction, involving a hybrid multi-hit mechanism. Furthermore, by using the C. neoformans-Galleria mellonella infection model, we demonstrated that DvAMP has significant therapeutic effects in vivo and that it significantly reduces the mortality and fungal burden of infected larvae. These results suggest that DvAMP may be a potential antifungal drug candidate for the treatment of cryptococcosis.

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

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 MIC80 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 MIC80 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.

Antimicrobial peptide AMP-17 exerts anti–Candida albicans effects through ROS-mediated apoptosis and necrosis / Péptido antimicrobiano AMP-17 ejerce efectos albicans anti-Candida a través de ROS-mediada apoptosis y necrosis

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. albicans effects 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. albicans cells 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. albicans cells.(AU)

Antimicrobial peptide AMP-17 exerts anti-Candida albicans effects through ROS-mediated apoptosis and necrosis.

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. albicans effects 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. albicans cells 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. albicans cells.

Antitumor Activity and Mechanism of Action of the Antimicrobial Peptide AMP-17 on Human Leukemia K562 Cells.

Cancer is one of the most common malignant diseases in the world. Hence, there is an urgent need to search for novel drugs with antitumor activity against cancer cells. AMP-17, a natural antimicrobial peptide derived from Musca domestica, has antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. However, its antitumor activity and potential mechanism of action in cancer cells remain unclear. In this study, we focused on evaluating the in vitro antitumor activity and mechanism of AMP-17 on leukemic K562 cells. The results showed that AMP-17 exhibited anti-proliferative activity on K562 cells with an IC50 value of 58.91 ± 3.57 µg/mL. The membrane integrity of K562 was disrupted and membrane permeability was increased after AMP-17 action. Further observation using SEM and TEM images showed that the cell structure of AMP-17-treated cells was disrupted, with depressions and pore-like breaks on the cell surface, and vacuolated vesicles in the cytoplasm. Furthermore, further mechanistic studies indicated that AMP-17 induced excessive production of reactive oxygen species and calcium ions release in K562 cells, which led to disturbance of mitochondrial membrane potential and blocked ATP synthesis, followed by activation of Caspase-3 to induce apoptosis. In conclusion, these results suggest that the antitumor activity of AMP-17 may be achieved by disrupting cell structure and inducing apoptosis. Therefore, AMP-17 is expected to be a novel potential agent candidate for leukemia treatment.

A Cecropin-4 Derived Peptide C18 Inhibits Candida albicans by Disturbing Mitochondrial Function.

Global burden of fungal infections and related health risk has accelerated at an incredible pace, and multidrug resistance emergency aggravates the need for the development of new effective strategies. Candida albicans is clinically the most ubiquitous pathogenic fungus that leads to high incidence and mortality in immunocompromised patients. Antimicrobial peptides (AMPs), in this context, represent promising alternatives having potential to be exploited for improving human health. In our previous studies, a Cecropin-4-derived peptide named C18 was found to possess a broader antibacterial spectrum after modification and exhibit significant antifungal activity against C. albicans. In this study, C18 shows antifungal activity against C. albicans or non-albicans Candida species with a minimum inhibitory concentration (MIC) at 4∼32 µg/ml, and clinical isolates of fluconazole (FLZ)-resistance C. tropicalis were highly susceptible to C18 with MIC value of 8 or 16 µg/ml. Additionally, C18 is superior to FLZ for killing planktonic C. albicans from inhibitory and killing kinetic curves. Moreover, C18 could attenuate the virulence of C. albicans, which includes damaging the cell structure, retarding hyphae transition, and inhibiting biofilm formation. Intriguingly, in the Galleria mellonella model with C. albicans infection, C18 could improve the survival rate of G. mellonella larvae to 70% and reduce C. albicans load from 5.01 × 107 to 5.62 × 104 CFU. For mechanistic action of C18, the level of reactive oxygen species (ROS) generation and cytosolic Ca2 + increased in the presence of C18, which is closely associated with mitochondrial dysfunction. Meanwhile, mitochondrial membrane potential (△Ψm) loss and ATP depletion of C. albicans occurred with the treatment of C18. We hypothesized that C18 might inhibit C. albicans via triggering mitochondrial dysfunction driven by ROS generation and Ca2 + accumulation. Our observation provides a basis for future research to explore the antifungal strategies and presents C18 as an attractive therapeutic candidate to be developed to treat candidiasis.

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

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.

Large-Scale Screening of Antifungal Peptides Based on Quantitative Structure-Activity Relationship.

Antifungal peptides are effective, biocompatible, and biodegradable, and thus, they are promising to be the next generation of drugs for treating infections caused by fungi. The identification processes of highly active peptides, however, are still time-consuming and labor-intensive. Quantitative structure-activity relationships (QSARs) have dramatically facilitated the discovery of many bioactive drug molecules without a priori knowledge. In this study, we have established an effective QSAR protocol for screening antifungal peptides. The screening protocol integrates an accurate antifungal peptide classification model and four activity prediction models against specified target fungi. A demonstrative application was performed on more than three million candidate peptides, and three outstanding peptides were identified. The whole screening took only a few days, which was much faster than our previous experimental screening works. In conclusion, the protocol is useful and effective for reducing repetitive laboratory efforts in antifungal peptide discovery. The prediction server (antifungal Web server) is freely available at www.chemoinfolab.com/antifungal.

TMT-based quantitative proteomic analysis of the effects of novel antimicrobial peptide AMP-17 against Candida albicans.

Candida albicans is the most common human fungal pathogen in immunocompromised individuals. With the emergence of clinical fungal resistance, there is an urgent need to develop novel antifungal agents. AMP-17, a novel antimicrobial peptide from Musca domestica, has an antifungal effect against C. albicans, but its mechanism of antifungal action remains unclear. In the current study, we performed a proteomics analysis in C. albicans using TMT technique under the treatment of AMP-17. A total of 3931 proteins were identified, of which 3600 included quantitative information. With a 1.5-fold change threshold and a t-test p-value < 0.05 as standard, 423 differentially expressed proteins (DEPs) were up-regulated and 180 DEPs were down-regulated in the AMP-17/control. Notably, GO enrichment revealed that DEPs associated with the cell wall, RNA and oxidative stress were significantly up-regulated, while DEPs involved in ergosterol metabolism and membrane were significantly down-regulated in the AMP-17/control. KEGG pathway enrichment revealed that DEPs involved seven significant metabolic pathways, mainly involved oxidative phosphorylation, RNA degradation, propanoate metabolism and fatty acid metabolism. These results show that AMP-17 induces a complex organism response in C. albicans, indicating that AMP-17 may inhibit growth by affecting multiple targets in C. albicans cells. SIGNIFICANCE: Antimicrobial peptides (AMPs) are an important part of the innate immune system of organisms and having broad range of activity against fungi, bacteria and viruses. These AMPs are considered as probable candidate for forthcoming drugs, due to their broad range of activity, lesser toxicity and decreased resistance development by target cells. AMP-17, a novel antimicrobial peptide from M. domestica, has significant antifungal activity against C. albicans. It has been confirmed that AMP-17 can play an antifungal effect by destroying the cell wall and cell membrane of C. albicans in previous studies, but its mechanism of action at the protein level is currently unclear. In the current study, using the TMT-based quantitative proteomics method, 603 differentially expressed proteins were identified in the cells of C. albicans treated with AMP-17 for 12 h, and these DEPs were closely related to cell wall, cell membrane, RNA degradation and oxidative stress. The results provide new insights into the potential mechanism of action of AMP- 17 against C. albicans. Meanwhile, it provides certain technical support and theoretical basis for the research and development of novel peptide drugs.