Antimicrobial peptide DvAMP combats carbapenem-resistant Acinetobacter baumannii infection.

Carbapenem-resistant Acinetobacter baumannii (CRAB), an important opportunistic pathogen, is a major cause of healthcare-associated infections. The polymyxins (colistin and polymyxin B) are the last line of defense in the treatment of CRAB infections, and there is an urgent need to develop novel alternative therapeutic strategies. In this study, we found that the antimicrobial peptide DvAMP exhibited satisfactory antibacterial and antibiofilm activity against CRAB. In addition, DvAMP showed tolerable stability in salt ions and serum and exhibited low toxicity in vivo. Investigation of the underlying mechanism demonstrated that DvAMP disrupts cell membrane structural integrity and specifically binds to exogenous lipopolysaccharides (LPS) and phospholipids (PG/CL), resulting in increased membrane permeability and dissipating proton motive force (PMF), further reducing intracellular ATP levels and inducing ROS accumulation, leading to bacterial death. Furthermore, DvAMP therapy efficiently improved survival rates and decreased the bacterial load in the lungs of mice in a mouse pneumonia model, showing that DvAMP administration reduced CRAB susceptibility to lung infection. These results indicate that the peptide DvAMP is a promising alternative therapeutic agent to combat CRAB infection.

The antibacterial activity and mechanism of a novel peptide MR-22 against multidrug-resistant Escherichia coli.

Introduction: Bacterial infections have become serious threats to human health, and the excessive use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria. E. coli is a human bacterial pathogen, which can cause severe infectious. Antimicrobial peptides are considered the most promising alternative to traditional antibiotics. Materials and methods: The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and hemolytic activity were determined by the microdilution method. The antimicrobial kinetics of MR-22 against E. coli were studied by growth curves and time-killing curves. The cytotoxicity of MR-22 was detected by the CCK-8 assay. The antimicrobial activity of MR-22 in salt, serum, heat and trypsin was determined by the microdilution method. The antimicrobial mechanism of MR-22 against drug-resistant E. coli was studied by Scanning Electron Microscope, laser confocal microscopy, and Flow Cytometry. The in vivo antibacterial activity of MR-22 was evaluated by the mice model of peritonitis. Results and discussion: In this study, MR-22 is a new antimicrobial peptide with good activity that has demonstrated against MDR E. coli. The antimicrobial activity of MR-22 exhibited stability under conditions of high temperature, 10% FBS, and Ca2+. However, a decline of the activity was observed in the presence of Na+, serum, and trypsin. MR-22 had no significant cytotoxicity or hemolysis in vitro. SEM and fluorescent images revealed that MR-22 could disrupt the integrity of cell membrane. DCFH-DA indicated that MR-22 increased the content of reactive oxygen species, while it decreased the content of intracellular ATP. In mice model of peritonitis, MR-22 exhibited potent antibacterial activity in vivo. These results indicated that MR-22 is a potential drug candidate against drug-resistant E. coli.

Function analysis and characterisation of a novel chitinase, MdCht9, in Musca domestica.

Insect chitinases have been proposed as potential targets for pest control. In this work, a novel group IV chitinase gene, MdCht9, from Musca domestica was found to have multiple functions in the physiological activity, including chitin regulation, development and antifungal immunity. The MdCht9 gene was cloned and sequenced, its phylogeny was analysed and its expression was determined in normal and 20E treated larvae. Subsequently, RNA interference (RNAi)-mediated MdCht9 knockdown was performed, followed by biochemical assays, morphological observations and transcriptome analysis. Finally, the recombinant protein MdCht9 (rMdCht9) was purified and tested for anti-microbial activity and enzyme characteristics. The results showed that MdCht9 consists of three domains, highly expressed in a larval salivary gland. RNAi silencing of MdCht9 resulted in significant down-regulation of chitin content and expression of 15 chitin-binding protein (CBP) genes, implying a new insight that MdCht9 might regulate chitin content by influencing the expression of CBPs. In addition, more than half of the lethality and partial wing deformity appeared due to the dsMdCht9 treatment. In addition, the rMdCht9 exhibited anti-microbial activity towards Candida albicans (fungus) but not towards Escherichia coli (G-) or Staphylococcus aureus (G+). Our work expands on previous studies of chitinase while providing a potential target for pest management.

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.

The Antimicrobial Peptide AMP-17 Derived from Musca domestica Inhibits Biofilm Formation and Eradicates Mature Biofilm in Candida albicans.

The biofilm formation of C. albicans represents a major virulence factor during candidiasis. Biofilm-mediated drug resistance has necessitated the search for a new antifungal treatment strategy. In our previous study, a novel antimicrobial peptide named AMP-17 derived from Musca domestica was confirmed to have significant antifungal activity and suppress hyphal growth greatly in C. albicans. In the current work, we aimed to investigate the antibiofilm property of AMP-17 in C. albicans and explore the underlying mechanism. An antifungal susceptibility assay showed that AMP-17 exerted a strong inhibitory efficacy on both biofilm formation and preformed biofilms in C. albicans. Furthermore, AMP-17 was found to block the yeast-to-hypha transition and inhibit the adhesion of biofilm cells with a reduction in cellular surface hydrophobicity. A morphological analysis revealed that AMP-17 indeed suppressed typical biofilm formation and damaged the structures of the preformed biofilm. The RNA-seq showed that the MAPK pathway, biosynthesis of antibiotics, and essential components of the cell were mainly enriched in the biofilm-forming stage, while the citrate cycle (TCA cycle), phenylamine metabolism, and propanoate metabolism were enriched after the biofilm matured. Moreover, the co-expressed DEGs in the two pairwise comparisons highlighted the terms of transmembrane transporter activity, regulation of filamentation, and biofilm formation as important roles in the antibiofilm effect of AMP-17. Additionally, qRT-PCR confirmed that the level of the genes involved in cell adhesion, filamentous growth, MAPK, biofilm matrix, and cell dispersal was correspondingly altered after AMP-17 treatment. Overall, our findings reveal the underlying antibiofilm mechanisms of AMPs in C. albicans, providing an interesting perspective for the development of effective antifungal agents with antibiofilm efficacy in Candida spp.

Identification and functional characterization of ORF19.5274, a novel gene involved in both azoles susceptibility and hypha development in Candida albicans.

Azole resistance is becoming increasingly serious due to the frequent recurrence of fungal infections and the need for long-term clinical prevention. In our previous study, we discovered ORF19.5274 with an unknown function by TMT™ quantitative proteomics technology after fluconazole (FLC) treatment of Candida albicans. In this study, we created the target gene deletion strain using CRISPR-Cas9 editing technology to see if ORF19.5274 regulates azole sensitivity. The data showed that ORF19.5274 was involved in hyphal development and susceptibility to antifungal azoles. Deleting this gene resulted in defective hyphal growth in solid medium, while only a weak lag in the initiation of hyphal development and restoring hyphal growth during the hyphal maintenance phase under liquid conditions. Moreover, intracellular reactive oxygen species (ROS) assay and propidium iodide staining assays showed increased endogenous ROS levels and membrane permeability, but decreased metabolic activity of biofilm in orf19.5274Δ/Δ after treatment with FLC in comparison with either SC5314 or orf19.5274Δ/Δ::ORF19.5274 strains. More importantly, orf19.5274Δ/Δ significantly enhanced the FLC efficacy against C. albicans in infected Galleria mellonella larvae. The above characteristics were fully or partially restored in the complemented strain indicating that the changes caused by ORF19.5274 deletion were specific. In summary, the ORF19.5274 gene is required for hyphal development of C. albicans, and is correlated with the response to antifungal azoles in vitro and in vivo. The identification of ORF19.5275 is promising to expand the potential candidate targets for azoles.

Ovomermis sinensis parasitism arrests midgut replacement by altering ecdysone and juvenile hormone in Helicoverpa armigera larvae.

Many entomopathogens regulate the development of their insect hosts. However, the influence of mermithid nematodes on the development of their host remains unclear. In the current study, we provide insights into how Ovomermis sinensis parasitism affects the development of Helicoverpa armigera. We observed that O. sinensis arrests host development, as evidenced by the reduced body size and failure of Helicoverpa armigera to pupate. Moreover, midgut replacement of the host was significantly blocked by parasitism. Furthermore, juvenile hormone (JHIII) titers of the host were dramatically elevated by parasitism, but JH esterase (JHE) activities were strongly inhibited. By contrast, steroid hormone (20-hydroxyecdysone, 20E) titers of the host were significantly depressed by parasitism on days 4-6. The expression profiles of hormone-related genes in the host also showed similar patterns with the hormone titer. For this reason, rescue experiments were performed by injecting 20E and JHIII into developmentally arrested hosts. Notably, the midgut replacement of the host was rescued by the injection of 20E, whereas JHIII injection resulted in negative effects. Altogether, O. sinensis arrests H. armigera midgut replacement by reducing 20E and maintaining JH, thereby causing developmental arrests. Our study is the first report of the possible mechanism of mermithid nematodes in regulating insect development.

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.

Identification and function analysis of chitinase 2 gene in housefly, Musca domestica.

Chitinases are hydrolytic enzymes that play important roles in chitin degradation during the insect development process, and thus are considered as the potential targets for pest management. Here, we identified and characterized the group VII chitinase gene from health pest Musca domestica (MdCht2). We found that MdCht2 was 1932 bp in length with an open reading frame of 1530 bp, which encodes a polypeptide of 509 amino acid residues. Phylogenetic analysis showed that MdCht2 gene was homologs with other closed insects, and belong to the group VII chitinases. Moreover, Real-time PCR analysis indicated that MdCht2 mRNA was highly expressed in pupa stage, as well as in integument and trachea. However, RNAi-mediated knockdown of MdCht2 resulted in high mortality rates and abnormal eclosion. Therefore, we hypothesized that MdCht2 was a crucial gene required for housefly development, which was supported by the transcription level of MdCht2 could be induced by 20-hydroxyecdysone (20E), and the dsMdCht2 could resulted in decrease of the chitinase activity and increase of the chitin content. Taken together, our findings suggested that MdCht2 regulated the chitin content via chitinases, thereby leading to abnormal development. Our results provide a potential target for M. domestica management.

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.

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

BACKGROUND: 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.

Antifungal Activity and Physicochemical Properties of a Novel Antimicrobial Protein AMP-17 from Musca domestica.

Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.375-18.75 µg/ml, and minimum fungicidal concentrations (MFC) of 18.75-37.5 µg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16× MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and -80°C, and NaCl and MgCl2 concentrations of 50-200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.375­18.75 µg/ml, and minimum fungicidal concentrations (MFC) of 18.75­37.5 µg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16× MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and ­80°C, and NaCl and MgCl2 concentrations of 50­200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.