Effect of tryptophan position and lysine/arginine substitution in antimicrobial peptides on antifungal action.

Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.

Functional Characterization of an Arabidopsis Profilin Protein as a Molecular Chaperone under Heat Shock Stress.

Profilins (PFNs) are actin monomer-binding proteins that function as antimicrobial agents in plant phloem sap. Although the roles of Arabidopsis thaliana profilin protein isoforms (AtPFNs) in regulating actin polymerization have already been described, their biochemical and molecular functions remain to be elucidated. Interestingly, a previous study indicated that AtPFN2 with high molecular weight (HMW) complexes showed lower antifungal activity than AtPFN1 with low molecular weight (LMW). These were bacterially expressed and purified to characterize the unknown functions of AtPFNs with different structures. In this study, we found that AtPFN1 and AtPFN2 proteins have LMW and HMW structures, respectively, but only AtPFN2 has a potential function as a molecular chaperone, which has never been reported elsewhere. AtPFN2 has better protein stability than AtPFN1 due to its higher molecular weight under heat shock conditions. The function of AtPFN2 as a holdase chaperone predominated in the HMW complexes, whereas the chaperone function of AtPFN1 was not observed in the LMW forms. These results suggest that AtPFN2 plays a critical role in plant tolerance by increasing hydrophobicity due to external heat stress.

Functional Mechanisms Underlying the Antimicrobial Activity of the Oryza sativa Trx-like Protein.

Plants are constantly subjected to a variety of environmental stresses and have evolved regulatory responses to overcome unfavorable conditions that might reduce or adversely change a plant's growth or development. Among these, the regulated production of reactive oxygen species (ROS) as a signaling molecule occurs during plant development and pathogen defense. This study demonstrates the possible antifungal activity of Oryza sativa Tetratricopeptide Domain-containing thioredoxin (OsTDX) protein against various fungal pathogens. The transcription of OsTDX was induced by various environmental stresses known to elicit the generation of ROS in plant cells. OsTDX protein showed potent antifungal activity, with minimum inhibitory concentrations (MICs) against yeast and filamentous fungi ranging between 1.56 and 6.25 and 50 and 100 µg/mL, respectively. The uptake of SYTOX-Green into fungal cells and efflux of calcein from artificial fungus-like liposomes suggest that its killing mechanism involves membrane permeabilization and damage. In addition, irregular blebs and holes apparent on the surfaces of OsTDX-treated fungal cells indicate the membranolytic action of this protein. Our results suggest that the OsTDX protein represents a potentially useful lead for the development of pathogen-resistant plants.

Anti-Biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-Resistant Pseudomonas aeruginosa and Staphylococcus aureus Planktonic Cells and Biofilm.

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.

Cell-selectivity of tryptophan and tyrosine in amphiphilic α-helical antimicrobial peptides against drug-resistant bacteria.

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, 'XWZX' basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure. Some peptides displayed a potent antibacterial activity via membranolytic action and high therapeutic index (toxic dose/minimum inhibitory concentration) in vitro. Furthermore, in vivo experiments using bacterial ear-skin infection models verified that these peptides have the potential to be powerful and safe antibiotics. The present study provides a lead sequence for designing peptide antibiotics against bacterial membranes and information for cell-selectivity of hydrophobic amino acids with aromatic side chains such as Trp and Tyr.

Hydrophilic Linear Peptide with Histidine and Lysine Residues as a Key Factor Affecting Antifungal Activity.

Increases in the numbers of immunocompromised patients and the emergence of drug-resistance fungal pathogens have led to the need for new, safe, efficacious antifungal agents. In this study, we designed a histidine-lysine-lysine (HKK) motif and synthesized six HKK peptides with repetitions of the motif. These peptides showed length-dependent antifungal activity against drug-susceptible and drug-resistant fungal pathogens via membranolytic or non-membranolytic action. None of the peptides were cytotoxic to rat erythrocytes or NIH3T3 mouse embryonic fibroblasts. Short-length peptides were directly translocated in fungal cytosol and reacted with mitochondria, resulting in apoptosis. Membrane-permeabilizing activity occurred in the presence of long peptides, and peptides were able to transfer to the cytosol and induce reactive oxygen species. Our results suggest that peptides composed only of cationic amino acids may be good candidates as antifungal agents.

Functional characterization of the Arabidopsis universal stress protein AtUSP with an antifungal activity.

An antifungal protein, AtUSP protein (At3g53990), was isolated from Arabidopsis thaliana leaves by ion and size chromatography and sequenced by N-terminal sequencing. The AtUSP gene amplified from an Arabidopsis leaf cDNA library was transformed to Escherichia coli to express the AtUSP protein. The recombinant protein inhibited the cell growth of various pathogenic fungal strains. The levels of the AtUSP transcripts were increased by various stresses, including pathogenic infection and salt stress. These results suggest that Arabidopsis AtUSP plays a critical role in the plant tolerance to diverse pathogenic infections. The potent antifungal action, which is a new function of AtUSP, was attributed to fungal reactive oxygen species (ROS) generation and mitochondrial potential alteration.

Functional characterization of alpha-synuclein protein with antimicrobial activity.

Alpha-synuclein (α-Syn), a small (14 kDa) protein associated with Parkinson's disease, is abundant in human neural tissues. α-Syn plays an important role in maintaining a supply of synaptic vesicles in presynaptic terminals; however, the mechanism by which it performs this function are not well understood. In addition, there is a correlation between α-Syn over-expression and upregulation of an innate immune response. Given the growing body of literature surrounding antimicrobial peptides (AMPs) in the brain, and the similarities between α-Syn and a previously characterized AMP, Amyloid-ß, we set out to investigate if α-Syn shares AMP-like properties. Here we demonstrate that α-Syn exhibits antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, we demonstrate a role for α-Syn in inhibiting various pathogenic fungal strains such as Aspergillus flavus, Aspergillus fumigatus and Rhizoctonia solani. We also analyzed localizations of recombinant α-Syn protein in E. coli and Candida albicans. These results suggest that in addition to α-Syn's role in neurotransmitter release, it appears to be a natural AMP.

Algicidal effect of hybrid peptides as potential inhibitors of harmful algal blooms.

OBJECTIVES: To biochemically characterize synthetic peptides to control harmful algal blooms (HABs) that cause red tides in marine water ecosystems. RESULTS: We present an analysis of several short synthetic peptides and their efficacy as algicidal agents. By altering the amino acid composition of the peptides we addressed the mode of algicidal action and determine the optimal balance of cationic and hydrophobic content for killing. In a controlled setting, these synthetic peptides disrupted both plasma and chloroplast membranes of several species known to result in HABs. This disruption was a direct result of the hydrophobic and cationic content of the peptide. Furthermore, by using an anti-HAB bioassay in scallops, we determined that these peptides were algicidal without being cytotoxic to other marine organisms. CONCLUSIONS: These synthetic peptides may prove promising for general marine ecosystem remediation where HABs have become widespread and resulted in serious economic loss.

Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 µM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent.

Antimicrobial action of water-soluble ß-chitosan against clinical multi-drug resistant bacteria.

Recently, the number of patients infected by drug-resistant pathogenic microbes has increased remarkably worldwide, and a number of studies have reported new antibiotics from natural sources. Among them, chitosan, with a high molecular weight and α-conformation, exhibits potent antimicrobial activity, but useful applications as an antibiotic are limited by its cytotoxicity and insolubility at physiological pH. In the present study, the antibacterial activity of low molecular weight water-soluble (LMWS) α-chitosan (α1k, α5k, and α10k with molecular masses of 1, 5, and 10 kDa, respectively) and ß-chitosan (ß1k, ß5k, and ß10k) was compared using a range of pathogenic bacteria containing drug-resistant bacteria isolated from patients at different pH. Interestingly, ß5k and ß10k exhibited potent antibacterial activity, even at pH 7.4, whereas only α10k was effective at pH 7.4. The active target of ß-chitosan is the bacterial membrane, where the leakage of calcein is induced in artificial PE/PG vesicles, bacterial mimetic membrane. Moreover, scanning electron microscopy showed that they caused significant morphological changes on the bacterial surfaces. An in vivo study utilizing a bacteria-infected mouse model found that LMWS ß-chitosan could be used as a candidate in anti-infective or wound healing therapeutic applications.

Anti-Biofilm Effects of Rationally Designed Peptides against Planktonic Cells and Pre-Formed Biofilm of Pseudomonas aeruginosa.

Biofilms are resistant to antibiotics and are a major source of persistent and recurring infections by clinically important pathogens. Drugs used for biofilm-associated infections are limited because biofilm-embedded or biofilm-matrix bacteria are difficult to kill or eradiate. Therefore, many researchers are developing new and effective antibiofilm agents. Among them, antimicrobial peptides have an attractive interest in the development of antibiofilm agents. The present study evaluated the effects of 10 synthetic peptides on growth inhibition, inhibition of biofilm formation, and biofilm elimination in drug-resistant Pseudomonas aeruginosa. The planktonic cell growth and biofilm formation were dose-dependently inhibited by most of the peptides. WIK-14 eliminated preformed biofilm masses by removing carbohydrates, extracellular nucleic acids, proteins, and lipids constituting extracellular polymeric substances. The results demonstrated that WIK-14 and WIKE-14 peptides might provide novel therapeutic drugs to overcome multidrug resistance in biofilm-associated infections.

Polyethylenimine grafted-chitosan based Gambogic acid copolymers for targeting cancer cells overexpressing transferrin receptors.

Recent advancements in gene delivery systems that specifically target a variety of cancer types have increased demand for tissue-specific gene therapy. The current study describes the synthesis of a copolymer (GPgWSC) composed of a polyethylenimine (PEI)-grafted water-soluble chitosan (WSC) and gambogic acid (GA). It was validated as a ligand capable of enabling targeted attachment to transferrin receptors in HCT116 cancer cell lines. GPgWSC demonstrated superior antitumor activity in vitro in HCT116 compared to LoVo or MCF-7 cell lines, facilitated by the apoptotic activity of psiRNA-hBCL2. Pre-incubation of transferrin significantly inhibited GFP expression in the GPgWSC polyplex, demonstrating that GA is an extremely effective transferrin receptor targeting molecule. Additionally, in the HCT116-bearing mouse model, the tumor mass of PBS-treated mice increased to 2270 mm2 after 22 days, but the injection of GPgWSC polyplex significantly reduced the mass-increasing rate as a mass size of 248 mm2.

Candidacidal and Antibiofilm Activity of PS1-3 Peptide against Drug-Resistant Candida albicans on Contact Lenses.

The recent emergence of antibiotic-resistant fungi has accelerated research on novel antifungal agents. In particular, Candida albicans infections are related to biofilm formation on medical devices, such as catheters, stents, and contact lenses, resulting in high morbidity and mortality. In this study, we aimed to elucidate the antifungal and antibiofilm effects of a peptide against drug-resistant C. albicans. α-Helical peptides in which the sequence of KWYK was repeated twice and four times, designated peptide series 1 (PS1)-1 and PS1-3, respectively, were generated, and the candidacidal activities of PS1-1, PS1-3, and fluconazole against drug-resistant C. albicans cells were assessed. The PS1-3 peptide showed higher killing activity than PS1-1 or fluconazole and acted via a membranolytic mechanism. In addition, the PS1-3 peptide exhibited more potent activity than PS1-1 and fluconazole in terms of fungal biofilm inhibition and reduction at the minimum fungicidal concentration on the contact lens surface. Overall, these findings established PS1-3 as a potential candidacidal agent for applications on contact lenses.

Design of Antimicrobial Peptides with Cell-Selective Activity and Membrane-Acting Mechanism against Drug-Resistant Bacteria.

Antimicrobial peptides (AMPs) can combat drug-resistant bacteria with their unique membrane-disruptive mechanisms. This study aimed to investigate the antibacterial effects of several membrane-acting peptides with amphipathic structures and positional alterations of two tryptophan residues. The synthetic peptides exhibited potent antibacterial activities in a length-dependent manner against various pathogenic drug-resistant and susceptible bacteria. In particular, the location of tryptophan near the N-terminus of AMPs simultaneously increases their antibacterial activity and toxicity. Furthermore, the growth inhibition mechanisms of these newly designed peptides involve cell penetration and destabilization of the cell membrane. These findings provide new insights into the design of peptides as antimicrobial agents and suggest that these peptides can be used as substitutes for conventional antibiotics.

Potent Anti-Inflammatory Effects of a Helix-to-Helix Peptide against Pseudomonas aeruginosa Endotoxin-Mediated Sepsis.

Although considerable scientific research data is available for sepsis and cytokine storm syndrome, there is a need to develop new treatments or drugs for sepsis management. Antimicrobial peptides (AMPs) possess anti-bacterial and anti-inflammatory activity, neutralizing toxins such as lipopolysaccharides (LPS, endotoxin). Most AMPs have been designed as a substitute for conventional antibiotics, which kill drug-resistant pathogens. The present study aimed to determine the anti-inflammatory potential of 10 designed XIW (X: lysine, arginine, or glutamic acid) α-helical peptides in macrophages and a mouse model in the presence of LPS. Among them, WIKE-14, a peptide with a helix-to-helix structure, having the 12th amino acid substituted with glutamic acid, suppressed pro-inflammatory cytokines in RAW 264.7 macrophages. This reaction was mediated by the inhibition of the binding between LPS and macrophages. In addition, the WIKE-14 peptide exhibited a potent anti-inflammatory activity in mice ears and lungs inflamed using LPS. Thus, our results may provide useful insights for the development of anti-sepsis agents via the sequence and structure information of the WIKE-14 peptide.

Antifungal and Anti-Inflammatory Activities of PS1-2 Peptide against Fluconazole-Resistant Candida albicans.

Clinically, fungal pneumonia rarely occurs in adults, and invasive fungal infections can cause substantial morbidity, and mortality due to sepsis and septic shock. In the present study, we have designed peptides that exhibit potent antifungal activities against fluconazole-resistant Candida albicans in physiological monovalent, and divalent ionic buffers, with minimal fungicidal concentrations ranging from 16 to 32 µM. None of these tested peptides resulted in the development of drug resistance similar to fluconazole. Among them, the PS1-2 peptide did not induce stimulation of macrophages by C. albicans, and it exerted antifungal and anti-inflammatory effects against C. albicans-induced intratracheal infection, in an acute lung injury mouse model. PS1-2 is likely a novel therapeutic agent for the control, and prevention of drug-resistant C. albicans infection, and our findings may be useful for designing antimicrobial peptides to combat fungal infection.

Antifungal Mechanism of Vip3Aa, a Vegetative Insecticidal Protein, against Pathogenic Fungal Strains.

Discovering new antifungal agents is difficult, since, unlike bacteria, mammalian and fungal cells are both eukaryotes. An efficient strategy is to consider new antimicrobial proteins that have variety of action mechanisms. In this study, a cDNA encoding Bacillus thuringiensis Vip3Aa protein, a vegetative insecticidal protein, was obtained at the vegetative growth stage; its antifungal activity and mechanism were evaluated using a bacterially expressed recombinant Vip3Aa protein. The Vip3Aa protein demonstrated various concentration- and time-dependent antifungal activities, with inhibitory concentrations against yeast and filamentous fungi ranging from 62.5 to 125 µg/mL and 250 to 500 µg/mL, respectively. The uptake of propidium iodide and cellular distributions of rhodamine-labeled Vip3Aa into fungal cells indicate that its growth inhibition mechanism involves its penetration within cells and subsequent intracellular damage. Furthermore, we discovered that the death of Candida albicans cells was caused by the induction of apoptosis via the generation of mitochondrial reactive oxygen species and binding to nucleic acids. The presence of significantly enlarged Vip3Aa-treated fungal cells indicates that this protein causes intracellular damage. Our findings suggest that Vip3Aa protein has potential applications in the development of natural antimicrobial agents.

Imaging and Targeted Antibacterial Therapy Using Chimeric Antimicrobial Peptide Micelles.

Infectious diseases induced by multidrug-resistant bacteria are a challenging problem in medicine because of global rise in the drug resistance to pathogenic bacteria. Despite great efforts on the development of antibiotics and antimicrobial agents, there is still a great need to develop a strategy to early detect bacterial infections and eradicate bacteria effectively and simultaneously. The innate immune systems of various organisms produce antimicrobial peptides, which kill a broad range of bacteria with minimal cytotoxicity to mammalian cells. Therefore, antimicrobial peptides have recently attracted increasing attention as an alternative to conventional antibiotics in antibacterial medications. Here, we report a new family of antibacterial agents, which is formulated from self-assembly of a chimeric antimicrobial lipopeptide (DSPE-HnMc) and amphiphilic biodegradable polymers. HnMc micelles could effectively bind the bacterial membrane to kill a wide spectrum of bacteria and bacterial biofilms. In the studies of mouse models of drug-resistant bacterial infections, HnMc micelles could target bacterial infections with high specificity and also kill drug-resistant bacteria effectively, demonstrating the great potential of HnMc micelles as imaging and targeted antibacterial agents. These findings also provide new insight into the design of antimicrobial peptide-based nanomedicine for detection and treatment of bacterial infections.

Improved Cell Selectivity of Pseudin-2 via Substitution in the Leucine-Zipper Motif: In Vitro and In Vivo Antifungal Activity.

Several antimicrobial peptides (AMPs) have been discovered, developed, and purified from natural sources and peptide engineering; however, the clinical applications of these AMPs are limited owing to their lack of abundance and side effects related to cytotoxicity, immunogenicity, and hemolytic activity. Accordingly, to improve cell selectivity for pseudin-2, an AMP from Pseudis paradoxa skin, in mammalian cells and pathogenic fungi, the sequence of pseudin-2 was modified by alanine or lysine at each position of two amino acids within the leucine-zipper motif. Alanine-substituted variants were highly selective toward fungi over HaCaT and erythrocytes and maintained their antifungal activities and mode of action (membranolysis). However, the antifungal activities of lysine-substituted peptides were reduced, and the compound could penetrate into fungal cells, followed by induction of mitochondrial reactive oxygen species and cell death. In vivo antifungal assays of analogous peptide showed excellent antifungal efficiency in a Candida tropicalis skin infection mouse model. Our results demonstrated the usefulness of selective amino acid substitution in the repeated sequence of the leucine-zipper motif for the design of AMPs with potent antimicrobial activities and low toxicity.