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The marine antifungal peptide epinecidin-1 (EPI) have been shown to inhibit Botrytis cinerea growth, while the molecular mechanism have not been explored based on omics technology. This study aimed to investigate the molecular mechanism of EPI against B. cinerea by transcriptome technology. Our findings indicated that a total of 1671 differentially expressed genes (DEGs) were detected in the mycelium of B. cinerea treated with 12.5 μmol/L EPI for 3 h, including 773 up-regulated genes and 898 down-regulated genes. Cluster analysis showed that DEGs (including steroid biosynthesis, (unsaturated) fatty acid biosynthesis) related to cell membrane metabolism were significantly down-regulated, and almost all DEGs involved in DNA replication were significantly inhibited. In addition, it also induced the activation of stress-related pathways, such as the antioxidant system, ATP-binding cassette transporter (ABC) and MAPK signaling pathways, and interfered with the tricarboxylic acid (TCA) cycle and oxidative phosphorylation pathways related to mitochondrial function. The decrease of mitochondrial related enzyme activities (succinate dehydrogenase, malate dehydrogenase and adenosine triphosphatase), the decrease of mitochondrial membrane potential and the increase content of hydrogen peroxide further confirmed that EPI treatment may lead to mitochondrial dysfunction and oxidative stress. Based on this, we speculated that EPI may impede the growth of B. cinerea through its influence on gene expression, and may lead to mitochondrial dysfunction and oxidative stress., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Innate defense regulators (IDRs) are synthetic host-defense peptides (HDPs) with broad-spectrum anti-infective properties, including immunomodulatory, anti-biofilm and direct antimicrobial activities. A lack of pharmacokinetic data about these peptides hinders their development and makes it challenging to fully understand how they work in vivo since their mechanism of action is dependent on tissue concentrations of the peptide. Here, we set out to define in detail the pharmacokinetics of a well-characterized IDR molecule, IDR-1018. To make the peptide traceable, it was radiolabeled with the long-lived gamma-emitting isotope gallium-67. After a series of bench-top characterizations, the radiotracer was administered to healthy mice intravenously (IV) or subcutaneously (SQ) at various dose levels (2.5-13 mg/kg). Nuclear imaging and ex-vivo biodistributions were used to quantify organ and tissue uptake of the radiotracer over time. When administered as an IV bolus, the distribution profile of the radiotracer changed as the dose was escalated. At 2.5 mg/kg, the peptide was well-tolerated, poorly circulated in the blood and was cleared predominantly by the reticuloendothelial system. Higher doses (7 and 13 mg/kg) as an IV bolus were almost immediately lethal due to respiratory arrest; significant lung uptake of the radiotracer was observed from nuclear scans of these animals, and histological examination found extensive damage to the pulmonary vasculature and alveoli. When administered SQ at a dose of 3 mg/kg, radiolabeled IDR-1018 was rapidly absorbed from the site of injection and predominately cleared renally. Apart from the SQ injection site, no other tissue had a concentration above the minimum inhibitory concentration that would enable this peptide to exert direct antimicrobial effects against most pathogenic bacteria. Tissue concentrations were sufficient, however, to disrupt microbial biofilms and alter the host immune response. Overall, this study demonstrated that the administration of synthetic IDR peptide in vivo is best suited to local administration which avoids some of the issues associated with peptide toxicity that are observed when administered systemically by IV injection, an issue that will have to be addressed through formulation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: EFH and REWH have invented and filed for patent protection on IDR-1018 and related peptide sequences. This patent has been assigned to their employer, the University of British Columbia, and has been licensed to ABT Innovations Inc., in which both have an ownership position. ABT Innovations Inc. is a subsidiary of ASEP Medical Holdings Inc. EFH is employed by ASEP and receives salary while REWH holds an executive position and is on the Board of ASEP. TVFE, CB, DP, CRR, KS and UOH report no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Antimicrobial peptides (AMPs) have attracted great attention as next generation antibiotics for the treatment of multidrug-resistant (MDR) bacterial infections. Poor proteolytic stability has however undermined clinical applications of AMPs. A novel peptide cyclization approach is described to enhance the in vivo antibacterial activity of AMPs. Bicyclic antimicrobial peptides were synthesized by cross-linking the ε-amino groups of three lysine residues with a 1,3,5-trimethylene benzene spacer. In a proof of principal study, four bicyclic peptides were synthesized from the cationic AMP OH-CM6. One bicyclic peptide retained strong antimicrobial activity and low toxicity but exhibited a prolonged half-life in serum. Antibacterial activity was consequently improved in vivo without renal or hepato-toxicity. The novel peptide cyclization approach represents an important tool for enhancing AMP proteolytic stability for improved treatment of bacterial infection.

Cationic biocides have been widely used as active ingredients in personal care and healthcare products for infection control and wound treatment for a long time, but there are concerns over their cytotoxicity and antimicrobial resistance. Designed lipopeptides are potential candidates for alleviating these issues because of their mildness to mammalian host cells and their high efficacy against pathogenic microbial membranes. In this study, antimicrobial and cytotoxic properties of a de novo designed lipopeptide, CH 3 (CH 2 ) 12 CO-Lys-Lys-Gly-Gly-Ile-Ile-NH 2 (C 14 KKGGII), were assessed against that of two traditional cationic biocides C n TAB ( n = 12 and 14), with different critical aggregation concentrations (CACs). C 14 KKGGII was shown to be more potent against both bacteria and fungi but milder to fibroblast host cells than the two biocides. Biophysical measurements mimicking the main features of microbial and host cell membranes were obtained for both lipid monolayer models using neutron reflection and small unilamellar vesicles (SUVs) using fluorescein leakage and zeta potential changes. The results revealed selective binding to anionic lipid membranes from the lipopeptide and in-membrane nanostructuring that is distinctly different from the co-assembly of the conventional C n TAB. Furthermore, C n TAB binding to the model membranes showed low selectivity, and its high cytotoxicity could be attributed to both membrane lysis and chemical toxicity. This work demonstrates the advantages of the lipopeptides and their potential for further development toward clinical application.

An "antibiotic-free strategy" provides a viable option to address bacterial infections, especially for the "superbug" challenge. However, the undesirable antibacterial activity of antibiotic-free agents hinders their practical applications. In this study, we developed a combination antibacterial strategy of coupling peptide-drug therapy with chemodynamic therapy (CDT) to achieve the effective bacterial inhibition. An amphiphilic oligopeptide (LAOOH-OPA) containing a therapeutic unit of D (KLAK) 2 peptide and a hydrophobic linoleic acid hydroperoxide (LAHP) was designed. The positively charged D (KLAK) 2 peptide with an α-helical conformation enabled rapid binding with microbial cells via electrostatic interaction and subsequent membrane insertion to deactivate the bacterial membrane. When triggered by Fe 2+ , moreover, LAHP could generate singlet oxygen ( 1 O 2 ) to elicit lipid bilayer leakage for enhanced bacteria inhibition. In vitro assays demonstrated that the combination strategy possessed excellent antimicrobial activity not only merely toward susceptible strains (Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli ) but also toward methicillin-resistant Staphylococcus aureus (MRSA). On the mouse skin abscess model induced by S. aureus , self-assembled LAOOH-OPA exhibited a more significant bacteria reduction (1.4 log 10 reduction) in the bioburden compared to that of the standard vancomycin (0.9 log 10 reduction) without apparent systemic side effects. This combination antibacterial strategy shows great potential for effective bacterial inhibition.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo . Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

In pursuit of ultrashort peptide-based antifungals, a new structural class, His(2-aryl)-Trp-Arg is reported. Structural changes were investigated on His-Trp-Arg scaffold to demonstrate the impact of charge and lipophilic character on the biological activity. The presence and size of the aryl moiety on imidazole of histidine modulated overall amphiphilic character, and biological activity. Peptides exhibited IC 50 of 0.37-9.66 μg/mL against C. neoformans. Peptide 14f [His(2-p-(n-butyl)phenyl)-Trp-Arg-OMe] exhibited two-fold potency (IC 50  = 0.37 μg/mL, MIC = 0.63 μg/mL) related to amphotericin B, without any cytotoxic effects up to 10 μg/mL. Peptide 14f act by nuclear fragmentation, membranes permeabilization, disruption and pore formations in the microbial cells as determined by the mechanistic studies employing Trp-quenching, CLSM, SEM, and HR-TEM. The amalgamation of short sequence, presence of appropriate aryl group on l-histidine, potent anticryptococcal activity, no cytotoxicity, and detailed mechanistic studies directed to the identification of 14f as a new antifungal structural lead., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Marine polysaccharides or oligosaccharides have potential to promote wound healing due to their biocompatibility and physicochemical properties. However, microbial infection delays wound healing process, and novel antimicrobial wound dressings are urgently needed. Here, agarose oligosaccharides (AGO) obtained from marine red algae were used as a reducing and stabilizer for green synthesis of silver nanoparticles (AgNPs), and further successfully connected with odorranain A (OA), one of antimicrobial peptides (AMPs), to obtain a novel composite nanomaterial (AGO-AgNPs-OA). Transmission electron microscopy (TEM) and Malvern particle size analyzer showed that AGO-AgNPs-OA was spherical or elliptic with average size of about 100 nm. Circular dichroism (CD) spectroscopy showed that AGO-AgNPs stabilized the α-helical structure of OA. AGO-AgNPs-OA showed stronger anti-bacterial activities than AGO-AgNPs, and had good biocompatibility and significant promoting effect on wound healing. Our data suggest that AMPs conjugated marine oligosaccharides and AgNPs may be effective and safe antibacterial materials for wound therapy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina , the classical example of peptides with a β-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.

Background: Metallic nanoparticles (NPs) are highly exploited in manufacturing and medical processes in a broad spectrum of industrial applications and in the academic sectors. Several studies have suggested that many metallic nanomaterials including those derived by silver (Ag) are entering the ecosystem to cause significant toxic consequences in cell culture and animal models. However, ecotoxicity studies are still receiving limited attention when designing functionalized and non.-functionalized AgNPs., Objective: This study aimed to investigate different ecotoxicological profiles of AgNPs, which were analyzed in two different states: in pristine form uncoated AgNPs and coated AgNPs with the antimicrobial peptide indolicidin. These two types of AgNPs are exploited for a set of different tests using Daphnia magna and Raphidocelis subcapitata , which are representatives of two different levels of the aquatic trophic chain, and seeds of Lepidium sativum, Cucumis sativus and Lactuca sativa ., Results: Ecotoxicological studies showed that the most sensitive organism to AgNPs was crustacean D. magna, followed by R. subcapitata and plant seeds, while AgNPs coated with indolicidin (IndAgNPs) showed a dose-dependent decreased toxicity for all three., Conclusion: The obtained results demonstrate that high ecotoxicity induced by AgNPs is strongly dependent on the surface chemistry, thus the presence of the antimicrobial peptide. This finding opens new avenues to design and fabricate the next generation of metallic nanoparticles to ensure the biosafety and risk of using engineered nanoparticles in consumer products., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Falanga et al.)

Antimicrobial peptides (AMP) are molecules with a broad spectrum of activities that have been identified in most living organisms. In addition, synthetic AMPs designed from natural polypeptides have been largely investigated. Here, we designed a novel AMP using the amino acid sequence of a plant trypsin inhibitor from Adenanthera pavonina seeds (ApTI) as a template. The 176 amino acid residues ApTI sequence was cleaved in silico using the Collection of Antimicrobial Peptides (CAMP R3 ), through the sliding-window method. Further improvements in AMP structure were carried out, resulting in adepamycin, an AMP designed from ApTI. Adepamycin showed antimicrobial activity from 0.9 to 3.6 μM against Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Moreover, this peptide also displayed activity against Candida albicans and Candida tropicalis. No toxic effects were observed on healthy human cells. Studies on the mechanism of action of adepamycin were carried out using an E. coli and C. tropicalis. Adepamycin triggers membrane disturbances, leading to intracellular nucleic acids release in E. coli. For C. tropicalis, an initial interference with the plasma membrane integrity is followed by the formation of intracellular reactive oxygen species (ROS), leading to apoptosis. Structurally, adepamycin was submitted to circular dichroism spectroscopy, molecular modeling and molecular dynamics simulations, revealing an environment-dependent α-helical structure in the presence of 2,2,2- trifluoroethanol (TFE) and in contact with mimetic vesicles/membranes. Therefore, adepamycin represents a novel lytic AMP with dual antibacterial and antifungal properties., (Copyright © 2020 Elsevier Inc. All rights reserved.)

There has been an increasing interest in the development of antimicrobial peptides (AMPs) and their synthetic mimics as a novel class of antibiotics to overcome the rapid emergence of antibiotic resistance. Recently, phenylglyoxamide-based small molecular AMP mimics have been identified as potential leads to treat bacterial infections. In this study, a new series of biphenylglyoxamide-based small molecular AMP mimics were synthesised from the ring-opening reaction of N -sulfonylisatin bearing a biphenyl backbone with a diamine, followed by the conversion into tertiary ammonium chloride, quaternary ammonium iodide and guanidinium hydrochloride salts. Structure-activity relationship studies of the analogues identified the octanesulfonyl group as being essential for both Gram-positive and Gram-negative antibacterial activity, while the biphenyl backbone was important for Gram-negative antibacterial activity. The most potent analogue was identified to be chloro-substituted quaternary ammonium iodide salt 15c , which possesses antibacterial activity against both Gram-positive (MIC against Staphylococcus aureus = 8 μM) and Gram-negative bacteria (MIC against Escherichia coli = 16 μM, Pseudomonas aeruginosa = 63 μM) and disrupted 35% of pre-established S. aureus biofilms at 32 μM. Cytoplasmic membrane permeability and tethered bilayer lipid membranes (tBLMs) studies suggested that 15c acts as a bacterial membrane disruptor. In addition, in vitro toxicity studies showed that the potent compounds are non-toxic against human cells at therapeutic dosages.

Cytotoxic frog antimicrobial peptide Temporin L (TempL) is an attractive molecule for the design of lead antimicrobial agents due to its short size and versatile biological activities. However, noncytotoxic TempL variants with desirable biological activities have rarely been reported. TempL analogue Q3K,TempL is water-soluble and possesses a significant antiendotoxin property along with comparable cytotoxicity to TempL. A phenylalanine residue, located at the hydrophobic face of Q3K,TempL and the "d" position of its phenylalanine zipper sequence, was replaced with a cationic lysine residue. This analogue, Q3K,F8K,TempL, showed reduced hydrophobic moment and was noncytotoxic with lower antimicrobial activity. Interestingly, swapping between tryptophan at the fourth and serine at the sixth positions turned Q3K,F8K,TempL totally amphipathic as reflected by its helical wheel projection with clusters of hydrophobic and hydrophilic residues and the highest hydrophobic moment among these peptides. Surprisingly, this analogue, SW,Q3K,F8K,TempL, was as noncytotoxic as Q3K,F8K,TempL but showed augmented antimicrobial and antiendotoxin properties, comparable to that of TempL and Q3K,TempL. SW,Q3K,F8K,TempL exhibited appreciable survival of mice against P. aeruginosa infection and a lipopolysaccharide (LPS) challenge. Unlike TempL and Q3K,TempL, SW,Q3K,F8K,TempL adopted an unordered secondary structure in bacterial membrane mimetic lipid vesicles and did not permeabilize them or depolarize the bacterial membrane. Overall, the results demonstrate the design of a nontoxic TempL analogue that possesses clusters of hydrophobic and hydrophilic residues with impaired secondary structure and shows a nonmembrane-lytic mechanism and in vivo antiendotoxin and antimicrobial activities. This paradigm of design of antimicrobial peptide with clusters of hydrophobic and hydrophilic residues and high hydrophobic moment but low secondary structure could be attempted further.

Trypanosoma cruzi, the etiological agent of Chagas disease, is responsible for the infection of millions of people worldwide and it is a public health problem, without an effective cure. Four fragments with antimicrobial potential from the hemocyanin of Penaeus monodon shrimp were identified using a computer software AMPA. The present study aimed to evaluate the antichagasic effect of these four peptides (Hmc364-382, Hmc666-678, Hmc185-197 and Hmc476-498). The peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Two fragments (Hmc364-382, Hmc666-678) showed activity against the epimastigote and trypomastigote forms and their selectivity index (SI) was calculated. The Hmc364-382 peptide was considered the most promising (SI > 50) one and it was used for further studies, using flow cytometry analyses with specific molecular probes and scanning electron microscopy (SEM). Hmc364-382 was able to induce cell death in T. cruzi through necrosis, observed by loss of membrane integrity in flow cytometry analyses and pore formation in SEM. Overall, Hmc364-382 open perspectives to the development of new antichagasic agents., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, financial or otherwise., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Antimicrobial peptides have attracted attention as alternatives to conventional antibiotics. Previously, a novel antimicrobial peptide, melectin, consisting of 18 amino acids was isolated from the venom of a bee, Melecta albifrons. Here, we investigated the antibacterial activity of melectin against drug-resistant bacteria. Melectin showed broad-spectrum antimicrobial activity but low cytotoxicity and no hemolytic activity. Melectin maintained its antimicrobial activity at physiological salt concentrations. Melectin is an α-helical structure that binds to the bacterial membrane via electrostatic interactions and kills bacteria in a short time by bacterial membrane targeting. Collectively, our results suggest that melectin has antibacterial activity and anti-inflammatory activity.

Because of their potential as novel antibiotic agents, antimicrobial peptides (AMPs) have generated considerable interest. The mechanism of bacterial toxicity of AMPs often involves the disruption and/or permeabilization of the bacterial membrane; even those that act intracellularly first have to traverse the membrane. In this work we have explored the incorporation of the fluorinated aromatic amino acids fluoro-Phe and fluoro-Tyr into the Trp- and Arg-rich AMP tritrpticin, and investigated their role in the membrane binding properties and the antimicrobial activity of the peptide. Fluorinated peptides were obtained with good yield by recombinant expression of tritrpticin as a calmodulin-fusion protein in Escherichia coli. Cells were grown in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, and the peptides were released by proteolysis from the purified fusion protein. By using SDS micelles, as a simplified model of the bacterial cytoplasmic membrane, we could study the peptide-membrane interactions and the preferred location of individual fluorinated residues in the micelles by 19 F NMR spectroscopy. Solvent-perturbation 19 F NMR measurements revealed that para-fluoro-Phe residues are embedded deeply in the hydrophobic region of the micelles. On the other hand, 3-fluoro-Tyr residues introduced in tritrpticin were located near the surface of the micelles with high solvent exposure, while 2-fluoro-Tyr sidechains were less solvent exposed. In combination with the outcome of determinations of their antimicrobial activity, our 19 F NMR results indicate that the higher solvent exposure of Tyr residues correlates with a decrease of the antimicrobial potency. This different role of Tyr can likely be extended from tritrpticin to other cationic AMPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increasing resistance in antibiotic and chemotherapeutic treatments has been pushing studies of design and evaluation of bioactive peptides. Designing relies on different approaches from minimalist sequences and endogenous peptides modifications to computational libraries. Evaluation relies on microbiological tests. Aiming a deeper understanding, we chose the octapeptide Jelleine-I (JI) for its selective and low toxicity profile, designed small modifications combining the substitutions of Phe by Trp and Lys/His by Arg and tested the antimicrobial and anticancer activity on melanoma cells. Biophysical methods identified environment-dependent modulation of aggregation, but critical aggregation concentrations of JI and analogs in buffer show that peptides start membrane interactions as monomers. The presence of model membranes increases or reduces the partial aggregation of peptides. Compared to JI, analog JIF2WR shows the lowest tendency to aggregation on bacterial model membranes. JI and analogs are lytic to model membranes. Their composition-dependent performance indicates preference for the higher charged anionic bilayers in line with their superior performance toward Staphylococcus aureus and Streptococcus pneumoniae. JIF2WR presented the higher partitioning, higher lytic activity and lower aggregated contents. Despite these increased membranolytic activities, JIF2WR exhibited comparable antimicrobial activity in relation to JI at the expenses of some loss in selectivity. We found that the substitution Phe/Trp (JIF2W) tends to decrease antimicrobial but to increase anticancer activity and aggregation on model membranes and the toxicity toward human cells. However, the concomitant substitution Lys/His by Arg (JIF2WR) modulates some of these tendencies, increasing both the antimicrobial and the anticancer activity while decreasing the aggregation tendency.

The antimicrobial peptide (AMP) piscidin was identified from Epinephelus lanceolatus and demonstrated to possess antimicrobial and immune-related functions. Supplementation of feed with recombinant Epinephelus lanceolatus piscidin (rEP)-expressing yeast pellets may minimize the excessive use of antibiotics and control pathogens in aquaculture or animal husbandry. However, before implementing rEP as a supplement, it is necessary to understand whether it harbors any toxicity. Since toxicological information on the topic is scarce, the present investigation was carried out to test whether rEP exhibits allergenic and/or toxic effects. In an oral acute toxicity test (OECD 425), Sprague Dawley (SD) rats were administered rEP dissolved in reverse osmosis water, yielding an LD 50 > 5000 mg/kg (no observed animal death). The compound was therefore classified as non-toxic by oral administration. In an acute respiratory toxicity test (OECD 403), heads and noses of SD rats were exposed to liquid aerosol for 4 h (the highest concentration that could be administered without causing any animal death), and a lethal concentration (LC 50 ) > 0.88 mg/L was obtained. The mass medium aerodynamics diameter (MMAD) of rEP aerosol particles was 8.18 μm and mass medium aerodynamics diameter (GSD) was 3.04, which meant that 25.90% could enter the airway (<4 μm) of a rat, and 58.06% (<10 μm) could be inhaled by humans. An ocular irritation test (OECD 405) with rEP powder was performed on New Zealand White (NZW) rabbits. Signs of irritation included conjunctival swelling and diffuse flushing 1 h after administration. The signs were less apparent after 24 h and disappeared after 72 h. The classification assigned to the powder was mild eye irritation. Skin sensitization was performed for a local lymphoproliferative test (OECD 442B) using BALB/c mice, with the highest soluble concentration of the rEP considered to be 100% test substance; formulations were diluted to 50% and 25%, and bromodeoxyuridine (BrdU) incorporation was used to measure the degree of lymphocyte proliferation. The stimulation indexes (SIs) were 1.06 (100%), 0.44 (50%), and 0.77 (25%), all of which were less than the cutoff value for a positive sensitization result (1.6). Negative response was also seen in the bacterial reverse mutation test (OECD 471), and no chromosomal effects on Chinese hamster ovary (CHO)-K1 cells were observed (OECD 487). Based on these six toxicity tests, rEP showed neither acute toxic effects in experimental animals nor mutagenicity. Thus, rEP can be considered safe for use in subsequent research on its application as a feed additive for poultry, cattle, or aquatic animals., Competing Interests: The authors declare no conflict of interest.

Parasitic diseases are a neglected and serious problem, especially in underdeveloped countries. Among the major parasitic diseases, Leishmaniasis figures as an urgent challenge due to its high incidence and severity. At the same time, the indiscriminate use of antibiotics by the population is increasing together with resistance to medicines. To address this problem, new antibiotic-like molecules that directly kill or inhibit the growth of microorganisms are necessary, where antimicrobial peptides (AMPs) can be of great help. In this work, the ferrocene molecule, one active compound with low levels of in vivo toxicity, was coupled to the N-terminus of the RP1 peptide (derived from the human chemokine CXCL4), aiming to evaluate how this change modifies the structure, biological activity, and toxicity of the peptide. The peptide and the conjugate were synthesized using the solid phase peptide synthesis (SPPS). Circular dichroism assays in PBS showed that the RP1 peptide and its conjugate had a typical spectrum for disordered structures. The Fc-RP1 presented anti-amastigote activity against Leishmania amazonensis (IC50 = 0.25 μmol L-1). In comparison with amphotericin B, a second-line drug approved for leishmaniasis treatment, (IC50 = 0.63 μmol L-1), Fc-RP1 was more active and showed a 2.5-fold higher selectivity index. The RP1 peptide presented a MIC of 4.3 μmol L-1 against S. agalactiae, whilst Fc-RP1 was four times more active (MIC = 0.96 μmol L-1), indicating that ferrocene improved the antimicrobial activity against Gram-positive bacteria. The Fc-RP1 peptide also decreased the minimum inhibitory concentration (MIC) in the assays against E. faecalis (MIC = 7.9 μmol L-1), E. coli (MIC = 3.9 μmol L-1) and S. aureus (MIC = 3.9 μmol L-1). The cytotoxicity of the compounds was tested against HaCaT cells, and no significant activity at the highest concentration tested (500 μg. mL-1) was observed, showing the high potential of this new compound as a possible new drug. The coupling of ferrocene also increased the vesicle permeabilization of the peptide, showing a direct relation between high peptide concentration and high carboxyfluorescein release, which indicates the action mechanism by pore formation on the vesicles. Several studies have shown that ferrocene destabilizes cell membranes through lipid peroxidation, leading to cell lysis. It is noteworthy that the Fc-RP1 peptide synthesized here is a prototype of a bioconjugation strategy, but it still is a compound with great biological activity against neglected and fish diseases., Competing Interests: The authors have declared that no competing interests exist.

Due to rapid development of bacterial resistance against antibiotics, an emerging health crisis is underway, where 'simple' infections may no longer be treatable. Antimicrobial peptides (AMPs) constitute a class of substances attracting interest in this context. So far, research on AMPs has primarily focused on the identification of potent and selective peptides, as well as on the action mode of such peptides. More recently, there has been an increasing awareness that the delivery of AMPs is challenging due to their size, net positive charge, amphiphilicity, and proteolytic susceptibility. Hence, successful development of AMP therapeutics will likely require also careful design of efficient AMP delivery systems. In the present brief review, we discuss microgels, as well as related polyelectrolyte complexes and macroscopic hydrogels, as delivery systems for AMPs. In doing so, key factors for peptide loading and release are outlined and exemplified, together with consequences of this for functional performance relating to antimicrobial effects and cell toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2020 Elsevier B.V. All rights reserved.)

The function and properties of peptide-based materials depend not only on the amino acid sequence but also on the molecular conformations. In this paper, we chose a series of peptides G m (XXKK) n X-NH 2 ( m = 0, 3; n = 2, 3; X = I, L, and V) as the model molecules and studied the conformation regulation through N-terminus lipidation and their formulation with surfactants. The structural and morphological transition of peptide self-assemblies have also been investigated via transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, and small-angle neutron scattering. With the terminal alkylation, the molecular conformation changed from random coil to β-sheet or α-helix. The antimicrobial activities of alkylated peptide were different. C 16 -G 3 (IIKK) 3 I-NH 2 showed antimicrobial activity against Streptococcus mutans, while C 16 -(IIKK) 2 I-NH 2 and C 16 -G 3 (IIKK) 2 I-NH 2 did not kill the bacteria. The surfactant sodium dodecyl sulfonate could rapidly induce the self-assemblies of alkylated peptides (C 16 -(IIKK) 2 I-NH 2 , C 16 -G 3 (IIKK) 2 I-NH 2 , C 16 -G 3 (VVKK) 2 V-NH 2 ) from nanofibers to micelles, along with the conformation changing from β-sheet to α-helix. The cationic surfactant hexadecyl trimethyl ammonium bromide made the lipopeptide nanofibers thinner, and nonionic surfactant polyoxyethylene (23) lauryl ether (C 12 EO 23 ) induced the nanofibers much more intensively. Both the activity and the conformation of the α-helical peptide could be modulated by lipidation. Then, the self-assembled morphologies of alkylated peptides could also be further regulated with surfactants through hydrophobic, electrostatic, and hydrogen-bonding interactions. These results provided useful strategies to regulate the molecular conformations in peptide-based material functionalization.

A strategy that has been gaining increased application for the study of the conformation, dynamics, orientation, and physicochemical properties of peptides is labeling with the paramagnetic amino acid TOAC. This approach was used to gain a deeper understanding on the mechanism of action of the antimicrobial peptide tritrpticin (TRP3). TRP3 was labeled with TOAC at the N-terminus (prior to V 1 , TOAC 0 -TRP3) or internally (replacing P 5 , TOAC 5 -TRP3). Functional studies showed that labeling led to peptides with higher activity against Gram-positive bacteria and lower hemolytic activity with respect to TRP3. Peptide-induced model membranes permeabilization and ion channel-like activity studies corroborated the functional assays qualitatively, showing higher activity of the peptides against negatively charged membranes, which had the purpose of mimicking bacterial membranes. TOAC presented a greater freedom of motion at the N-terminus than at the internal position, as evinced by EPR spectra. EPR and fluorescence spectra reported on the peptides conformational properties, showing acquisition of a more packed conformation in the presence of the secondary structure-inducing solvent, TFE. CD studies showed that TOAC 0 -TRP3 acquires a conformation similar to that of TRP3, both in aqueous solution and in TFE, while TOAC 5 -TRP3 presents a different conformation in all environments. While the mechanism of action of TRP3 was impacted to some extent by TOAC labeling at the N-terminus, it did change upon replacement of P 5 by TOAC. The results demonstrated that TOAC-labeling could be used to modulate TRP3 activity and mechanism of action and, more importantly, the critical role of P 5 for TRP3 pore formation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Typically, antimicrobial peptides (AMPs) are short positive charged peptides serving a key role in innate immunity as well as antimicrobial activity. Discovering novel therapeutic agents is considered as an undeniable demand due to increasing microbial species with antibiotic resistance. In this direction, the unique ability of AMPs to modulate immune responses highlighted them as novel drug candidates in the field of microbiology. Patients affected by leishmaniasis; a neglected tropical disease, confront serious problems for their treatment including resistance to common drugs as well as toxicity and high cost of therapy. So, there is a need for development of new drug candidates to control the diseases. Jellein, a peptide derived from royal jelly of honeybee has been shown to have promising effect against several bacterial and fungal species. In current study, anti-leishmanial effect of Jellein and its lauric acid conjugated form was investigated against two forms of Leishmania major (L. major) parasite. Moreover, cytotoxic effect of these peptides was studied in THP1 cell line and human Red Blood Cells (RBCs). Furthermore, the mechanism of action of peptides on L. major promastigotes was assessed through different methods. The results demonstrated that, conjugation of lauric acid to Jellein not only had no effect on the elevation of antimicrobial activity but also halted it completely. Moreover, Jellein caused a limitation in the number of L. major promastigotes by pore formation as well as changing the membrane potential rather than induction of apoptosis or activation of caspases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the past few years the increasing incidence of hospital infections with Acinetobacter baumannii, especially in immunocompromised patients, and its proneness to develop multidrug resistance have been raising considerable concern. This study examines the antimicrobial and antibiofilm activity of protegrin 1 (PG-1), an antimicrobial peptide from porcine leukocytes, against A. baumannii strains isolated from surgical wounds. PG-1 was tested both alone and combined with the antibiotics commonly used in clinical settings. Its antimicrobial activity was evaluated by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), checkerboard assays, and time-kill experiments. Its effects on biofilm inhibition/eradication were tested with crystal violet staining. The strains were grown in subinhibitory or increasing PG-1 concentrations to test the development of resistance. Mammalian cell toxicity was tested by XTT assays. PG-1 MICs and MBCs ranged from 2 to 8 µg/ml. PG-1 was most active and demonstrated a synergistic interaction with colistin, a last resort antibiotic. Interestingly, antagonism was never observed. In time-kill experiments, incubation with 2 × MIC for 30 min suppressed all viable cells. PG-1 did not select resistant strains and showed a limited effect on cell viability, but it did exert a strong activity against multidrug-resistant A. baumannii. In contrast, in our experimental conditions it had no effect on biofilm inhibition/eradication. PG-1 thus seems to be a promising antimicrobial agent against multidrug-resistant Gram-negative infections.

Non-natural amino acids can be incorporated into synthetic host defense peptides (HDPs) to modulate their susceptibility to proteolytic degradation. However, the impact of non-natural amino acids on the antibiofilm and immunomodulatory activities of synthetic HDPs remains unclear. Using SPOT-synthesized peptide arrays, non-natural cationic amino acids of varying side chain lengths were incorporated into a synthetic HDP, IDR-1018, and the impact of these substitutions on the antibiofilm activity toward methicillin resistant Staphylococcus aureus biofilms was assessed. Multiply-substituted derivatives were designed that incorporated favorable non-natural cationic amino acid moieties throughout IDR-1018. The antibiofilm and immunomodulatory activities of these derivatives were assessed in vitro , revealing that the incorporation of non-natural amino acids modulated (either positively or negatively) these activities of IDR-1018. Furthermore, the tryptic stability of the IDR-1018 derivatives was assessed revealing that proteolytic stability was favored for shorter cationic side chains and was influenced by the primary peptide sequence.

Antibiotic resistance and infection recurrence are critical issues in treating bacterial vaginosis, the most common vaginal disorder in women of reproductive age. Novel alternatives to traditional antibiotics, such as peptidomimetics, have the potential to address this challenge. Previously, two series of cationic amphiphiles (CAms) were developed with both hydrophilic head groups and non-polar domains, giving them the ability to self-assemble into supramolecular nanostructures with membrane-lytic properties. Those CAms were shown to be effective against biofilms of Gardnerella vaginalis while preserving the commensal microbiota. Two new series of CAms were designed with varying levels of flexibility between the hydrophilic head groups and the hydrophobic domains. Activities against the vaginal pathogen G. vaginalis ranged from 1.3 to 18.5 µM, while the tested vaginal lactobacilli were significantly more tolerant of CAms, with minimal inhibitory concentration values as high as 208 µM. Minimal biofilm bactericidal concentrations of the tested CAms ranged from 21.47 to <388.3 µM, and were lowest against resistant forms of G. vaginalis, while Lactobacillus biofilms were tolerant of concentrations ≥687 µM. Safety aspects of the CAms were also investigated, and they were found to be safe for use against vaginal ectocervical tissue., (© FEMS 2019.)

It is important to control immediate hemorrhage and prevent infection simultaneously in the wound management. However, most of hemostatic materials are associated with low efficiency of hemostasis, poor biocompatibility and lack of antimicrobial properties. A kind of starch-based macroporous sponges (KR-Sps) immobilized covalently with antimicrobial peptide KR12 using highly efficient thiol-ene photo click reaction were developed. The physical properties of these sponges could be fine-tuned by varying the ratio of modified starch/HS-PEG-SH and the polymer concentration. The in vitro and vivo results demonstrated that KR-Sps induced thrombosis, shortened clotting time and reduced the blood loss at bleeding site. Besides, KR12 immobilized sponge exhibited inherent antimicrobial properties against Gram (+) and (-) bacteria and methicillin-resistant Staphylococcus aureus (MRSA), which could maintain at least 5 days. Therefore, KR-Sps were believed to be an excellent candidate as hemostatic and antimicrobial product for the intraoperative wound management., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

VmCT1 is a cationic antimicrobial peptide (AMP) from the venom of the scorpion Vaejovis mexicanus. VmCT1 and analogs were designed with single substitutions for verifying the influence of changes in physicochemical features described as important for AMPs antimicrobial and hemolytic activities, as well as their effect on VmCT1 analogs resistance against proteases action. The increase of the net positive charge by the introduction of an arginine residue in positions of the hydrophilic face of the helical structure affected directly the antimicrobial activity. Arg-substituted analogs presented activity against Gram-negative bacteria from the ESKAPE list of pathogens that were not observed for VmCT1. Additionally, peptides with higher net positive charge presented increased antimicrobial activity with values ranging from 0.39 to 12.5 μmol L -1 against Gram-positive and Gram-negative bacteria and fungi. The phenylalanine substitution by glycine (position 1), and the valine substitution by a proline residue (position 8) led to analogs with lower hemolytic activity (at concentrations 50 and 100 μmol L -1 , respectively). These results revealed that it is possible to modulate the biological activities of VmCT1 derivatives by designing single substituted-analogs as prospective therapeutics against bacteria and fungi., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts, retinal cells, embryonic kidney cells, neuroblastoma cells, alveolar macrophage cells, and neutrophils. The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells and neutrophils being the most significantly affected. A circular dichroism analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell type. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane's anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.

Natural α-helical cationic antimicrobial peptide (CAP) sequences are predominantly amphipathic, with only ca. 2% containing four or more consecutive positively charged amino acids (Lys/Arg). We have designed synthetic CAPs that deviate from these natural sequences, as typified by the charge-clustered peptide KKKKKKAAFAAWAAFAA-NH 2, (termed 6K-F17), which displays high antimicrobial activity with no toxicity to mammalian cells. We created a series of peptides varying in charge patterning, increasing the amphipathic character of 6K-F17 to mimic the design of natural CAPs (e.g., KAAKKFAKAWAKAFAA-NH 2 ). Amphipathic sequences displayed increased antimicrobial activity against bacteria but were significantly more toxic to mammalian cells and more susceptible to protease degradation than their corresponding charge-clustered variants, suggesting that amphipathic sequences may be desirable in nature to allow for more versatile functions (i.e., antibacterial, antifungal, antipredator) and rapid clearance from vulnerable host cells. Our approach to clustering of charges may therefore allow for specialization against bacteria, in concert with prolonged peptide half-life.

The antibiotic-resistant viable but non-culturable (VBNC) pathogenic bacteria are considered as a new threat to public health. Antimicrobial peptides (AMPs), possessing bactericidal effects in a rapid membrane attacking mode, are supposed to be effective against bacteria entering the VBNC state. In the current study, the activity of grouper AMP piscidin killing the VBNC state cells of pathogenic bacteria Escherichia coli O157, Staphylococcus aureus, and Vibrio parahaemolyticus OS4 was studied. After entering the VBNC state, cells of E. coli O157, S. aureus, and V. parahaemolyticus OS4 developed resistance to the antibiotics Ampicillin and Kanamycin. Rather than truncated form of Malabar grouper piscidin 1 (EmPis-1S), full-length Malabar grouper piscidin 1 (EmPis-1L) showed strong activity to kill the above VBNC bacteria. The VBNC state cells (1 × 10 5  CFU/mL) of the three species of bacteria could be totally lysed by 10 μmol/L of EmPis-1L in 1 h. The VBNC state cells of S. aureus were most susceptible to EmPis-1L, which killed the cells by 100% in 30 min at the low concentration of 2.0 μmol/L. In addition, EmPis-1L at the concentration of no more than 10 μmol/L showed no observed toxicity to human lung carcinoma epithelial cells (A549) and mouse neuroblastoma cells (N2a). Accordingly, EmPis-1L could be a promisingly safe and efficient agent for eliminating the traditional antibiotic-resistant VBNC state cells of pathogenic bacteria, E. coli, S. aureus, and V. parahaemolyticus.

The alarming burden of antibiotic resistance in nosocomial pathogens warrants the discovery and development of new and effective antimicrobial compounds. Small cationic antimicrobial peptides seem to be a promising therapeutic alternative to fight multi-drug resistance. This study investigated the in-vitro potential of a previously reported lantibiotic, paenibacillin, from the clinical perspective. An antimicrobial peptide, M152-P4, was isolated, purified and characterized from a mud isolate, and its susceptibility was determined in clinical isolates of Staphylococcus aureus and Enterococcus spp. Time-kill kinetics, resistance, probable mode of action, haemolytic activity and mammalian cytotoxicity were investigated. M152-P4 was identified as paenibacillin based on mass spectroscopy data, amino acid analysis and biosynthetic gene cluster analysis. It had potent antibacterial activity against the Gram-positive pathogens tested, with minimum inhibitory concentrations from 0.1 to 1.56 µM. It appeared very challenging for S. aureus to develop resistance to this compound. Also, paenibacillin penetrated the outer layer of bacteria, and depolarized the membrane completely by creating pores in the plasma membrane with better potential than nisin. Paenibacillin showed no haemolysis up to 60 µM, and the half maximal inhibitory concentration on mammalian cell lines was >100 µM. These results highlight the excellent antibacterial properties of paenibacillin in clinically relevant pathogens. It is stable in the presence of serum, and non-haemolytic and non-cytotoxic even above the therapeutic concentration. Further research efforts regarding toxicity and in-vivo efficacy are necessary to develop paenibacillin as a next-generation therapeutic drug to overcome multi-drug resistance in Gram-positive pathogens., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

K4 is a de novo peptide with antibacterial activity on human pathogens. It has a short sequence (14 amino acids), with a cationic N-terminal moiety and an amphipathic ɑ-helix structure. The present paper demonstrates its activity on Vibrio bacteria in a marine environment. It was found non-toxic on marine organisms including Artemia salina, Dicentrarchus labrax, and Magallana gigas at different developmental stages, but influenced the growth of unicellular organisms like microalgae, depending on the algal strain and on K4 concentration. Furthermore, an original approach coupling liquid chromatography (RP-HPLC) and mass spectrometry (MS/MS) allowed us to monitor the degradation time course of the peptide for the first time in conditions close to a hatchery environment, i.e., in the presence of oyster spat. We detected truncated forms over time, and the full K4 was gradually no longer found in these filter-feeder oysters. Finally, using an automated optical density meter, we monitored the growth of several aquatic bacteria identified as pathogenic on animals. K4 had a bactericidal effect on Aeromonas salmonicida and Vibrio splendidus LGP32 at concentrations below 45 μg mL -1 . Our results show that K4 could be an environment-friendly alternative to antibiotics, non-toxic to several marine organisms. The use of K4 would be particularly useful to decrease the bacterial load associated with food intake in the early developmental stages of marine animals reared in hatcheries.

Broad-spectrum antibiotics have, until now, been the mainstay of antibiotic therapy. However, the increasing threat of drug-resistant bacteria and the ecological imbalance of normal microbial communities have forced a reconsideration of the best strategies to treat such pathogens. Therefore, antibacterial agents with specific abilities of eliminating pathogens may provide long-term protection. Antimicrobial peptides (AMPs), which can be optimized by modifying their primary sequences, are regarded as potentially valuable in development of pathogen-specific agents. To obtain efficient narrow-spectrum AMPs, database-filtering technology, which filters the most probable amino acid composition, positive charge, sequence length and hydrophobic content of peptides against Gram-negative bacteria, was taken as the first step. Then, the filtered parameters were distributed and modified into an α-helical symmetrical structure by considering the structure-function relationship of synthesized antimicrobial peptides. Finally, short, safe and stable peptides against Escherichia coli, Salmonella pullorum and Pseudomonas aeruginosa were successfully identified. The potential peptides F1 and F4 showed low cell toxicity, low resistance potential and low salt sensitivity. CD spectroscopy of the peptides illustrated that F1 and F4 exhibited a tendency towards an α-helical structure in a membrane-mimetic environment. Indeed, fluorescence spectroscopy and electron microscopy analyses indicated that the shorter potential sequence F4 killed the bacteria by causing physical destruction of the bacterial membrane and cytosol leakage. In the mouse model test, F4 reduced the bacterial load in major organs and the cytokine (TNF-α, IL-6, and IL-1β) levels in serum significantly (P < 0.05). Collectively, this symmetric-helical distribution, dependent on database-filtering parameters, is a promising strategy for designing effective smart AMPs with high cell selectivity, and it also provides new insights into the design and optimization of pathogen-specific biomaterials.

Commensalism coinfection of pathogens has seriously jeopardized human health. Currently, Kunitzin-RE, as an amphibian-derived bioactivity peptide, is regarded as a potential antimicrobial candidate. However, its antimicrobial properties were unsatisfactory. In this study, a set of shortened variants of Kunitzin-RE was developed by the interception of a peptide fragment and single-site mutation to investigate the effect of chain length, positive charge, hydrophobicity, amphipathicity, and secondary structure on antimicrobial properties. Among them, W8 (AARIILRWRFR) significantly broadened the antimicrobial spectrum and showed the highest antimicrobial activity (GM all = 2.48 μM) against all the fungi and bacteria tested. Additionally, W8 showed high cell selectivity and salt tolerance in vitro, whereas it showed high effectiveness against mice keratitis cause by infection by C. albicans 2.2086. Additionally, it also had obviously lipopolysaccharide-binding ability and a potent membrane-disruptive mechanism. Overall, these findings contributed to the design of short antimicrobial peptides and to combat the serious threat of commensalism coinfection of pathogens.

Antimicrobial peptides have been evaluated as possible alternatives to traditional antibiotics. The translational potential of the antimicrobial peptide DGL13K was tested with focus on peptide toxicity and in vivo activity in two animal models. DGL13K was effective against Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus with minimal bactericidal concentrations similar to the minimal inhibitory concentration. The peptide showed low toxicity to human red blood cells and HEK cells with median lethal dose around 1 mg/ml. The median lethal dose in greater wax moth larvae (Galleria mellonella) was about 125mg/kg while the peptide caused no skin toxicity in a mouse model. A novel high-throughput luminescence assay was used to test peptide activity in infected G. mellonella, thus reducing vertebrate animal use. DGL13K killed P. aeruginosa in both the G. mellonella model and a mouse burn wound infection model, with bacterial viability 3-10-fold lower than in untreated controls. Future experiments will focus on optimizing peptide delivery, dose and frequency to further improve the antibacterial effect., Competing Interests: Dr. Gorr holds two linked patents related to the research reported in this manuscript: Synthetic peptides and peptide mimetics. U.S. Patent number: US9914750. Date of Patent: March 13, 2018. Assignee: University of Louisville Research Foundation Inventor: Sven-Ulrik Gorr. Synthetic peptides and peptide mimetics, U.S. Patent number: US8569449, Date of Patent: October 29, 2013, Assignee: University of Louisville Research Foundation, Inc. Inventor: Sven-Ulrik Gorr. Dr. Gorr declares no commercial funding, employment, consultancy, or commercial products in development related to these patents. This does not alter our adherence to PLoS ONE policies on sharing data and materials. None of the authors declare any competing interests, as defined by PLoS One.

Rapidly growing resistance of pathogenic bacteria to conventional antibiotics leads to inefficiency of traditional approaches of countering infections and determines the urgent need for a search of fundamentally new anti-infective drugs. Antimicrobial peptides (AMPs) of the innate immune system are promising candidates for a role of such novel antibiotics. However, some cytotoxicity of AMPs toward host cells limits their active implementation in medicine and forces attempts to design numerous structural analogs of the peptides with optimized properties. An alternative route for the successful AMPs introduction may be their usage in combination with conventional antibiotics. Synergistic antibacterial effects have been reported for a number of such combinations, however, the molecular mechanisms of the synergy remain poorly understood and little is known whether AMPs cytotoxicy for the host cells increases upon their application with antibiotics. Our study is directed to examination of a combined action of natural AMPs with different structure and mode of action (porcine protegrin 1, caprine bactenecin ChBac3.4, human alpha- and beta-defensins (HNP-1, HNP-4, hBD-2, hBD-3), human cathelicidin LL-37), and egg white lysozyme with varied antibiotic agents (gentamicin, ofloxacin, oxacillin, rifampicin, polymyxin B, silver nanoparticles) toward selected bacteria, including drug-sensitive and drug-resistant strains, as well as toward some mammalian cells (human erythrocytes, PBMC, neutrophils, murine peritoneal macrophages and Ehrlich ascites carcinoma cells). Using "checkerboard titrations" for fractional inhibitory concentration indexes evaluation, it was found that synergy in antibacterial action mainly occurs between highly membrane-active AMPs (e.g., protegrin 1, hBD-3) and antibiotics with intracellular targets (e.g., gentamicin, rifampcin), suggesting bioavailability increase as the main model of such interaction. In some combinations modulation of dynamics of AMP-bacterial membrane interaction in presence of the antibiotic was also shown. Cytotoxic effects of the same combinations toward normal eukaryotic cells were rarely synergistic. The obtained data approve that combined application of antimicrobial peptides with antibiotics or other antimicrobials is a promising strategy for further development of new approach for combating antibiotic-resistant bacteria by usage of AMP-based therapeutics. Revealing the conventional antibiotics that increase the activity of human endogenous AMPs against particular pathogens is also important for cure strategies elaboration.

The increasing emergence of multidrug-resistant microorganisms represents one of the greatest challenges in the clinical management of infectious diseases, and requires the development of novel antimicrobial agents. To this aim, we de novo designed a library of Arg-rich ultra-short cationic antimicrobial lipopeptides (USCLs), based on the Arg-X-Trp-Arg-NH2 peptide moiety conjugated with a fatty acid, and investigated their antibacterial potential. USCLs exhibited an excellent antimicrobial activity against clinically pathogenic microorganisms, in particular Gram-positive bacteria, including multidrug resistant strains, with MIC values ranging between 1.56 and 6.25 μg/mL. The capability of the two most active molecules, Lau-RIWR-NH2 and Lau-RRIWRR-NH2, to interact with the bacterial membranes has been predicted by molecular dynamics and verified on liposomes by surface plasmon resonance. Both compounds inhibited the growth of S. aureus even at sub MIC concentrations and induced cell membranes permeabilization by producing visible cell surface alterations leading to a significant decrease in bacterial viability. Interestingly, no cytotoxic effects were evidenced for these lipopeptides up to 50-100 μg/mL in hemolysis assay, in human epidermal model and HaCaT cells, thus highlighting a good cell selectivity. These results, together with the simple composition of USCLs, make them promising lead compounds as new antimicrobials., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: A. Romani is co-founder and CEO of ArtaPeptidion srls, but the study was in no way funded by this company, which had no influence on the study design or the content of this manuscript. I confirm that this commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. A. Romani, M.Scocchi and M. Benincasa are co-inventors of the Ultra-Short Cationic Lipopeptides described in this manuscript (European patent application No. 17425081.1 -1453; Title: Short and ultra-short antimicrobial lipopeptides and use thereof). I confirm that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Rosacea is a chronic inflammatory cutaneous disease characterized by immune system anomalies and vascular hyperreactivity. Recently, therapy of rosacea has improved substantially with the approval of Tranexamic acid (TXA), an antifibrinolytic agent. However, we know little about the underlying mechanism. In this study, we evaluated the effects of TXA and its molecular mechanism on rosacea by using LL37-induced mouse model and HaCaT cell model. Rosacea-like symptoms including skin erythema and histopathological alterations, as well as the elevated pro-inflammatory cytokines (IL-6 and TNFα) and MMP9 expression were significantly ameliorated by TXA treatment. In addition, TXA reduced the expression levels of innate immune gene (TLR2, KLK5 and Camp) and neutrophils relative gene in rosacea-like lesion. For adaptive immune, CD4 + T cell infiltration and the gene expression of Th cytokines and chemokines were regulated by TXA in skin lesion. Furthermore, the anti-inflammatory effects of TXA were associated with the inhibition of TLR2, pro-inflammatory cytokines (IL-6 and TNFα) and chemokines (CCL10) expression in LL37-activated HaCaT cells. Finally, TXA repressed the angiogenesis by reducing the number of CD31 + cell and downregulating the expression levels of VEGF in rosacea. In conclusion, our finding defines a treatment mechanism by which TXA ameliorates rosacea symptoms by regulating the immune response and angiogenesis., (Copyright © 2018. Published by Elsevier B.V.)

In recent years, antimicrobial peptides have received increased interest and are potential substitutes for antibiotics. However, natural antimicrobial peptides are always toxic to mammalian cells and usually exhibit weak antibacterial activity, which restrict their wide application. In this study, a novel antibacterial peptide named PEW300 was designed with three mutations to the parental peptide cecropin A. As predicted by bioinformatic programs, the positive charge of PEW300 increased from + 6 to + 9 compared with cecropin A, and the grand average of hydropathicity increased from - 0.084 to - 0.008. Expression of PEW300 resulted in serious inhibition of Escherichia coli BL21(DE3) cells, indicating designed PEW300 may have stronger antibacterial activity. A simple, fast, and low-cost approach without tedious protein purification steps was selected for the efficient production of PEW300 by fusion with ELK16 and about 7.38 μg/mg wet cell weight PEW300 was eventually obtained. Purified PEW300 exhibited strong antibacterial activity against various Gram-positive and Gram-negative bacteria which was enhanced four- to sevenfold compared with the parental peptide cecropin A. Besides, PEW300 had no hemolytic activity toward mammalian cells even at high concentration (224 ng/μl). PEW300 showed good stability in neutral and alkaline solutions. Moreover, PEW300 was thermally stable even at up to 100 °C and resistant to proteinase K, pepsin, snailase, and trypsin. The incubation with human serum had no effect on the antibacterial activity of PEW300. All these results demonstrated that PEW300 designed in this work may have good potential as a candidate pharmaceutical agent.

Considering the worldwide increasing prevalence of resistance to traditional antibiotics, it is necessary to find new antibiotics to deal with this issue. Recently, antimicrobial peptides (AMPs) have been proposed as new antimicrobial agents. Aureins are a family of AMPs that are isolated from Green and Golden Bell Frogs. These peptides have a favorable antibacterial activity against Gram-positive bacteria. We designed two peptides derived from natural Aurein enjoying alignment-based design method. After synthesis of the peptides, their secondary structure was checked by circular dichroism. Consequently, the antibacterial effects of these peptides were investigated by determining the minimum inhibitory concentration (MIC) and bactericidal concentration. Eventually, the toxicity of these peptides was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay on normal human skin cells (Hu02 cell line). Natural Aurein1.2 was used as a natural control to compare the properties in all stages. The results indicated that these new peptides had medium-upward antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis (MIC of 8-64 μg/mL) and weak bactericidal activity against Staphylococcus aureus (MIC of 128-256 μg/mL). Also, MTT assays results showed that AureinN2 is less toxic than AureinN1 and Aurein1.2. Toxicity of AureinN2 for Hu02 cell lines was between 20 and 40% at the concentration of 8-500 μg/mL. In this study, we were able to improve antimicrobial activity of two synthetic derivatives of the Aurein family against Gram-negative bacteria by using machine-learning algorithm and other in silico methods., (© 2018 Wiley Periodicals, Inc.)

Antimicrobial peptides (AMPs) are regarded as next-generation antibiotics to replace conventional antibiotics due to their rapid and broad-spectrum antimicrobial properties and far less sensitivity to the development of pathogen resistance. However, they are susceptible to proteolysis in vivo by endogenous or bacterial proteases as well as induce the lysis of red blood cells, which prevent their intravenous applications. In this work, polyion complex (PIC) micelles of the cationic AMP MSI-78 and the anionic copolymer methoxy poly(ethylene glycol)-b-poly(α-glutamic acid) (mPEG-b-PGlu) were prepared to develop novel antimicrobial agents for potential application in vivo. With an increase in molar ratio of mPEG-b-PGlu to MSI-78, the complexation ability of the PIC micelles increased. FITC-labeled MSI-78 showed a sustained release from the PIC micelles. More importantly, these PIC micelles greatly decreased the hemolytic toxicity of MSI-78 to human red blood cells, without influencing its antimicrobial activity. Thus, this approach could be used as a suitable in vivo delivery method of AMPs in the future., (Copyright © 2018. Published by Elsevier B.V.)

The biological activities and the structural arrangement of adevonin, a novel antimicrobial peptide, were investigated. The trypsin inhibitor ApTI, isolated from Adenanthera pavonina seeds, was used as a template for screening 18-amino acid peptides with predicted antimicrobial activity. Adevonin presented antimicrobial activity and minimum inhibitory concentrations (MIC) ranging from 1.86 to 7.35 µM against both Gram-positive and - negative bacterial strains. Moreover, adevonin exerted time-kill effects within 10 min and both susceptible and drug-resistant bacterial strains were affected by the peptide. In vitro and in vivo assays showed that, at MIC concentration, adevonin did not affect human fibroblasts (MRC-5) viability or Galleria mellonella survival, respectively. Hemolytic activity was observed only at high peptide concentrations. Additionally, nucleic acid efflux assays, gentian violet uptake and time-kill kinetics indicate that the antimicrobial activity of adevonin may be mediated by bacterial membrane damage. Furthermore, molecular dynamic simulation in the presence of SDS micelles and anionic membrane bilayers showed that adevonin acquired a stable α-helix secondary structure. Further studies are encouraged to better understand the mechanism of action of adevonin, as well as to investigate the anti-infective activity of this peptide.

Over the last decade, global increase in antibiotic consumption is a major concern in the word. Antimicrobial peptides (AMPs) known as potential alternative and were considered as a safe antimicrobial agent. However, current approaches for production and purification of AMPs are costly and time-consuming. Here we show that heterologous expression of a chimeric peptide was successfully developed in Lactococcus lactis as a safe and cost-effective recombinant protein expression platform. Minimum inhibitory concentrations (MICs) of His-tag purified peptide was determined against a broad spectrum of human pathogenic bacteria consistence of Gram-positive, Gram-negative and resistance strains in deferent range from 7.24 ± 0.4 to 156.24 ± 3.0 μg/mL. Furthermore, our results showed that the peptide was not toxic to HEK and HeLa cells and even at concentrations as high as 250 μg/mL exhibited minimal hemolysis against RBCs. Additional characteristics such as thermal, protease and 50% human plasma stability were determined for cLFchimera. Molecular modeling analysis demonstrated that fusion of His-tag to the C-terminal of chimeric peptide increased peptide stability during 10 ns simulation in water. Overall, the chimeric peptide has a considerable antibacterial activity with low hemolysis, low or none in toxicity and good temperature resistance and also high stability in serum. We anticipate the established expression system could be developed and used more effectively in probiotic strains in future studies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacterial colonization and biofilm formation is a growing challenge in the biomedical field. Although nanotechnology has emerged as an alternative strategy to combat biofilm formation, the toxicity of nanomaterials is a major concern. In this study, we report a safe-by-design strategy for the synthesis of a poly-cationic peptide functionalized graphene-silver nanocomposite (designated as GAPP) and its enhanced biofilm inhibition and disruption properties to eliminate the biofilm development of Gram-negative bacteria. The graphene-silver (rGOAg) nanocomposite was synthesized by microwave reduction, and subsequently functionalized with an antimicrobial poly-cationic peptide through covalent bonding. The results demonstrated that GAPP effectively killed the planktonic cells and biofilms of Escherichia coli and Pseudomonas aeruginosa depending upon the concentration and duration of the interaction. The complete eradication of preformed biofilm was achieved when treated with 10 μg mL-1 of GAPP for 5 h. The GAPP exerted bactericidal and biofilm inhibition activity through a "contact-kill-release" mode of action, wherein the electrostatic interaction of GAPP with the bacterial cells induced physical disruption accompanied by ROS-mediated biochemical changes. The internalization of GAPP into the cytoplasm through the damaged membrane led to metabolic imbalance in the cells. The peptide functionalization further prevented the dissolution of Ag+ ions, thus minimizing the cytotoxicity of GAPP to adult zebrafish. More importantly, the poly-cationic peptide functionalization enhanced the bioavailability, biofilm inhibition and disruption activities of GAPP, while minimizing its toxicological impact. The results obtained thereby provide an effective strategy in the design of alternative antibacterial agents for fighting biofilms of Gram-negative bacteria.

In recent years, the overuse of antibiotics has become very serious. Many pathogenic bacteria have become resistant to them, with serious potential health consequences. Thus, it is urgent that we develop new antibiotic drugs. Antimicrobial peptides (AMPs) are important endogenous antibacterial molecules that contribute to immunity. Most have spectral antibacterial properties and do not confer drug resistance. In this paper, an 11-residue peptide (LFcinB18⁻28) with a sequence of KCRRWQWRMKK was modified by amino acid substitution to form a symmetrical amino acid sequence. The antibacterial activities and mechanisms of action of engineered peptides including KW-WK (KWRRWQWRRWK), FP-PF (FPRRWQWRRPF), FW-WF (FWRRWQWRRWF), and KK-KK (KKRRWQWRRKK) were investigated. The four engineered peptides could more effectively inhibit bacteria than the original peptide, LFcinB18⁻28. This suggested that a symmetrical amino acid sequence might enhance the antibacterial activity of AMPs. However, only peptides KW-WK, FP-PF, and KK-KK were safe; FW-WF displayed hemolytic activity. The engineered peptides shared cationic and amphipathic characteristics that facilitated interactions with the anionic microbial membranes, leading to disruption of membrane integrity and permeabilizing microbial membranes, resulting in cell death. Therefore, a symmetrical amino acid sequence and related structural parameters offer an alternative approach to the design of AMPs. This will provide a scientific basis for the design and synthesis of new AMPs.

Novicidin (NVC), is a membrane-penetrating peptide, which forms a stable complex with Zn-Schiff base with interesting antitumor selectivity. We studied NVC derivatives to determine functional roles of key amino acids in toxicity, helicity, and binding of the Zn-Schiff base complex. Trimmed derivatives highlighted the role of peptide length and helicity in toxicity and membrane penetration. The removal of Lys from position 1 and 2 strongly increases the ability to disrupt the membranes. The trimming of the N-terminal residues significantly increases the stability of peptide helicity enhancing penetrating properties. Gly residue derivatives undermined a role of peptide bending in membrane penetration and toxicity. After the substitution of the central Gly derivatives with Ile or Lys, the peptides retained toxicity. These results illustrate the minor role of central helix bending in NVC toxicity. Binding-site-peptide derivatives identified His residue as the sole Zn-Schiff base binding site and eliminated the role of other aromatic residues.

The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca 2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mycobacterium massiliense is a rapid growing, multidrug-resistant, non-tuberculous mycobacteria that is responsible for a wide spectrum of skin and soft tissue infections, as well as other organs, such as the lungs. Antimicrobial peptides had been described as broad-spectrum antimicrobial, chemotactic, and immunomodulator molecules. In this study we evaluated an antimicrobial peptide derived from scorpion Tityus obscurus as an anti-mycobacterial agent in vitro and in vivo. Bioinformatics analyses demonstrated that the peptide ToAP2 have a conserved region similar to several membrane proteins, as well as mouse cathelicidin. ToAP2 inhibited the growth of four M. massiliense strains (GO01, GO06, GO08, and CRM0020) at a minimal bactericidal concentration (MBC) of 200 µM. MBC concentration used to treat infected macrophages was able to inhibit 50% of the bacterial growth of all strains. ToAP2 treatment of infected mice with bacilli reduced the bacterial load in the liver, lung, and spleen, similarly to clarithromycin levels (90%). ToAP2 alone recruited monocytes (F4/80 low Gr1), neutrophils (F4/80 - Gr1), and eosinophils (F4/80+ Gr1+). ToAP2, together with M. massiliense infection, was able to increase F4/80 low and reduce the percentage of F4/80 high macrophages when compared with infected and untreated mice. ToAP2 has in vitro anti-microbial activity that is improved in vivo due to chemotactic activity.

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)