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The negative membrane potential within bacterial cells is crucial in various essential cellular processes. Sustaining a hyperpolarised membrane could offer a novel strategy to combat antimicrobial resistance. However, it remains uncertain which molecules are responsible for inducing hyperpolarization and what the underlying molecular mechanisms are. Here, we demonstrate that chemically diverse antimicrobial peptides (AMPs) trigger hyperpolarization of the bacterial cytosolic membrane when applied at subinhibitory concentrations. Specifically, these AMPs adopt a membrane-induced amphipathic structure and, thereby, generate hyperpolarization in Escherichia coli without damaging the cell membrane. These AMPs act as selective ionophores for K + (over Na + ) or Cl - (over H 2 PO 4 - and NO 3 - ) ions, generating diffusion potential across the membrane. At lower dosages of AMPs, a quasi-steady-state membrane polarisation value is achieved. Our findings highlight the potential of AMPs as a valuable tool for chemically hyperpolarising bacteria, with implications for antimicrobial research and bacterial electrophysiology., (© 2024. The Author(s).)

Many factors contribute to bacterial membrane stabilization, including steric effects between lipids, membrane spontaneous curvature, and the difference in the number of neighboring molecules. This forum provides an overview of the physicochemical properties associated with membrane curvature and how this parameter can be tuned to design more effective antimicrobial peptides., Competing Interests: Declaration of interests C.F-N. provides consulting services to Invaio Sciences and is a member of the Scientific Advisory Boards of Nowture S.L., Peptidus, and Phare Bio. C.F-N. is also on the Advisory Board of the Peptide Drug Hunting Consortium (PDHC). The de la Fuente Lab has received research funding or in-kind donations from United Therapeutics, Strata Manufacturing PJSC, and Procter & Gamble, none of which were used in support of this work. C.F-N. is on the Advisory Board of Cell Reports Physical Science., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Temporins are a family of antimicrobial peptides (AMPs) isolated from frog skin, which are very short, weakly charged, and highly hydrophobic. They execute bactericidal activities in different ways from many other AMPs. This work investigated morphological changes of planar bilayer membranes composed of mixed zwitterionic and anionic phospholipids induced by temporin B and L (TB and TL) using all-atom and coarse-grained molecular dynamics simulations. We found that TB and TL fold to α-helices at the membrane surface and penetrate shallowly into the bilayer. These short AMPs have low propensity to induce membrane pore formation but possess high ability to extract lipids out. At relatively high peptide concentrations, the strong hydrophobicity of TB and TL promotes them to aggregate into clusters on the membrane surface. These aggregates attract a large amount of lipids out of the membrane to release compression induced by other dispersed peptides binding to the membrane. The extruded lipids mix evenly with the peptides in the cluster and form tubule-like protrusions. Certain water molecules follow the movement of lipids, which not only fill the cavities of the protrusion but also assist in maintaining the tubular structures. In contrast, the peptide-free leaflet remains intact. The present results unravel distinctive antimicrobial mechanisms of temporins disturbing membranes.

In the emerging era of antimicrobial resistance, the susceptibility to co-infections of patients suffering from either acquired or inherited hemolytic disorders can lead to dramatic increase in mortality rates. Closely related, heme liberated during hemolysis is one of the major sources of iron, which is vital for both host and invading microorganisms. While recent intensive research in the field has demonstrated that heme exerts diverse local effects including impairment of immune cells functions, it is almost completely unknown how it may compromise key molecules of our innate immune system, such as antimicrobial host defense peptides (HDPs). Since HDPs hold great promise as natural therapeutic agents against antibiotic-resistant microbes, understanding the effects that may modulate their action in microbial infection is crucial. Here we explore how hemin can interact directly with selected HDPs and influence their structure and membrane activity. It is revealed that induced helical folding, large assembly formation, and altered membrane activity is promoted by hemin. However, these effects showed variations depending mainly on peptide selectivity toward charged lipids, and the affinity of the peptide and hemin to lipid bilayers. Hemin-peptide complexes are sought to form semi-folded co-assemblies, which are present even with model membranes resembling mammalian or bacterial lipid compositions. In vitro cell-based toxicity assays supported that toxic effects of HDPs could be attenuated due to their assembly formation. These results are in line with our previous findings on peptide-lipid-small molecule systems suggesting that small molecules present in the complex in vivo milieu can regulate HDP function. Inversely, diverse effects of endogenous compounds could also be manipulated by HDPs., (© 2021. The Author(s).)

PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG). Although both PMAP-PA and PMAP-PG led to effective membrane depolarization and permeabilization, they showed less antimicrobial activity than wild-type PMAP-23. Interestingly, we observed that PMAP-23 crossed lipid bilayers much more efficiently than its Pro-substituted derivatives. The fact that the Gly-induced hinge was unable to replace the PXXP motif in PMAP-23 translocation suggests that the PXXP motif has unique structural properties other than the central hinge. Surface plasmon resonance sensorgrams showed that the running buffer almost entirely dissociated PMAP-23 from the membrane surface, while its Pro-substituted derivatives remained significantly bound to the membrane. In addition, kinetic analysis of the sensorgrams revealed that the central PXXP motif allows PMAP-23 to rapidly translocate at the interface between the hydrophilic and hydrophobic phases. Taken together, we propose that the structural and kinetic understanding of the PXXP motif in peptide translocation could greatly aid the development of novel antimicrobial peptides with intracellular targets by promoting peptide entry into bacterial cells.

Peptides with a combination of high positive charge and high hydrophobicity have high antimicrobial activity, as epitomized by peptide venoms, which are designed by nature as disruptors of host membranes yet also display significant efficacy against pathogens. To investigate this phenomenon systematically, here we focus on ponericin W1, a peptide venom isolated from Pachycondyla goeldii ants (WLGSALKIGAKLLPSVVGLFKKKKQ) to examine whether Lys positioning can be broadly applied to optimize the functional range of existing natural sequences. We prepared sets of ponericin W1 analogues, where Lys residues were either distributed in an amphipathic manner throughout the sequence (PonAmp), clustered at the N-terminus (PonN), or clustered at the C-terminus (PonC), along with their counterparts of reduced hydrophobicity through 2-4 Leu-to-Ala replacements. We found that wild-type ponericin W1 and all three variants displayed toxicity against human erythrocytes, but hemolysis was eliminated by the replacement of two or more Leu residues by Ala residues. As well, peptides containing up to 3 Leu-to-Ala replacements retained antimicrobial activity against E. coli bacteria. Biophysical analyses of peptide-membrane interaction patterns by circular dichroism spectroscopy revealed a novel mode of cluster-dependent peptide positioning vis-à-vis the water-membrane interface, where PonAmp and PonC peptides displayed full or partial helical structures, while PonN peptides were unstructured, likely due, in part, to dynamic interchange between aqueous and membrane surface environments. The overall findings suggest that the lower membrane penetration of N-terminal charge-clustered constructs coupled with moderate sequence hydrophobicity may be advantageous for conferring enhanced target selectivity for bacterial versus mammalian membranes.

Anticancer peptides (ACPs) could potentially offer many advantages over other cancer therapies. ACPs often target cell membranes, where their surface mechanism is coupled to a conformational change into helical structures. However, details on their binding are still unclear, which would be crucial to reach progress in connecting structural aspects to ACP action and to therapeutic developments. Here we investigated natural helical ACPs, Lasioglossin LL-III, Macropin 1, Temporin-La, FK-16, and LL-37, on model liposomes, and also on extracellular vesicles (EVs), with an outer leaflet composition similar to cancer cells. The combined simulations and experiments identified three distinct binding modes to the membranes. Firstly, a highly helical structure, lying mainly on the membrane surface; secondly, a similar, yet only partially helical structure with disordered regions; and thirdly, a helical monomeric form with a non-inserted perpendicular orientation relative to the membrane surface. The latter allows large swings of the helix while the N-terminal is anchored to the headgroup region. These results indicate that subtle differences in sequence and charge can result in altered binding modes. The first two modes could be part of the well-known carpet model mechanism, whereas the newly identified third mode could be an intermediate state, existing prior to membrane insertion.

The global increase in cancer mortality and economic losses necessitates the cautious quest for therapeutic agents with compensatory advantages over conventional therapies. Anticancer peptides (ACPs) are a subset of host defense peptides, also known as antimicrobial peptides, which have emerged as therapeutic and diagnostic candidates due to several compensatory advantages over the non-specificity of the current treatment regimens. This review aimed to highlight the ravaging incidence of cancer, the use of ACPs in cancer treatment with their mechanisms, ACP discovery and delivery methods, and the limitations for their use. This would create awareness for identifying more ACPs with better specificity, accuracy and sensitivity towards the disease. It would also promote their efficacious utilization in biotechnology, medical sciences and molecular biology to ease the severity of the disease and enable the patients living with these conditions to develop an accommodating lifestyle.

The human cathelicidin LL-37 is a multifunctional peptide of the human innate immune system. Among the various functions of LL-37, its antimicrobial activity is important in controlling the microorganisms of the human body. The target molecules of LL-37 in bacteria include membrane lipids, lipopolysaccharides (LPS), lipoteichoic acid (LTA), proteins, DNA and RNA. In this mini-review, we summarize the entity of LL-37 structural data determined over the last 15 years and specifically discuss features implicated in the interactions with lipid-like molecules. For this purpose, we discuss partial and full-length structures of LL-37 determined in the presence of membrane-mimicking detergents. This constantly growing structural database is now composed of monomers, dimers, tetramers, and fiber-like structures. The diversity of these structures underlines an unexpected plasticity and highlights the conformational and oligomeric adaptability of LL-37 necessary to target different molecular scaffolds. The recent co-crystal structures of LL-37 in complex with detergents are particularly useful to understand how these molecules mimic lipids and LPS to induce oligomerization and fibrillation. Defined detergent binding sites provide deep insights into a new class of peptide scaffolds, widening our view on the fascinating world of the LL-37 structural factotum. Together, the new structures in their evolutionary context allow for the assignment of functionally conserved residues in oligomerization and target interactions. Conserved phenylalanine and arginine residues primarily mediate those interactions with lipids and LPS. The interactions with macromolecules such as proteins or DNA remain largely unexplored and open a field for future studies aimed at structures of LL-37 complexes. These complexes will then allow for the structure-based rational design of LL-37-derived peptides with improved antibiotic properties.

Peptide stapling chemistry represents an attractive strategy to promote the clinical translation of protein epitope mimetics, but its use has not been applied to natural cytotoxic peptides (NCPs) to produce new oncolytic peptides. Based on a wasp venom peptide, a series of stapled anoplin peptides (StAnos) were prepared. The optimized stapled Ano-3/3s were shown to be protease-resistant and exerted superior tumor cell-selective cytotoxicity by rapid membrane disruption. In addition, Ano-3/3s induced tumor ablation in mice through the direct oncolytic effect and subsequent stimulation of immunogenic cell death. This synergistic oncolytic-immunotherapy effect is more remarkable on melanoma than on triple-negative breast cancer in vivo . The efficacies exerted by Ano-3/3s on melanoma were further characterized by CD8+ T cell infiltration, and the addition of anti-CD8 antibodies diminished the long-term antitumor effects. In summary, these results support stapled peptide chemistry as an advantageous method to enhance the NCP potency for oncolytic therapy.

Cationic antimicrobial peptides have been investigated for their potential use in combating infections by targeting the cell membrane of microbes. Their unique chemical structure has been investigated to understand their mode of action and optimize their dose-response by rationale design. One common feature among cationic AMPs is an amidated C-terminus that provides greater stability against in vivo degradation. This chemical modification also likely modulates the interaction with the cell membrane of bacteria yet few studies have been performed comparing the effect of the capping groups. We used maculatin 1.1 (Mac1) to assess the role of the capping groups in modulating the peptide bacterial efficiency, stability and interactions with lipid membranes. Circular dichroism results showed that C-terminus amidation maintains the structural stability of the peptide (α-helix) in contact with micelles. Dye leakage experiments revealed that amidation of the C-terminus resulted in higher membrane disruptive ability while bacteria and cell viability assays revealed that the amidated form displayed higher antibacterial ability and cytotoxicity compared to the acidic form of Mac1. Furthermore, 31 P and 2 H solid-state NMR showed that C-terminus amidation played a greater role in disturbance of the phospholipid headgroup but had little effect on the lipid tails. This study paves the way to better understand how membrane-active AMPs act in live bacteria.

The membrane interaction and damage caused by C-terminally amidated esculentin-2 peptides identified from the frog skin is illustrated in the present study using Staphylococcus aureus and Vibrio cholerae . Double staining with fluorescent probes SYTOX and DAPI proved the concentration-dependent bacterial membrane damage induced by the peptides. It was found that the sub-MIC of both peptides induced transient pores on the bacterial membrane. These peptides also caused depolarisation on the bacterial membrane during their interaction. The physical changes on bacterial cells like blebbing, elongation, fusion, and so forth upon peptide treatment were visualized through SEM images. The antimicrobial activity of the peptides against S. aureus and V. cholerae was not altered at physiological concentrations of divalent and monovalent cations, which is advantageous in a therapeutic context. The increase of MIC against V. cholerae at higher concentrations of Mg 2+ and Ca 2+ (>5 µM) is due to the concentration-dependent antagonism exhibited by these ions for the cation binding sites on the bacterial membrane, which facilitates the process of 'self-promoted uptake.' The study emphasizes to utilize the ability of these peptides to produce transient pores at sub-MICs in combinatorial therapy.

Antimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling., (Copyright © 2021. Published by Elsevier Inc.)

Multidrug resistant (MDR) bacteria have adapted to most clinical antibiotics and are a growing threat to human health. One promising type of candidates for the everlasting demand of new antibiotic compounds constitute antimicrobial peptides (AMPs). These peptides act against different types of microbes by permeabilizing pathogen cell membranes, whereas being harmless to mammalian cells. Contrarily, another class of membrane-active peptides, namely cell-penetrating peptides (CPPs), is known to translocate in eukaryotic cells without substantially affecting the cell membrane. Since CPPs and AMPs share several physicochemical characteristics, we hypothesized if we can rationally direct the activity of a CPP towards antimicrobial activity. Herein, we describe the screening of a synthetic library, based on the CPP sC18, including structure-based design to identify the active residues within a CPP sequence and to discover novel AMPs with high activity. Peptides with increased hydrophobicity were tested against various bacterial strains, and hits were further optimized leading to four generations of peptides, with the last also comprising fluorinated amino acid building blocks. Interestingly, beside strong antibacterial activities, we also detected activity in cancer cells, while non-cancerous cells remained unharmed. The results highlight our new candidates, particularly those from generation 4, as a valuable and promising source for the development of future therapeutics with antibacterial activity and beyond., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Anticancer chemo- and targeted therapies are limited in some cases due to strong side effects and/or drug resistance. Peptides have received renascent interest as anticancer therapeutics and are currently being considered as alternatives and/or as complementary to biologics and small-molecule drugs. Gomesin, a disulfide-rich host defense peptide expressed in the Brazilian spider Acanthoscurria gomesiana selectively targets and disrupts cancer cell membranes. In the current study, we employed a range of biophysical methodologies with model membranes and bioassays to investigate the use of a cyclic analogue of gomesin as a drug scaffold to internalize cancer cells. We found that cyclic gomesin can internalize cancer cells via endocytosis and direct membrane permeation. In addition, we designed an improved non-disruptive and non-toxic cyclic gomesin analogue by incorporating D-amino acids within the scaffold. This improved analogue retained the ability to enter cancer cells and can be used as a scaffold to deliver drugs. Efforts to investigate the internalization mechanism used by host defense peptides, and to improve their stability, potency, selectivity and ability to permeate cancer cell membranes will increase the opportunities to repurpose peptides as templates for designing alternative anticancer therapeutic leads., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human cathelicidin LL-37 has recently attracted interest as a potential therapeutic agent, mostly because of its ability to kill a wide variety of pathogens and cancer cells. In this study, we used molecular dynamics simulation aimed to get insights that help to correlate with the antibacterial activity of previously designed LL-37 anticancer derivative (i.e. GF-17). Two independent molecular dynamics simulation involving four units of GF-17 peptide in the mixture (9:1) of 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and the pure DPPG lipids were performed. Various properties of membranes such as mass density distributions, area per lipid, bilayer thickness, and lateral diffusion were examined in both systems. The results showed that the thickness of the bilayer was not affected by the presence of GF-17, while the area per lipid and lateral diffusion of lipids showed an increase. Moreover, the potential of the mean force (PMF) method was used to calculate the free energy profile for transferring GF-17 from the bulk water into both kinds of membranes. It revealed that penetration of GF-17 into the DPPG membrane was more favorable than the DPPE/DPPG membrane, and there was no energy barrier for crossing through the bilayer center. Investigation of the radius of gyration (Rg) and root mean square fluctuation (RMSF) of peptides in two membranes showed that GF-17 had more compactness and rigidity in the pure DPPG system. By examining the secondary structure of GF-17 peptide, it was seen that the α-helix, and coil structures in both DPPE/DPPG and pure DPPG membranes are dominant.

Antimicrobial peptides are small molecules that display antimicrobial activity against a wide range of pathogens. In a previous work, by using model membranes we studied P6, a peptide that shows no antimicrobial activity, and P6.2, which exhibits antibacterial activity. In the present work we aimed to unravel the mode of action of these peptides by studying their interaction in vivo with Escherichia coli and Staphylococcus aureus. In this sense, to study the interactions with bacterial cells and their effect on the bacterial surface, zeta potential, spectroscopic, and microscopic methodologies were applied. P6.2 exhibits a higher affinity toward both bacterial envelopes. The ability of both peptides to disrupt afterwards the bacterial membrane was also studied. Both peptides were able to induce bacterial membrane damage, but higher concentrations of P6 were needed to obtain results comparable to those obtained for P6.2. Additionally, P6.2 exhibited faster damage kinetics. Altogether, these data allow postulating, in a physiologic model, that the lower affinity of P6 for bacterial envelope results in a minor final concentration of the peptide in the bacterial membrane unable to trigger the antimicrobial activity. Finally, the fact that the active P6.2 has the same MIC value for the Gram-positive and Gram-negative bacteria tested, but not the same profile in the permeabilization assays, reinforces the question of whether cell wall components act as electrostatic barriers preventing or minimizing membrane-active AMPs lethal action at the membrane level., 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 human cathelicidin LL-37 serves a critical role in the innate immune system defending bacterial infections. LL-37 can interact with molecules of the cell wall and perforate cytoplasmic membranes resulting in bacterial cell death. To test the interactions of LL-37 and bacterial cell wall components we crystallized LL-37 in the presence of detergents and obtained the structure of a narrow tetrameric channel with a strongly charged core. The formation of a tetramer was further studied by cross-linking in the presence of detergents and lipids. Using planar lipid membranes a small but defined conductivity of this channel could be demonstrated. Molecular dynamic simulations underline the stability of this channel in membranes and demonstrate pathways for the passage of water molecules. Time lapse studies of E. coli cells treated with LL-37 show membrane discontinuities in the outer membrane followed by cell wall damage and cell death. Collectively, our results open a venue to the understanding of a novel AMP killing mechanism and allows the rational design of LL-37 derivatives with enhanced bactericidal activity.

Background: The increase in bacterial resistance phenotype cases is a global health problem. New strategies must be explored by the scientific community in order to create new treatment alternatives. Animal venoms are a good source for antimicrobial peptides (AMPs), which are excellent candidates for new antimicrobial drug development. Cathelicidin-related antimicrobial peptides (CRAMPs) from snake venoms have been studied as a model for the design of new antimicrobial pharmaceuticals against bacterial infections., Results: In this study we present an 11 amino acid-long peptide, named pseudonajide, which is derived from a Pseudonaja textilis venom peptide and has antimicrobial and antibiofilm activity against Staphylococcus epidermidis. Pseudonajide was selected based on the sequence alignments of various snake venom peptides that displayed activity against bacteria. Antibiofilm activity assays with pseudonajide concentrations ranging from 3.12 to 100 μM showed that the lowest concentration to inhibit biofilm formation was 25 μM. Microscopy analysis demonstrated that pseudonajide interacts with the bacterial cell envelope, disrupting the cell walls and membranes, leading to morphological defects in prokaryotes., Conclusions: Our results suggest that pseudonajide's positives charges interact with negatively charged cell wall components of S. epidermidis, leading to cell damage and inhibiting biofilm formation.

The search for novel compounds to combat multi-resistant bacterial infections includes exploring the potency of antimicrobial peptides and derivatives thereof. Complementary to high-throughput screening techniques, biophysical and biochemical studies of the biological activity of these compounds enable deep insight, which can be exploited in designing antimicrobial peptides with improved efficacy. This approach requires the combination of several techniques to study the effect of such peptides on both bacterial cells and simple mimics of their cell envelope, such as lipid-only vesicles. These efforts carry the challenge of bridging results across techniques and sample systems, including the proper choice of membrane mimics. This review describes some important concepts toward the development of potent antimicrobial peptides and how they translate to frequently applied experimental techniques, along with an outline of the biophysics pertaining to the killing mechanism of antimicrobial peptides., 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.)

All antibiotics have to engage bacterial amphiphilic barriers such as the lipopolysaccharide-rich outer membrane or the phospholipid-based inner membrane in some manner, either by disrupting them outright and/or permeating them and thereby allow the antibiotic to get into bacteria. There is a growing class of cyclic antibiotics, many of which are of bacterial origin, that exhibit activity against Gram-negative bacteria, which constitute an urgent problem in human health. We examine a diverse collection of these cyclic antibiotics, both natural and synthetic, which include bactenecin, polymyxin B, octapeptin, capreomycin, and Kirshenbaum peptoids, in order to identify what they have in common when they interact with bacterial lipid membranes. We find that they virtually all have the ability to induce negative Gaussian curvature (NGC) in bacterial membranes, the type of curvature geometrically required for permeation mechanisms such as pore formation, blebbing, and budding. This is interesting since permeation of membranes is a function usually ascribed to antimicrobial peptides (AMPs) from innate immunity. As prototypical test cases of cyclic antibiotics, we analyzed amino acid sequences of bactenecin, polymyxin B, and capreomycin using our recently developed machine-learning classifier trained on α-helical AMP sequences. Although the original classifier was not trained on cyclic antibiotics, a modified classifier approach correctly predicted that bactenecin and polymyxin B have the ability to induce NGC in membranes, while capreomycin does not. Moreover, the classifier was able to recapitulate empirical structure-activity relationships from alanine scans in polymyxin B surprisingly well. These results suggest that there exists some common ground in the sequence design of hybrid cyclic antibiotics and linear 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. Published by Elsevier B.V.)

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.

Mixtures of Magainin 2 and PGLa are simulated with 94 nm-sized bilayers composed of phospholipids and lyso-phospholipids for 3 μs using coarse-grained force fields. Calculation of the bilayer bending modulus shows that bilayers become more flexible in the presence of lyso-lipids or peptides, in agreement with experiments. Starting with the initial configuration of peptides randomly distributed on the bilayer surface, peptides aggregate, insert to the bilayer, and form pores. Aggregated peptides do not retain side-by-side heterodimeric structure but instead show the anchoring between C-terminal groups of magainin 2 and PGLa, which allows the deeper insertion of PGLa into the bilayer. In particular, due to the anchoring of magainin 2 and PGLa, the deeply inserted PGLa pull magainin 2 into contact with the edge of the opposite leaflet of the bilayer, which stabilizes the pore. In addition to these biophysical insights, anionic unsaturated-phospholipid bilayers are also simulated to mimic bacterial cell membranes, showing less extent of PGLa insertion and no pore formation. These simulation findings indicate that these synergistic heterodimers have the anchoring structure rather than the side-by-side structure, which supports the experimental observations suggesting the deeper insertion of PGLa and pore formation via the anchoring between anionic C-terminus of magainin 2 and cationic C-terminus of PGLa., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Antimicrobial peptides are widely studied as an alternative to traditional antibiotics. However, they are difficult to develop, as multiple factors influence their potency and selectivity toward bacterial cells. In this paper, we investigate three simplified model peptides that bear crown ethers, and the effects of simple structural modifications (peptide length and crown ether ring size) on their secondary structures and their permeabilizing activity on living cells and model membranes made with egg yolk phosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol. Circular dichroism studies show that the peptide length and the crown ether ring size do influence the conformation, but no trend could be determined from the results. Permeabilization studies with model membranes and with red blood cells demonstrated that from 13 residues to 16 residues, there is a gradual increase in activity as the peptides get longer. However, the shortest tested analogs, with 12 residues, also exhibited an increase in activity caused by the removal of one amino acid that was bearing a crown ether. Permeabilization assays showed that larger ring size analogs showed higher hemolytic activities. Altogether, the results reported help design new and more selective antimicrobial peptides., 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.

Antimicrobial Peptides (AMPs) are host defense molecules that initiate microbial death by binding to the membrane. On membrane binding, AMPs undergo changes in conformation and aggregation state to enable killing action. Depending on the AMP and cell membrane characteristics, the nature of binding can be aggregating or non-aggregating, with high/low cooperativity, at single or multiple sites with high/low affinity leading to a unique killing action that needs to be studied individually. In the present study, a steady-state model that simulates AMP-membrane interaction was developed and was used to predict the mechanism of AMP binding. The predictions obtained from the model were validated with experimentally deciphered values available in literature. The model was further used to predict the mechanism for a set of designed AMPs with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family. Depending on the predicted mechanism, a unique half saturation constant and steepness of response (Hill coefficient) was obtained which was further validated with available data from literature. The model could reliably predict the mechanism, the half saturation constant and the Hill coefficient values. Further based on the analysis, it was observed that aggregation and oligomerization result in drastic killing action in a short range of peptide concentration owing to high Hill coefficient values. Mechanisms such as monomers binding at multiple sites with/without cooperativity result in antimicrobial activity at low half saturation constant though the killing action may not be steep. Thus, the methodology developed here can be used to develop hypothesis for studying AMP-membrane interaction mechanisms., 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 widespread abuse of antibiotics has led to the use of antimicrobial peptides (AMPs) as a replacement for the existing conventional therapeutic agents for combating microbial infections. The broad-spectrum activity and the resilient nature of AMPs has mainly aggrandized their utilization. Here, we report the design of non-toxic, non-hemolytic and salt tolerant undecapeptides (AMP21-24), derived by modification of a peptide P5 (NH2-LRWLRRLCONH 2 ) reported earlier by our group. Our results depict that the designed peptides show potency against several bacterial as well as fungal strains. Circular dichroism (CD) spectroscopy in combination with molecular dynamic (MD) simulations confirm that the peptides are unstructured. Intrinsic tryptophan fluorescence quenching as well as interaction studies using isothermal calorimetry (ITC) of these peptides in the presence of biological microbial membrane mimics establish the strong microbial membrane affinity of these AMPs. Membrane permeabilization assay and cytoplasmic membrane depolarization studies of Pseudomonas aeruginosa and Candida albicans in the presence of AMPs also hint towards the AMP-membrane interactions. Leakage of calcein dye from membrane mimic liposomes, live cell NMR and field emission scanning electron microscopy (FESEM) studies suggest that the AMPs may be primarily involved in membrane perturbation leading to release of intracellular substances resulting in subsequent microbial cell death. Confocal laser scanning microscopy (CLSM) shows localization of the peptides throughout the cell, indicating the possibility of secondary mode of actions. Electrostatic interactions seem to govern the preferential binding of the AMPs to the microbial membranes in comparison to the mammalian membranes as seen from the MD simulations., Competing Interests: Declaration of competing interest The authors declare that they do not have any conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The amphipathic α-helical antimicrobial peptide MSI-103 (aka KIA21) can form stable transmembrane pores when the bilayer takes on a positive spontaneous curvature, e.g. by the addition of lyso-lipids. Solid-state 31 P- and 15 N-NMR demonstrated an enrichment of lyso-lipids in these toroidal wormholes. Anionic lyso-lipids provided additional stabilization by electrostatic interactions with the cationic peptides. The remaining lipid matrix did not affect the nature of the pore, as peptides maintained the same orientation independent of lipid charge, and a change in membrane thickness did not considerably affect their tilt angle. Under optimized conditions (i.e. in the presence of lyso-lipids and appropriate bilayer thickness), stable and well-aligned pores could be obtained for solid-state 2 H-NMR analysis. These data revealed for the first time the complete 3D alignment of this representative amphiphilic peptide in fluid membranes, which is compatible with either monomeric helices as constituents, or left-handed supercoiled dimers as building blocks from which the overall toroidal wormhole is assembled.

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins., Competing Interests: AT, IK, TK, LS, ŠP, MH, RV No competing interests declared, (© 2020, Tuerkova et al.)

Cellular membranes are complex structures and simplified analogues in the form of model membranes or biomembranes are used as platforms to understand fundamental properties of the membrane itself as well as interactions with various biomolecules such as drugs, peptides and proteins. Model membranes at the air-liquid and solid-liquid interfaces can be studied using a range of complementary surface-sensitive techniques to give a detailed picture of both the structure and physicochemical properties of the membrane and its resulting interactions. In this review, we will present the main planar model membranes used in the field to date with a focus on monolayers at the air-liquid interface, supported lipid bilayers at the solid-liquid interface and advanced membrane models such as tethered and floating membranes. We will then briefly present the principles as well as the main type of information on molecular interactions at model membranes accessible using a Langmuir trough, quartz crystal microbalance with dissipation monitoring, ellipsometry, atomic force microscopy, Brewster angle microscopy, Infrared spectroscopy, and neutron and X-ray reflectometry. A consistent example for following biomolecular interactions at model membranes is used across many of the techniques in terms of the well-studied antimicrobial peptide Melittin. The overall objective is to establish an understanding of the information accessible from each technique, their respective advantages and limitations, and their complementarity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Solid-state 19 F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the 19 F-reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non-hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene-based dipeptide isostere, Val-Ψ[(Z)-CF=CH]-Gly, and inserted it in the sequence of two well-studied antimicrobial peptides: PGLa and (KIGAKI) 3 are representatives of an α-helix and a β-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for 19 F NMR structure analysis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Brevinin-1BYa is a 24-amino acid residue host-defense peptide, first isolated from skin secretions of the foothill yellow-legged frog Rana boylii. The peptide is of interest, as it shows broad-spectrum antimicrobial activity, and is particularly effective against opportunistic yeast pathogens. Its potential for clinical use, however, is hindered by its latent haemolytic activity. The structures of two analogues, the less haemolytic [C18S,C24S]brevinin-1BYa and the more potent cis-dicarba-brevinin-1BYa, were investigated in various solution and membrane-mimicking environments by [Formula: see text] spectroscopy and molecular modelling techniques. Neither peptide possesses a secondary structure in aqueous solution. In both the membrane-mimicking sodium dodecyl sulphate micelles and 33% 2,2,2-trifluoroethanol ([Formula: see text] solvent mixture, the peptides' structures are characterised by two [Formula: see text]-helices connected by a flexible hinge located at the [Formula: see text] residues. With the aid of molecular dynamics simulations and paramagnetic probes, it was determined that the peptides' helical segments lie parallel to the micellar surface, with their hydrophobic residues facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that both peptides exert their biological activity by a non-pore-forming mechanism. Unlike that of the dicarba analogue, the C-terminus of the acyclic peptide is only weakly associated with the micellar surface and is in direct contact with the surrounding aqueous solvent.

The development of antimicrobial agents that target and selectively disrupt biofilms is a pressing issue since, so far, no antibiotics have been developed that achieve this effectively. Previous experimental work has found a promising set of antibacterial peptides: β 2,2 -amino acid derivatives, relatively small molecules with common structural elements composed of a polar head group and two non-polar hydrocarbon arms. In order to develop insight into possible mechanisms of action of these novel antibacterial agents, we have performed an in silico investigation of four leading β 2,2 -amino acid derivatives, interacting with models of both bacterial (target) and eukaryotic (host) membranes, using molecular dynamics simulation with a model with all-atom resolution. We found an unexpected result that could shed light on the mechanism of action of these antimicrobial agents: the molecules assume a conformation where one of the hydrophobic arms is directed downward into the membrane core while the other is directed upwards, out of the membrane and exposed above the position of the membrane headgroups; we dubbed this conformation the "can-can pose". Intriguingly, the can-can pose was most closely linked to the choice of headgroup. Also, the compound previously found to be most effective against biofilms displayed the strongest extent of this behavior and, additionally, this behavior was more pronounced for this compound in the bacterial than in the eukaryotic membrane. We hypothesize that adopting the can-can pose could possibly disrupt the protective peptidoglycan macronet found on the exterior of the bacterial membrane., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

The resistance of pathogens to traditional antibiotics is currently a global issue of enormous concern. As the discovery and development of new antibiotics become increasingly challenging, synthetic antimicrobial lipopeptides (AMLPs) are now receiving renewed attention as a new class of antimicrobial agents. In contrast to traditional antibiotics, AMLPs act by physically disrupting the cell membrane (rather than targeting specific proteins), thus reducing the risk of inducing bacterial resistance. In this study, we use microsecond-timescale atomistic molecular dynamics simulations to quantify the interaction of a short AMLP (C16-KKK) with model bacterial lipid bilayers. In particular, we investigate how fundamental transmembrane properties change in relation to a range of lipopeptide concentrations. A number of structural, mechanical, and dynamical features are found to be significantly altered in a non-linear fashion. At 10 mol% concentration, lipopeptides have a condensing effect on bacterial bilayers, characterized by a decrease in the area per lipid and an increase in the bilayer order. Higher AMLP concentrations of 25 and 40 mol% destabilize the membrane by disrupting the bilayer core structure, inducing membrane thinning and water leakage. Important transmembrane properties such as the lateral pressure and dipole potential profiles are also affected. Potential implications on membrane function and associated proteins are discussed.

Antimicrobial peptides (AMPs) are effector molecules of the innate immune system and have been isolated from multiple organisms. Their antimicrobial properties are due to the fact that they interact mainly with the anionic membrane of the microorganisms, permeabilizing it and releasing the cytoplasmic content. Alyteserin 1c (+2), an AMP isolated from Alytes obstetricans and its more cationic and hydrophilic analogue (+5) were synthesized using the solid phase method, in order to study the interaction with model membranes by calorimetric and spectroscopic assays. Differential scanning calorimetry (DSC) showed that both peptides had a strong effect when the membrane contained phosphatidylcholine (PC) alone or was mixed with phosphatidylglycerol (PG), increasing membrane fluidization. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the secondary structure of the peptide. Peptide +2 exhibited a transition from β-sheet/turns to β-sheet/α-helix structures after binding with model membranes, whereas peptide +5 had a transition from aggregation/unordered to β-sheet/α-helix structures after binding with membrane-contained PC. Interestingly, the latter showed a β-sheet structure predominantly in the presence of PG lipids. Additionally, molecular dynamics (MD) results showed that the carboxy-terminal of the peptide +5 has the ability to insert into the surface of the PC/PG membranes, resulting in the increase of the membrane fluidity.

Antimicrobial peptides (AMPs) are naturally-occurring peptide antibiotics. AMPs are typically cationic and utilize their electrostatic interactions with the bacterial membrane to selectively attack bacteria. The way they work has inspired a vigorous search for optimized peptides for fighting resistant bacteria. Here, we present a physical model of membrane selectivity of AMPs. The challenge for theoretical modeling of membrane-peptide systems arises from the simultaneous presence of several competing effects, including lipid demixing and peptide-peptide interactions on the membrane surface. We first examine critically a number of models of peptide-membrane interactions and map out one, which incorporates adequately these competing effects as well as the geometry of various regions in membranes, occupied by bound peptides, anionic lipids within the interaction range of each peptide, and those outside this range. This effort leads to a systematically-improved model for peptide selectivity. Using the model, we relate peptide's intrinsic (Ccell-independent) selectivity to an apparent, Ccell-dependent one, and clarify the relative roles of peptide parameters and cell densities in determining their selectivity. This relationship suggests that the selectivity is more sensitive to peptide parameters at low cell densities; as a result, the optimal peptide charge, at which the selectivity is maximized, increases with the cell density in such a manner that this notion becomes less meaningful at high cell densities.

In the present scenario, the invention of bacteria-selective antimicrobial agent comprising negligible toxicity and hemolytic effect is a great challenge. To surmount this challenge, here, a series of polypeptide nanogels (PNGs) have been fabricated by a coordination-assisted self-assembly of a mannose-conjugated antimicrobial polypeptide, poly(arginine- r -valine)-mannose (poly(Arg- r -Val)-M 2 ), with Zn 2+ ions. The fabricated PNGs are spherical in shape with a unique structural appearance similar to that of Taxus baccata fruits. PNGs, with a unique structural arrangement and threshold surface charge density, selectively interact with the bacterial membrane and exhibit potent antimicrobial activity, as reflected in their lower minimum inhibitory concentration values (varies from 2 to 16 μg/mL). PNGs show a remarkably high binding constant, 6.02 × 105 M -1 (from isothermal titration calorimetry, ITC), with the bacterial membrane which manifests its potent bactericidal effect. PNGs are nontoxic against mammalian and red blood cells as reflected from their higher cell viability and insignificant hemolytic effect. PNGs are taken up by the bacterial membrane and selectively undergo structural deformation (scrutinized by ITC) followed by an exposure of free poly(Arg- r -Val)-M 2 molecules. The free poly(Arg- r -Val)-M 2 molecules are enforced to lyse the bacterial membrane (visualized by cryo-transmission electron microscopy) followed by the diffusion of the cytoplasmic component out of the membrane which culminates in the final death of the bacterium.

The rapid emergence of resistant bacterial strains has made the search for new antibacterial agents an endeavor of paramount importance. Cationic antimicrobial peptides (AMPs) have the ability to kill resistant pathogens while diminishing the development of resistance. Citropin 1.1 (Cit 1.1) is an AMP effective against a broad range of pathogens. 20 analogues of Cit 1.1 were prepared to understand how sequence variations lead to changes in structure and biological activity. Various analogues exhibited an increased antimicrobial activity relative to Cit 1.1. The two most promising, AMP-016 (W3F) and AMP-017 (W3F, D4R, K7R) presented a 2- to 8-fold increase in activity against MRSA (both = 4 μg/mL). AMP-017 was active against E. coli (4 μg/mL), K. pneumoniae (8 μg/mL), and A. baumannii (2 μg/mL). NMR studies indicated that Cit 1.1 and its analogues form a head-to-tail helical dimer in a membrane environment, which differs from a prior study by Sikorska et al. Active peptides displayed a greater tendency to form α-helices and to dimerize when in contact with a negatively-charged membrane. Antimicrobial activity was observed to correlate to the overall stability of the α-helix and to a positively charged N-terminus. Biologically active AMPs were shown by SEM and flow cytometry to disrupt membranes in both Gram-positive and Gram-negative bacteria through a proposed carpet mechanism. Notably, active peptides exhibited typical serum stabilities and a good selectivity for bacterial cells over mammalian cells, which supports the potential use of Cit 1.1 analogues as a novel broad-spectrum antibiotic for drug-resistant bacterial infections., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A design template for membrane active antibiotics against microbial and tumor cells is described. The template is an amino acid sequence that combines the properties of helminth defense molecules, which are not cytolytic, with the properties of host-defense peptides, which disrupt microbial membranes. Like helminth defense molecules, the template folds into an amphipathic helix in both mammalian host and microbial phospholipid membranes. Unlike these molecules, the template exhibits antimicrobial and anticancer properties that are comparable to those of antimicrobial and anticancer antibiotics. The selective antibiotic activity of the template builds upon a functional synergy between three distinctive faces of the helix, which is in contrast to two faces of membrane-disrupting amphipathic structures. This synergy enables the template to adapt pore formation mechanisms according to the nature of the target membrane, inducing the lysis of microbial and tumor cells.

A novel antimicrobial peptide named NP-6 was identified in our previous work. Here, the mechanisms of the peptide against Escherichia coli (E. coli) were further investigated, as well as the peptide's resistance to temperature, pH, salinity, and enzymes. The transmission electron microscopy (TEM), confocal laser scanning microcopy (CLSM), and flow cytometric (FCM) analysis, combined with measurement of released K + , were performed to evaluate the effect of NP-6 E. coli cell membrane. The influence of NP-6 on bacterial DNA/RNA and enzyme was also investigated. The leakage of K + demonstrated that NP-6 could increase the permeability of E. coli cell membrane. The ATP leakage, FCM, and CLSM assays suggested that NP-6 caused the disintegration of bacterial cell membrane. The TEM observation indicated that NP-6 could cause the formation of empty cells and debris. Besides, the DNA-binding assay indicated that NP-6 could bind with bacterial genomic DNA in a way that ethidium bromide (EB) did, and suppress the migration of DNA/RNA in gel retardation. Additionally, NP-6 could also affect the activity of β-galactosidase. Finally, the effect of different surroundings such as heating, pH, ions, and protease on the antimicrobial activity of NP-6 against E. coli was also investigated. Results showed that the peptide was heat stable in the range of 60~100 °C and performed well at pH 6.0~8.0. However, the antimicrobial activity of NP-6 decreased significantly in the presence of Mg 2+ /Ca 2+ , and after incubation with trypsin/proteinase K. The results will provide a theoretical support in the further application of NP-6.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.

Mel4 is a novel cationic peptide with potent activity against Gram-positive bacteria. The current study examined the anti-staphylococcal mechanism of action of Mel4 and its precursor peptide melimine. The interaction of peptides with lipoteichoic acid (LTA) and with the cytoplasmic membrane using DiSC(3)-5, Sytox green, Syto-9 and PI dyes were studied. Release of ATP and DNA/RNA from cells exposed to the peptides were determined. Bacteriolysis and autolysin-activated cell death were determined by measuring decreases in OD620nm and killing of Micrococcus lysodeikticus cells by cell-free media. Both peptides bound to LTA and rapidly dissipated the membrane potential (within 30 seconds) without affecting bacterial viability. Disturbance of the membrane potential was followed by the release of ATP (50% of total cellular ATP) by melimine and by Mel4 (20%) after 2 minutes exposure (p<0.001). Mel4 resulted in staphylococcal cells taking up PI with 3.9% cells predominantly stained after 150 min exposure, whereas melimine showed 34% staining. Unlike melimine, Mel4 did not release DNA/RNA. Cell-free media from Mel4 treated cells hydrolysed peptidoglycan and produced greater zones of inhibition against M. lysodeikticus lawn than melimine treated samples. These findings suggest that pore formation is unlikely to be involved in Mel4-mediated membrane destabilization for staphylococci, since there was no significant Mel4-induced PI staining and DNA/RNA leakage. It is likely that the S. aureus killing mechanism of Mel4 involves the release of autolysins followed by cell death. Whereas, membrane interaction is the primary bactericidal activity of melimine, which includes membrane depolarization, pore formation, release of cellular contents leading to cell death., Competing Interests: The authors have declared that no competing interests exist.

Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide-lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling-at the biophysical level-the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading microorganisms. Although AMPs are known to act on bacterial membranes and increase membrane permeability, the action mechanism of most AMPs still remains unclear. In this report, we found that the TP4 peptides from Nile tilapia anchored on E. coli cells and enabled them permeable to SYTOX Green in few minutes after TP4 addition. TP4 peptides existed in small dots either on live or glutaraldehyde-fixed cells. TP4 peptides were driven into oligomers either in soluble or insoluble form by a membrane-mimicking anionic surfactant, sarkosyl, depending on the concentrations employed. The binding forces among TP4 components were mediated through hydrophobic interaction. The soluble oligomers were negatively charged on surface, while the insoluble oligomers could be fused with each other or piled on existing particles to form larger particles with diameters 0.1 to 20 μm by hydrophobic interactions. Interestingly, the morphology and solubility of TP4 particles changed with the concentration of exogenous sarkosyl or trifluoroethanol. The TP4 peptides were assembled into oligomers on or in bacterial membrane. This study provides direct evidence and a model for the oligomerization and insertion of AMPs into bacterial membrane before entering into cytosol., Competing Interests: The authors have declared that no competing interests exist.

Membrane permeabilizing peptides (MPPs) are as ubiquitous as the lipid bilayer membranes they act upon. Produced by all forms of life, most membrane permeabilizing peptides are used offensively or defensively against the membranes of other organisms. Just as nature has found many uses for them, translational scientists have worked for decades to design or optimize membrane permeabilizing peptides for applications in the laboratory and in the clinic ranging from antibacterial and antiviral therapy and prophylaxis to anticancer therapeutics and drug delivery. Here, we review the field of membrane permeabilizing peptides. We discuss the diversity of their sources and structures, the systems and methods used to measure their activities, and the behaviors that are observed. We discuss the fact that "mechanism" is not a discrete or a static entity for an MPP but rather the result of a heterogeneous and dynamic ensemble of structural states that vary in response to many different experimental conditions. This has led to an almost complete lack of discrete three-dimensional active structures among the thousands of known MPPs and a lack of useful or predictive sequence-structure-function relationship rules. Ultimately, we discuss how it may be more useful to think of membrane permeabilizing peptides mechanisms as broad regions of a mechanistic landscape rather than discrete molecular processes.

Antimicrobial peptides (AMPs), as a key component of the immune defense systems of organisms, are a promising solution to the serious threat of drug-resistant bacteria to public health. As one of the most representative and extensively studied AMPs, melittin has exceptional broad-spectrum activities against microorganisms, including both Gram-positive and Gram-negative bacteria. Unfortunately, the action mechanism of melittin with bacterial membranes, especially the underlying physics of peptide-induced membrane poration behaviors, is still poorly understood, which hampers efforts to develop melittin-based drugs or agents for clinical applications. In this mini-review, we focus on recent advances with respect to the membrane insertion behavior of melittin mostly from a computational aspect. Membrane insertion is a prerequisite and key step for forming transmembrane pores and bacterial killing by melittin, whose occurrence is based on overcoming a high free-energy barrier during the transition of melittin molecules from a membrane surface-binding state to a transmembrane-inserting state. Here, intriguing simulation results on such transition are highlighted from both kinetic and thermodynamic aspects. The conformational changes and inter-peptide cooperation of melittin molecules, as well as melittin-induced disturbances to membrane structure, such as deformation and lipid extraction, are regarded as key factors influencing the insertion of peptides into membranes. The associated intermediate states in peptide conformations, lipid arrangements, membrane structure, and mechanical properties during this process are specifically discussed. Finally, potential strategies for enhancing the poration ability and improving the antimicrobial performance of AMPs are included as well., Competing Interests: The authors declare no conflicts of interest.

Antimicrobial peptides (AMPs) are plausible candidates for the development of novel classes of antibiotics with a low tendency to elicit resistance. They often form lesions in the bacterial membrane making it hard for bacteria to develop permanent resistance. However, a potent antibacterial activity is often accompanied by excessive cytotoxicity towards host cells. Modifying known natural sequences, based on desirable biophysical properties, is expensive and time-consuming and often with limited success. 'Mutator' is a freely available web-based computational tool for suggesting residue variations that potentially increase a peptide's selectivity, based on the use of quantitative structure activity relationship (QSAR) criteria. Although proven to be successful, it has never been used to analyze multiple sequences simultaneously. Modifying the Mutator algorithm allowed screening of many sequences in the dedicated Database of Anuran Defense Peptides (DADP) and by implementing limited amino acid substitutions on appropriate candidates, propose 8 potentially selective AMPs called Dadapins. Two were chosen for testing, confirming the prediction and validating this approach. They were shown to efficiently inactivate bacteria by disrupting their membranes but to be non-toxic for host cells, as determined by flow cytometry and confirmed by atomic force microscopy (AFM)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Many short cationic peptides are amphiphilic and are often termed antimicrobial peptides (AMPs) as they can kill various microorganisms. These AMPs have largely been discovered from nature, but over the past two decades many biomimetic and de novo designed AMPs have been reported, offering a huge variety of attractive properties for further exploitation. Under the current global endeavour of fighting against antimicrobial resistance, it is useful to introduce AMPs to the biointerface research community and compare their modes of action with conventional antibiotics. Because natural AMPs often have long sequences and other biological functions implicated, they can't be used as antimicrobial agents. However, rational AMP design helps eliminate their shortcomings and more importantly, optimise their structure-function relationship. This review will first introduce the key approaches recently utilised in structural design of AMPs and then introduce the main lipid membrane models such as spread lipid monolayers and vesicles together with the characterisation techniques adopted in early AMP design and development. These studies are crucial towards understanding key factors affecting their efficacy and toxicity. Thus, various interfacial measurements facilitated by different forms of lipid monolayers and bilayers provide valuable support to the selective responses of AMPs to different cell types used in bactericidal assays and cytotoxicity tests, emphasising the link between molecular models and cell models. A number of clinical trials of AMPs have been either under way or completed, demonstrating the huge potential of AMPs in a range of applications., (Copyright © 2018. Published by Elsevier Inc.)

Synthetic peptidomimetics of antimicrobial peptides (AMPs) are promising antimicrobial drug candidates because they promote membrane disruption and exhibit greater structural and proteolytic stability than natural AMPs. We previously reported selective antifungal 14-helical β-peptides, but the mechanism of antifungal toxicity of β-peptides remains unknown. To provide insight into the mechanism, we studied antifungal β-peptide binding to artificial membranes and living Candida albicans cells. We investigated the ability of β-peptides to interact with and permeate small unilamellar vesicle models of fungal membranes. The partition coefficient supported a pore-mediated mechanism characterized by the existence of a critical β-peptide concentration separating low- and high-partition coefficient regimes. Live cell intracellular tracking of β-peptides showed that β-peptides translocated into the cytoplasm, and then disrupted the nucleus and vacuole sequentially, leading to cell death. This understanding of the mechanisms of antifungal activity will facilitate design and development of peptidomimetic AMPs, including 14-helical β-peptides, for antifungal applications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)