Membrane-permeabilizing Peptides Research Articles

Arg-biodynamers as antibiotic potentiators through interacting with Gram-negative outer membrane lipopolysaccharides

Antimicrobial resistance is becoming more prominent day after day due to a number of mechanisms by microbes, especially the sophisticated biological barriers of bacteria, especially in Gram-negatives. There, the lipopolysaccharides (LPS) layer is a unique component of the outer leaflet of the outer membrane which is highly impermeable and prevents antibiotics from passing passively into the intracellular compartments. Biodynamers, a novel class of dynamically bio-responsive polymers, may open new perspectives to overcome this particular barrier by accommodating various secondary structures and form supramolecular structures in such bacterial microenvironments. Generally, bio-responsive polymers are not only candidates as bio-active molecules against bacteria but also carriers via their interactions with the cargo. Based on their dynamicity, design flexibility, biodegradability, biocompatibility, and pH-responsiveness, we investigated the potential of two peptide-based biodynamers for improving antimicrobial drug delivery. By a range of experimental methods, we discovered a greater affinity of Arg-biodynamers for bacterial membranes than for mammalian membranes as well as an enhanced LPS targeting on the bacterial membrane, opening perspectives for enhancing the delivery of antimicrobials across the Gram-negative bacterial cell envelope. This could be explained by the change of the secondary structure of Arg-biodynamers into a predominant β-sheet character in the LPS microenvironment, by contrast to the α-helical structure typically observed for most lipid membrane-permeabilizing peptides. In comparison to poly-L-arginine, the intrinsic antibacterial activity of Arg-biodynamers was nearly unchanged, but its toxicity against mammalian cells was >128-fold reduced. When used in bacterio as an antibiotic potentiator, however, Arg-biodynamers improved the minimum inhibitory concentration (MIC) against Escherichia coli by 32 times compared to colistin alone. Similar effect has also been observed in two stains of Pseudomonas aeruginosa. Arg-biodynamers may therefore represent an interesting option as an adjuvant for antibiotics against Gram-negative bacteria and to overcome antimicrobial resistance.

An Extensive Review on Production, Purification, and Bioactive Application of Different Classes of Bacteriocin

Lactic Acid Bacteria (LAB) synthesize various metabolites during their growth phase and are Generally Recognized as­­ Safe (GRAS) and Qualified Presumption of Safety (QPS). Ribosomally synthesized Antimicrobial Peptides (AMP) or Bacteriocins from the genera of Lactic Acid Bacteria and other prokaryotic genera are cationic, heat-stable, amphiphilic and the membrane permeabilizing peptides built with an excess amount of lysyl and arginyl residues. Antimicrobial compounds produced by LAB depend on the physical and biological conditions of microbial culture. Different classes of bacteriocin are produced by both Gram-positive and Gram-negative bacteria. The production of bacteriocin is influenced by various environmental factors. Bacteriocin has a wide variety of applications in various fields. The application spectrum of bacteriocins can be expanded in various domains such as food processing, biomedical, and personal care due to the increase in the number of newly discovered bacteriocins. Bacteriocins acquire a wide spectrum of antimicrobial activity with minimal level of cytotoxicity. In addition, bacteriocins were studied for their anticancer activity against different cancer cell lines. Selective binding of bacteriocins (cationic) towards cancer cells (anionic) increases the cytotoxicity of cancer cells. Bacteriocin peptides initiate necrosis by communicating with the cell surface which selectively targets and kills the cells with tumor formation and does not cause any damage to the normal healthy cells. In this review, the bacteriocins synthesized from lactic acid bacteria along with their interaction with cancer cell lines and other applications are discussed along with a few examples of other bioactive compounds produced by LAB.

Membrane-Permeable Antibacterial Enzyme against Multidrug-Resistant Acinetobacter baumannii.

Bacteriophage endolysins (lysins, or murein hydrolases) are enzymes that bacteriophages utilize to degrade the cell wall peptidoglycans (PG) and subsequently disintegrate bacterial cells from within. Due to their muralytic activity, lysins are considered as potential candidates to battle against antibiotic resistance. However, most lysins in their native form lack the capability of trespassing the outer membrane (OM) of Gram-negative (G-ve) bacteria. To turn the bacteriophage enzymes into antibacterial weapons against G-ve bacteria, endowing these enzymes the capability of accessing the PG substrate underneath the OM is critical. Here we show that fusing a membrane-permeabilizing peptide CeA at the C-terminus of a muralytic enzyme LysAB2 renders a two-step mechanism of bacterial killing and increases the activity of LysAB2 against the multidrug resistant Acinetobacter baumannii by up to 100 000-folds. The engineered LysAB2, termed LysAB2-KWK here, also shows remarkable activity against A. baumannii at the stationary phase and a prominent capability to disrupt biofilm formation. In addition, the enzyme shows a broad antibacterial spectrum against G-ve bacteria, a decent tolerance to serum, and a prolonged storage life. LysAB2-KWK rescues the larva of the greater wax moth Galleria mellonella from A. baumannii infection through systemic administration. Altogether, our work equips a globular lysin with OM permeabilization activity to enable effective killing of G-ve bacteria, reveals the critical role of the C-terminus of a globular lysin in the antibacterial activity, and points toward a viable route to engineer globular lysins as antibacterial enzymes for potential clinical use against multidrug resistant G-ve bacteria.

Rapid and High-Throughput Evaluation of Diverse Configurations of Engineered Lysins Using the VersaTile Technique.

Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targeting Klebsiella pneumoniae. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth of Klebsiella pneumoniae could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP–linker–CBD–EAD) and the inverse configuration (CBD–EAD–linker–OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions.

Atomic force microscopy for quantitative understanding of peptide-induced lipid bilayer remodeling.

A number of peptides are known to bind lipid bilayer membranes and cause these natural barriers to leak in an uncontrolled manner. Though membrane permeabilizing peptides play critical roles in cellular activity and may have promising future applications in the therapeutic arena, significant questions remain about their mechanisms of action. The atomic force microscope (AFM) is a single molecule imaging tool capable of addressing lipid bilayers in near-native fluid conditions. The apparatus complements traditional assays by providing local topographic maps of bilayer remodeling induced by membrane permeabilizing peptides. The information garnered from the AFM includes direct visualization and statistical analyses of distinct bilayer remodeling modes such as highly localized pore-like voids in the bilayer and dispersed thinned membrane regions. Colocalization of distinct remodeling modes can be studied. Here we examine recent work in the field and outline methods used to achieve precise AFM image data. Experimental challenges and common pitfalls are discussed as well as techniques for unbiased analysis including the Hessian blob detection algorithm, bootstrapping, and the Bayesian information criterion. When coupled with robust statistical analyses, high precision AFM data is poised to advance understanding of an important family of peptides that cause poration of membrane bilayers.

Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs.

Mechanism of Action of Peptides That Cause the pH-Triggered Macromolecular Poration of Lipid Bilayers.

Using synthetic molecular evolution, we previously discovered a family of peptides that cause macromolecular poration in synthetic membranes at low peptide concentration in a way that is triggered by acidic pH. To understand the mechanism of action of these "pHD peptides", here we systematically explored structure-function relationships through measurements of the effect of pH and peptide concentration on membrane binding, peptide structure, and the formation of macromolecular-sized pores in membranes. Both AFM and functional assays demonstrate the peptide-induced appearance of large pores in bilayers. Pore formation has a very steep pH dependence and is also dependent on peptide concentration. In vesicles, 50% leakage of 40 kDa dextrans occurs at 1 bound peptide per 1300 lipids or only 75 peptides per vesicle, an observation that holds true across a wide range of acidic pH values. The major role of pH is to regulate the amount of peptide bound per vesicle. The physical chemistry and sequence of the pHD peptides affect their potency and pH dependence; therefore, the sequence-structure-function relationships described here can be used for the future design and optimization of membrane permeabilizing peptides for specific applications.

Mechanistic Landscape of Membrane-Permeabilizing Peptides.

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.

Potent Macromolecule-Sized Poration of Lipid Bilayers by the Macrolittins, A Synthetically Evolved Family of Pore-Forming Peptides.

Pore-forming peptides with novel functions have potential utility in many biotechnological applications. However, the sequence-structure-function relationships of pore forming peptides are not understood well enough to empower rational design. Therefore, in this work, we used synthetic molecular evolution to identify a novel family of peptides that are highly potent and cause macromolecular poration in synthetic lipid vesicles at low peptide concentration and at neutral pH. These unique 26-residue peptides, which we call macrolittins, release macromolecules from lipid bilayer vesicles made from zwitterionic PC lipids at peptide to lipid ratios as low as 1:1000, a property that is almost unprecedented among known membrane permeabilizing peptides. The macrolittins exist as membrane-spanning α-helices. They cause dramatic bilayer thinning and form large pores in planar supported bilayers. The high potency of these peptides is likely due to their ability to stabilize bilayer edges by a process that requires specific electrostatic interactions between peptides.

Ebola Virus Delta-Peptide Acts as an Enterotoxic Viroporin In Vivo

Ebola virus (EBOV), part of the filovirus family, has a genome that encodes for a partly conserved, 40-residue polypeptide, called the delta peptide, which is produced during Ebola virus disease pathogenesis. Sequence-structure analysis and a wealth of in vitro data suggest that the delta peptide is a viroporin, a diverse family of pore-forming peptides and proteins involved in replication and pathogenesis of numerous viruses. Specifically, many filovirus delta peptides have a high abundance of cationic and aromatic residues while maintaining amphipathicity; these are properties that are common in membrane-permeabilizing peptides. Full-length and conserved C-terminal EBOV delta peptide fragments were shown to permeabilize synthetic lipid bilayers and multiple cell types in vitro. Here, we follow up those results with an established mouse model of diarrheal pathology focusing on the small intestine. We hypothesized that the lytic activity observed against numerous cell types in vitro would also be apparent when testing the delta peptide in vivo. We found that the delta peptide exerts potent, dose-dependent enterotoxic activity against intestinal cells upon introduction to a closed intestinal loop. The peak activity of the peptide, which results in a diarrheal syndrome in the mice, occurs 9-12 hours after introduction to the small intestine. This conclusion is confirmed with histology studies in which we examined intestinal architecture and found that it was severely damaged by the delta peptide. Further, the density of mucin-producing goblet cells in villi was diminished by the peptide. These results show that the EBOV delta peptide is a potential novel therapeutic target, and neutralizing it could ameliorate some of the severe enterotoxic burden characteristic of EBOV disease pathogenesis.

The Delta Peptide of Ebola Virus has Potent Viroporin Activity

The Delta peptide, cleaved from the soluble glycoprotein (sGP) of Ebola virus (EBOV), is a partially conserved, 40-residue nonstructural polypeptide that has no known function. This peptide shows high similarity in amino acid composition to membrane permeabilizing peptides, including a peptide in the diarrhea-inducing viroporin, NSP4, of Rotavirus. In addition, EBOV disease (EVD) survivors from West Africa have antibodies against the Delta peptide. Therefore, we hypothesize that the Delta peptide has an important role as a viroporin in EVD pathology. Here we show that a peptide corresponding to the serum-stable, conserved C-terminal sequence of Delta peptide permeabilizes and kills nucleated mammalian cells, and potently increasing ion permeability across synthetic lipid bilayers. Importantly, this permeabilizing activity is eliminated by the reduction of the disulfide bond between conserved cysteines. We conclude that EBOV Delta peptide does not form large unregulated pores through membranes, but rather increases ionic permeation, which is similar to other viroporins. By this mechanism, it could contribute to EVD pathology in human through vascular leakage, enterotoxic effects, or other cytotoxic pathways.

PH-Triggered, Macromolecule-Sized Poration of Lipid Bilayers by Synthetically Evolved Peptides.

pH-triggered membrane-permeabilizing peptides could be exploited in a variety of applications, such as to enable cargo release from endosomes for cellular delivery, or as cancer therapeutics that selectively permeabilize the plasma membranes of malignant cells. Such peptides would be especially useful if they could enable the movement of macromolecules across membranes, a rare property in membrane-permeabilizing peptides. Here we approach this goal by using an orthogonal high-throughput screen of an iterative peptide library to identify peptide sequences that have the following two properties: (i) little synthetic lipid membrane permeabilization at physiological pH 7 at high peptide concentration and (ii) efficient formation of macromolecule-sized defects in synthetic lipid membranes at acidic pH 5 and low peptide concentration. The peptides we selected are remarkably potent macromolecular sized pore-formers at pH 5, while having little or no activity at pH 7, as intended. The action of these peptides likely relies on tight coupling between membrane partitioning, α-helix formation, and electrostatic repulsions between acidic side chains, which collectively drive a sharp pH-triggered transition between inactive and active configurations with apparent pKa values of 5.5-5.8. This work opens new doors to developing applications that utilize peptides with membrane-permeabilizing activities that are triggered by physiologically relevant decreases in pH.

Laterosporulin10: a novel defensin like Class IId bacteriocin from Brevibacillus sp. strain SKDU10 with inhibitory activity against microbial pathogens.

Bacteriocins are antimicrobial peptides (AMPs) produced by bacteria to acquire survival benefits during competitive inter- and intra-species interactions in complex ecosystems. In this study, an AMP-producing soil bacterial strain designated SKDU10 was isolated and identified as a member of the genus Brevibacillus. The AMP produced by strain SKDU10 identified as a class IId bacteriocin with 57.6 % homology to laterosporulin, a defensin-like class IId bacteriocin. However, substantial differences were observed in the antimicrobial activity spectrum of this bacteriocin named laterosporulin10 when compared to laterosporulin. Laterosporulin10 effectively inhibited the growth of Staphylococcus aureus and Mycobacterium tuberculosis (Mtb H37Rv) with LD50 values of 4.0 µM and 0.5 µM, respectively. Furthermore, laterosporulin10 inhibited the growth of Mtb H37Rv strain at about 20 times lower MIC value compared to S. aureus MTCC 1430 or M. smegmatis MC2 155 in vitro and ex vivo. Electron micrographs along with membrane permeabilization studies using FACS analysis revealed that laterosporulin10 is a membrane-permeabilizing peptide. Interestingly, laterosporulin10 was able to efficiently kill Mtb H37Rv strain residing inside the macrophages and did not show haemolysis up to 40 µM concentration.

Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.

To better understand the sequence–structure–function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding–folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the “negative” peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens.

The therapeutic potential of α-helical anti-microbial peptides (AH-AMP) to combat pathogens is fast gaining prominence. Based on recently published open access software for characterizing α-helical peptides (PAGAL), we elucidate a search methodology (SCALPEL) that leverages the massive structural data pre-existing in the PDB database to obtain AH-AMPs belonging to the host proteome. We provide in vitro validation of SCALPEL on plant pathogens ( Xylella fastidiosa, Xanthomonas arboricola and Liberibacter crescens) by identifying AH-AMPs that mirror the function and properties of cecropin B, a well-studied AH-AMP. The identified peptides include a linear AH-AMP present within the existing structure of phosphoenolpyruvate carboxylase (PPC20), and an AH-AMP mimicing the properties of the two α-helices of cecropin B from chitinase (CHITI25). The minimum inhibitory concentration of these peptides are comparable to that of cecropin B, while anionic peptides used as control failed to show any inhibitory effect on these pathogens. Substitute therapies in place of conventional chemotherapies using membrane permeabilizing peptides like these might also prove effective to target cancer cells. The use of native structures from the same organism could possibly ensure that administration of such peptides will be better tolerated and not elicit an adverse immune response. We suggest a similar approach to target Ebola epitopes, enumerated using PAGAL recently, by selecting suitable peptides from the human proteome, especially in wake of recent reports of cationic amphiphiles inhibiting virus entry and infection.

The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens

The therapeutic potential of α-helical anti-microbial peptides (AH-AMP) to combat pathogens is fast gaining prominence. Based on recently published open access software for characterizing α-helical peptides (PAGAL), we elucidate a search methodology (SCALPEL) that leverages the massive structural data pre-existing in the PDB database to obtain AH-AMPs belonging to the host proteome. We provide in vitro validation of SCALPEL on plant pathogens ( Xylella fastidiosa, Xanthomonas arboricola and Liberibacter crescens) by identifying AH-AMPs that mirror the function and properties of cecropin B, a well-studied AH-AMP. The identified peptides include a linear AH-AMP present within the existing structure of phosphoenolpyruvate carboxylase (PPC20), and an AH-AMP mimicing the properties of the two α-helices of cecropin B from chitinase (CHITI25). The minimum inhibitory concentration of these peptides are comparable to that of cecropin B, while anionic peptides used as control failed to show any inhibitory effect on these pathogens. Substitute therapies in place of conventional chemotherapies using membrane permeabilizing peptides like these might also prove effective to target cancer cells. The use of native structures from the same organism could possibly ensure that administration of such peptides will be better tolerated and not elicit an adverse immune response. We suggest a similar approach to target Ebola epitopes, enumerated using PAGAL recently, by selecting suitable peptides from the human proteome, especially in wake of recent reports of cationic amphiphiles inhibiting virus entry and infection.

The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens

The therapeutic potential ofα-helical anti-microbial peptides (AH-AMP) to combat pathogens is fast gaining prominence. Based on recently published open access software for characterizingα-helical peptides (PAGAL), we elucidate a search methodology (SCALPEL) that leverages the massive structural data pre-existing in the PDB database to obtain AH-AMPs belonging to the host proteome. We providein vitrovalidation of SCALPEL on plant pathogens (Xylella fastidiosa,Xanthomonas arboricolaandLiberibacter crescens) by identifying AH-AMPs that mirror the function and properties of cecropin B, a well-studied AH-AMP. The identified peptides include a linear AH-AMP present within the existing structure of phosphoenolpyruvate carboxylase (PPC20), and an AH-AMP mimicing the properties of the twoα-helices of cecropin B from chitinase (CHITI25). The minimum inhibitory concentration of these peptides are comparable to that of cecropin B, while anionic peptides used as control failed to show any inhibitory effect on these pathogens. Substitute therapies in place of conventional chemotherapies using membrane permeabilizing peptides like these might also prove effective to target cancer cells. The use of native structures from the same organism largely ensures that administration of such peptides will be better tolerated and not elicit an adverse immune response. We suggest a similar approach to target Ebola epitopes, enumerated using PAGAL recently, by selecting suitable peptides from the human proteome, especially in wake of recent reports of cationic amphiphiles inhibiting virus entry and infection.

Toward the de novo design of antimicrobial peptides: Lack of correlation between peptide permeabilization of lipid vesicles and antimicrobial, cytolytic, or cytotoxic activity in living cells

We previously performed a lipid vesicle-based, high-throughput screen on a 26-residue combinatorial peptide library that was designed de novo to yield membrane-permeabilizing peptides that fold into β-sheets. The most active and soluble library members that were identified permeabilized lipid vesicles detectably, but not with high potency. Nonetheless, they were broad-spectrum, membrane-permeabilizing antibiotics with minimum sterilizing activity at low µM concentrations. In an expansion of that work, we recently performed an iterative screen in which an active consensus sequence from that first-generation library was used as a template to design a second-generation library which was then screened against lipid vesicles at very high stringency. Compared to the consensus sequence from the first library, the most active second-generation peptides are highly potent, equilibrium pore-formers in synthetic lipid vesicles. Here, we use these first- and second-generation families of peptides to test the hypothesis that a large increase in potency in bacteria-like lipid vesicles will correlate with a large improvement in antimicrobial activity. The results do not support the hypothesis. Despite a 20-fold increase in potency against bacteria-like lipid vesicles, the second-generation peptides are only slightly more active against bacteria, and at the same time, are also more toxic against mammalian cells. The results suggest that a "pipeline" strategy toward the optimization of antimicrobial peptides could begin with a vesicle-based screen for identifying families with broad-spectrum activity, but will also need to include screening or optimization steps that are done under conditions that are more directly relevant to possible therapeutic applications.

A Modular Approach to the Histone H2A Family of Antimicrobial Peptides

Hipposin and buforin II (BF2) are naturally occurring antimicrobial peptides (AMPs). Although BF2 is essentially identical to the middle section of hipposin, BF2 is a membrane translocating peptide and hipposin is a membrane permeabilizing peptide. To investigate this difference, we compared the structure, antimicrobial activity, translocating ability, and membrane permeabilizing ability of hipposin, BF2, parasin (analogous to the N‐terminal section of hipposin), and buforin I (essentially parasin plus BF2). Hipposin and BF2 had the highest activities against bacteria, followed by buforin I then parasin. Cellular permeabilization assays demonstrated that hipposin, buforin I, and parasin all display membrane permeabilizing ability, while translocation assays showed that only BF2 was membrane translocating. These observations imply that the addition of the parasin fragment to BF2 alters its antibacterial mechanism. Ongoing studies are examining the activity and mechanisms of other portions of the hipposin peptide.

Synthetic Molecular Evolution of Pore-Forming Peptides by Iterative Combinatorial Library Screening

We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have β-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not α-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic α-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.