Evan F. Haney

The expanding scope of antimicrobial peptide structures and their modes of action

Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protect a host from invading pathogenic bacteria. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as potential alternatives for antibiotics. Although extremely variable in length, amino acid composition and secondary structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent studies demonstrate that they achieve their antimicrobial activity by disrupting various key cellular processes. Some peptides can even use multiple mechanisms. Moreover, several intact proteins or protein fragments are now being shown to have inherent antimicrobial activity. A better understanding of the structure-activity relationships of AMPs is required to facilitate the rational design of novel antimicrobial agents.

Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

The immunology of host defence peptides: beyond antimicrobial activity

Host defence peptides (HDPs) are short, cationic amphipathic peptides with diverse sequences that are produced by various cells and tissues in all complex life forms. HDPs have important roles in the bodys response to infection and inflammation. This Review focuses on human HDPs and explores the diverse immunomodulatory effects of HDPs from a systems biology perspective, which highlights the interconnected nature of the effect (or effects) of HDPs on the host. Studies have demonstrated that HDPs are expressed throughout the body and mediate a broad range of activities, which explains their association with various inflammatory diseases and autoimmune disorders. The diverse actions of HDPs, such as their roles in wound healing and in the maintenance of the microbiota, are also explored, in addition to potential therapeutic applications.

Solution NMR studies of amphibian antimicrobial peptides: linking structure to function?

The high-resolution three-dimensional structure of an antimicrobial peptide has implications for the mechanism of its antimicrobial activity, as the conformation of the peptide provides insights into the intermolecular interactions that govern the binding to its biological target. For many cationic antimicrobial peptides the negatively charged membranes surrounding the bacterial cell appear to be a main target. In contrast to what has been found for other classes of antimicrobial peptides, solution NMR studies have revealed that in spite of the wide diversity in the amino acid sequences of amphibian antimicrobial peptides (AAMPs), they all adopt amphipathic alpha-helical structures in the presence of membrane-mimetic micelles, bicelles or organic solvent mixtures. In some cases the amphipathic AAMP structures are directly membrane-perturbing (e.g. magainin, aurein and the rana-box peptides), in other instances the peptide spontaneously passes through the membrane and acts on intracellular targets (e.g. buforin). Armed with a high-resolution structure, it is possible to relate the peptide structure to other relevant biophysical and biological data to elucidate a mechanism of action. While many linear AAMPs have significant antimicrobial activity of their own, mixtures of peptides sometimes have vastly improved antibiotic effects. Thus, synergy among antimicrobial peptides is an avenue of research that has recently attracted considerable attention. While synergistic relationships between AAMPs are well described, it is becoming increasingly evident that analyzing the intermolecular interactions between these peptides will be essential for understanding the increased antimicrobial effect. NMR structure determination of hybrid peptides composed of known antimicrobial peptides can shed light on these intricate synergistic relationships. In this work, we present the first NMR solution structure of a hybrid peptide composed of magainin 2 and PGLa bound to SDS and DPC micelles. The hybrid peptide adopts a largely helical conformation and some information regarding the inter-helix organization of this molecule is reported. The solution structure of the micelle associated MG2-PGLa hybrid peptide highlights the importance of examining structural contributions to the synergistic relationships but it also demonstrates the limitations in the resolution of the currently used solution NMR techniques for probing such interactions. Future studies of antimicrobial peptide synergy will likely require stable isotope-labeling strategies, similar to those used in NMR studies of proteins.

Peptide design for antimicrobial and immunomodulatory applications

The increasing threat of antibiotic resistance in pathogenic bacteria and the dwindling supply of antibiotics available to combat these infections poses a significant threat to human health throughout the world. Antimicrobial peptides (AMPs) have long been touted as the next generation of antibiotics capable of filling the anti-infective void. Unfortunately, peptide-based antibiotics have yet to realize their potential as novel pharmaceuticals, in spite of the immense number of known AMP sequences and our improved understanding of their antibacterial mechanism of action. Recently, the immunomodulatory properties of certain AMPs have become appreciated. The ability of small synthetic peptides to protect against infection in vivo has demonstrated that modulation of the innate immune response is an effective strategy to further develop peptides as novel anti-infectives. This review focuses on the screening methods that have been used to assess novel peptide sequences for their antibacterial and immunomodulatory properties. It will also examine how we have progressed in our ability to identify and optimize peptides with desired biological characteristics and enhanced therapeutic potential. In addition, the current challenges to the development of peptides as anti-infectives are examined and the strategies being used to overcome these issues are discussed.

Induction of non-lamellar lipid phases by antimicrobial peptides: a potential link to mode of action

Antimicrobial peptides are naturally produced by numerous organisms including insects, plants and mammals. Their non-specific mode of action is thought to involve the transient perturbation of bacterial membranes but the molecular mechanism underlying the rearrangement of the lipid molecules to explain the formation of pores and micelles is still poorly understood. Biological membranes mostly adopt planar lipid bilayers; however, antimicrobial peptides have been shown to induce non-lamellar lipid phases which may be intimately linked to their proposed mechanisms of action. This paper reviews antimicrobial peptides that alter lipid phase behavior in three ways: peptides that induce positive membrane curvature, peptides that induce negative membrane curvature and peptides that induce cubic lipid phases. Such structures can coexist with the bilayer structure, thus giving rise to lipid polymorphism induced upon addition of antimicrobial peptides. The discussion addresses the implications of induced lipid phases for the mode of action of various antimicrobial peptides.

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.

Mechanism of action of puroindoline derived tryptophan-rich antimicrobial peptides.

A tryptophan (Trp)-rich region in the wheat endosperm protein, puroindoline A, was previously shown to possess potent antimicrobial activity against Gram-positive and Gram-negative bacteria and this was attributed to the peptide inducing membrane instability. In the present work, the antimicrobial activity of the corresponding Trp-rich region in the puroindoline B isoform was examined and its antimicrobial activity was characterized. Unexpectedly, the puroindoline B Trp-rich peptide (PuroB) was relatively inactive compared to the related puroindoline A peptide (PuroA), despite strong sequence similarity. Using the sequence of PuroA as a template, a series of PuroB variants were synthesized and the antimicrobial activity was restored. Interestingly, all of these PuroB peptides preferentially interacted with negatively charged phospholipids, but unlike PuroA, they did not disrupt the integrity of lipid bilayers. This suggests that the primary mode of action of the PuroB peptides involves an antimicrobial target other than the bacterial membrane. Further tests revealed that all of the puroindoline derived peptides bind deoxyribonucleic acid (DNA) and block macromolecular synthesis in vivo. Based on these results, it appears that the interaction between puroindoline derived peptides and membranes is only an initial step in the mode of action and that binding to intracellular targets, such as DNA and ribonucleic acid (RNA), contributes significantly to their antimicrobial mode of action.

High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides.

The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favorably modulating the immune response would be particularly advantageous for treating serious skin infections caused by Staphylococcus aureus. In the present work, using SPOT-synthesized peptide arrays on cellulose membranes, we outline a strategy for systematically assessing the antibiofilm activity of hundreds of IDR-1002 (VQRWLIVWRIRK-NH2) and IDR-HH2 (VQLRIRVAVIRA-NH2) peptide variants against MRSA biofilms. In addition, the ability of these peptides to stimulate production of a monocyte chemoattractant protein (MCP-1) and suppress LPS-induced interleukin (IL)-1β production in human peripheral blood mononuclear cells (PBMCs) was evaluated. These results informed the synthesis of second-generation peptides resulting in a new peptide, IDR-2009 (KWRLLIRWRIQK-NH2), with enhanced MCP-1 stimulatory activity, favorable IL-1β suppression characteristics and strong antibiofilm activity against MRSA and Pseudomonas aeruginosa biofilms. This work provides a proof-of-concept that multiple peptide activities can be optimized simultaneously to generate novel sequences that possess a variety of biological properties.

Structural and biophysical characterization of an antimicrobial peptide chimera comprised of lactoferricin and lactoferrampin

Lactoferricin and lactoferrampin are two antimicrobial peptides found in the N-terminal lobe of bovine lactoferrin with broad spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacteria as well as Candida albicans. A heterodimer comprised of lactoferrampin joined to a fragment of lactoferricin was recently reported in which these two peptides were joined at their C-termini through the two amino groups of a single Lys residue (Bolscher et al., 2009, Biochimie 91(1):123-132). This hybrid peptide, termed LFchimera, has significantly higher antimicrobial activity compared to the individual peptides or an equimolar mixture of the two. In this work, the underlying mechanism behind the increased antibacterial activity of LFchimera was investigated. Differential scanning calorimetry studies demonstrated that all the peptides influenced the thermotropic phase behaviour of anionic phospholipid suspensions. Calcein leakage and vesicle fusion experiments with anionic liposomes revealed that LFchimera had enhanced membrane perturbing properties compared to the individual peptides. Peptide structures were evaluated using circular dichroism and NMR spectroscopy to gain insight into the structural features of LFchimera that contribute to the increased antimicrobial activity. The NMR solution structure, determined in a miscible co-solvent mixture of chloroform, methanol and water, revealed that the Lys linkage increased the helical content in LFchimera compared to the individual peptides, but it did not fix the relative orientations of lactoferricin and lactoferrampin with respect to each other. The structure of LFchimera provides insight into the conformation of this peptide in a membranous environment and improves our understanding of its antimicrobial mechanism of action.