Structure-function relationships of antimicrobial peptides

Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides (6-50 residues) are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. All of the peptides are highly cationic and hydrophobic. It is widely believed that they act through nonspecific binding to biological membranes, even though the exact nature of these interactions is presently unclear. High-resolution nuclear magnetic resonance (NMR) has contributed greatly to knowledge in this field, providing insight about peptide structure in aqueous solution, in organic cosolvents, and in micellar systems. Solid-state NMR can provide additional information about peptide-membrane binding. Here we review our current knowledge about the structure of antimicrobial peptides. We also discuss studies pertaining to the mechanism of action. Despite the different three-dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-parallel orientation, interacting only with one face of the bilayer. This may be sufficient for antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity. An understanding of the key features of the secondary and tertiary structures of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the rational design of improved analogs for clinical use.     

Design and synthesis of amphipathic antimicrobial peptides

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic alpha-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no alpha-helicity in buffer or liposomes, exhibits a modest degree of alpha-helical structure in the presence of the alpha-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an alpha-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.     

Self-assembly of designed antimicrobial peptides in solution and micelles

Hydrophobic interactions are responsible for stabilizing leucine zippers in peptides containing heptad repeats. The effects of substituting leucine by phenylalanine and alanine by glycine on the self-assembly of coiled-coils were examined in minimalist antimicrobial peptides designed to form amphipathic alpha-helices. The secondary structure of these peptides was monitored in solution and in diphosphocholine (DPC) micelles using circular dichroism spectroscopy. The leucine peptides (KLAKLAK)3 and (KLAKKLA)n (n = 3, 4) become alpha-helical with increasing concentrations of salt, peptide, and DPC. The aggregation state and equilibrium constant for self-association of the peptides were measured by sedimentation equilibrium. The glycine peptide (KLGKKLG)3 does not self-associate. The leucine peptides and phenylalanine peptides (KFAKFAK)3 and (KFAKKFA)n (n = 3, 4) are in a monomer-tetramer equilibrium in solution, with the phenylalanine zippers being 2-4 kcal/mol less stable than the equivalent leucine zippers. Thermodynamic parameters for the association reaction were calculated from the temperature dependence of the association constants. Leucine zipper formation has DeltaCp = 0, whereas phenylalanine zipper formation has a small negative DeltaCp, presumably due to the removal of the larger surface area of phenylalanine from water. Self-association of the peptides is coupled to formation of a hydrophobic core as detected using 1-anilino-naphthalene-8-sulfonate fluorescence. Carboxyfluorescein-labeled peptides were used to determine the aggregation state of (KLAKKLA)3 and (KLGKKLG)3 in DPC micelles. (KLAKKLA)3 forms dimers, and (KLGKKLG)3 is a monomer. Aggregation appears to correlate with the cytotoxicity of these peptides.     

Mechanism of synergism between antimicrobial peptides magainin 2 and PGLa

The antimicrobial peptides magainin 2 and PGLa, discovered in the skin of the African clawed frog, Xenopus laevis, exhibit marked synergism [Westerhoff, H. V., Zasloff, M., Rosner, J. L., Hendler, R. W., de Waal, A., Vaz Gomes, A., Jongsma, A. P. M., Riethorst, A., and Juretic, D., Eur. J. Biochem. 228, 257-264 (1995)], although the mechanism is not yet clear. They are believed to kill bacteria by permeabilizing membranes. In this study, we examined the interactions of these peptides in lipid bilayers. PGLa, like magainin 2, preferentially interacts with acidic lipids, forming an amphipathic helix. The peptide induces the release of a water-soluble dye, calcein, entrapped within liposomes. The coexistence of magainin 2 enhances membrane permeabilization, which is maximal at a 1:1 molar ratio. Fluorescence experiments using L18W-PGLa revealed that both peptides form a stoichiometric 1:1 complex in the membrane phase with an association free energy of -15 kJ/mol. Single amino acid mutations in magainin 2 significantly altered the synergistic activity, suggesting that precise molecular recognition is involved in complex formation. The complex as well as each component peptide form peptide-lipid supramolecular complex pores, which mediate the mutually coupled transbilayer transport of dye, lipid, and the peptide per se. The rate of pore formation rate is in the order complex >/= PGLa > magainin 2, whereas the pore lifetime is in the order magainin 2 > complex > PGLa. Therefore, the synergism is a consequence of the formation of a potent heterosupramolecular complex, which is characterized by fast pore formation and moderate pore stability.     

Radiolabelled antimicrobial peptides for imaging of infections: a review

Risk factors suggestive of relatively late exposure to EBV have been consistently associated with Hodgkin's disease (HD) in younger adults. In addition, evidence of EBV infection has been found in the Reed-Sternberg cells themselves in about one-third to one-half of all HD cases. However, no study yet published has correlated these childhood social environment risk factors with the presence of EBV in Hodgkin's tumor cells. We examined whether EBV-positive HD occurs in those patients whose childhood environment would predispose them to relatively late exposure to EBV. The study population consisted of 102 cases of mixed cellularity (MC; n = 25) or nodular sclerosing (n = 77) HD. Samples that tested positive for either EBV-encoded RNA or latent membrane protein or both were considered EBV-positive. Of the 102 cases, 83 completed a questionnaire regarding childhood social environment. The association with EBV-positivity was estimated by the odds ratio (OR) with 95% confidence intervals (CI). Twenty-two percent of the cases were EBV-positive. These cases were more likely to be MC (OR, 6.2; CI, 2.3-16.3) and male (OR, 3.4; CI, 1.3-9.0). History of infectious mononucleosis (IM) was not predictive of EBV-positivity, with only 3 of 14 such patients being EBV-positive (P = 0.82). Contrary to our hypothesis, no association between EBV and childhood environment risk factors was identified. The association of EBV with MC histology and male gender agrees with previous reports. The most intriguing finding was the dissociation between IM history and EBV-positivity, in that almost all of the cases with a history of IM were EBV-negative.     

Epithelial antimicrobial peptides: review and significance for oral applications

Epithelial tissues provide the first line of defense between an organism and the environment. Disruption of this barrier leads to bacterial invasion and subsequent inflammation. This is precisely the situation existing in the human oral cavity, where tissues are constantly exposed to a variety of microbial challenges that can lead to bacterially induced periodontal diseases, and to infections of the oral mucosa by bacteria, fungi, and viruses. With the recent discoveries of host-derived peptide antibiotics in mammalian mucosal epithelium, a new line of investigation is emerging to test the hypothesis that one class of these peptides, called "beta-defensins", functions to protect the host against microbial pathogenesis at these critical, confrontational sites. In that light, impairment of beta-defensin activity has recently been implicated in chronic bacterial infections in cystic fibrosis patients. The first direct evidence of expression of defensin peptides in the oral mucosa was the identification of a novel epithelial beta-defensin in mammalian tongue. It was shown to be upregulated in inflammation, suggesting that it participates in host defense. It is theorized that epithelial cell-derived antimicrobial peptides function to keep the natural flora of micro-organisms in a steady state in different niches such as the skin, the intestines, the airway, the endocervix, and the mouth. There is now evidence indicating that normal gingival epithelial cells and tissues express two beta-defensins, hBD-1 and the newly described hBD-2. In addition, a cathelin-class antimicrobial peptide, designated LL-37 and found in human neutrophils, is also expressed in skin and gingiva. It is highly likely that these and/or other epithelial antimicrobial peptides play an important role in determining the outcome of the host-pathogen interaction at the oral mucosal barrier, and that they may have important future applications in antibiotic treatment.     

Detection of anionic antimicrobial peptides in ovine bronchoalveolar lavage fluid and respiratory epithelium

Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.     

Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity

Novel alpha-helical antimicrobial peptides have been devised by comparing the N-terminal sequences of many of these peptides from insect, frog and mammalian families, extracting common features, and creating sequence templates with which to design active peptides. Determination of the most frequent amino acids in the first 20 positions for over 80 different natural sequences allowed the design of one peptide, while a further three were based on the comparison of the sequences of alpha-helical antimicrobial peptides derived from the mammalian cathelicidin family of precursors. These peptides were predicted to assume a highly amphipathic alpha-helical conformation, as indicated by high mean hydrophobic moments. In fact, circular dichroism experiments showed clear transitions from random coil in aqueous solution to an alpha-helical conformation on addition of trifluoroethanol. All four peptides displayed a potent antibacterial activity against selected gram-positive and gram-negative bacteria (minimum inhibitory concentrations in the range 1-8 microM), including some antibiotic resistant strains. Permeabilization of both the outer and cytoplasmic membranes of the gram-negative bacterium, Escherichia coli, by selected peptides was quite rapid and a dramatic drop in colony forming units was observed within 5 min in time-killing experiments. Permeabilization of the cytoplasmic membrane of the gram-positive bacterium, Staphylococcus aureus, was instead initially quite slow, gathering speed after 45 min, which corresponds to the time required for significant inactivation in time-killing studies. The cytotoxic activity of the peptides, determined on several normal and transformed cell lines, was generally low at values within the minimum inhibitory concentration range.     

Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides

Cathelicidins are a novel family of antimicrobial peptide precursors from mammalian myeloid cells. They are characterized by a conserved N-terminal region while the C-terminal antimicrobial domain can vary considerably in both primary sequence and length. Four cathelicidins, proBac5, proBac7, prododecapeptide and proBMAP-28, have been concurrently purified from bovine neutrophils, using simple and rapid methodologies. The correlation of ES-MS data from the purified proteins with their cDNA-deduced sequences has revealed several common features of their primary sequence, such as the presence of N-terminal 5-oxoproline (pyroglutamate) residues and two disulfide bridges in a 1-2, 3-4 arrangement. The N-terminal domains of the cathelicidins present one or two Asp-Pro bonds, which are particularly acid-labile in proBac5 and proBac7, but stable in prododecapeptide. This suggests that the spatial organization around these bonds may vary in different cathelicidins, and favour hydrolysis in some cases. An unexpected feature of the prododecapeptide is that it exists as dimers formed by three possible combinations of its two isoforms. The isolation of a truncated, monomeric form of this protein, lacking the cysteine-containing antimicrobial dodecapeptide, indicates that dimerization occurs via disulfide bridge formation at the level of the C-terminal domain and that the dodecapeptide is likely released as a dimer from its precursor. Sequence-based secondary structure predictions and CD results indicate for cathelicidins a 30-50% content of extended conformation and <20% content of alpha-helical conformation, with the alpha-helical segment placed near the N-terminus. Finally, similarity searching and topology-based structure prediction underline a significant sequential and structural similarity between the conserved N-terminal domain of cathelicidins and cystatin-like domains, placing this family within the cystatin superfamily. When assayed against cathepsin L, unlike the potent cystatin inhibitors, three of the four cathelicidins show only a poor inhibitory activity (Ki = 0.6-3 microM).     

Staphylococcal strains involved in bovine mastitis are inhibited by Staphylococcus aureus antimicrobial peptides

The inhibitory activity of five bacteriocin (Bac)-producer strains of Staphylococcus aureus was tested against bacteria pathogenic for cattle. Sixty-five epidemiologically unrelated strains of Staph. aureus involved in bovine mastitis were used as indicators in an agar diffusion test. Bacteriocins produced by four strains could inhibit only a limited number of test organisms. However, all 65 indicator strains proved to be susceptible to the combined action of both bacteriocins encoded by pRJ9, a Bac plasmid found in strain A53. Therefore, the bacteriocins produced by this strain may represent new antimicrobial peptides with potential applications in the prevention and treatment of bovine mastitis.     

Chemostat production of plantaricin C by Lactobacillus plantarum LL441

Plantaricin C, a bacteriocin synthesized by Lactobacillus plantarum LL441, was optimally produced in chemostats kept at pH 5.0, 30 degreesC, 150 rpm, and a dilution rate of 0.05 h-1 when glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h-1 when sucrose or fructose was used instead. Production was abolished at high dilution rates, i.e., when the cells grew rapidly in all carbon sources.     

The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.     

Synthetic peptides corresponding to the beta-hairpin loop of rabbit defensin NP-2 show antimicrobial activity

Mammalian defensins, a class of antibacterial peptides, are composed of 29-35 amino acids with six cysteines which form three disulfide bonds. Structural studies indicate a triple stranded beta-sheet structure with a well defined beta-hairpin loop at the C-terminal region. It is demonstrated in this report that 18 and 26 residue synthetic peptides corresponding to the beta-hairpin region, constrained by a single disulfide bond, have potent antimicrobial activity without hemolytic activity. Circular dichroism spectroscopy indicates that the single S-S bridge appears to constrain the peptides to a beta-structure. Peptides corresponding to the beta-hairpin region of defensins could thus be attractive candidates as therapeutic agents as well as good model compounds for investigation of the various physiological actions of defensins.     

Ranatuerins: antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana

Nine peptides, termed ranatuerins 1-9, with antimicrobial activity towards Staphylococcus aureus, were isolated from an extract of the skin of the adult American bullfrog, Rana catesbeiana. In common with other cytolytic peptides from Ranid frogs, (e.g. ranalexin, gaegurins, brevinins), ranatuerins 1 and 4 contain an intramolecular disulfide bridge forming a heptapeptide ring whereas in ranatuerins 2 and 3 the disulfide bridge forms a hexapeptide ring. The structurally related ranatuerins 5-9 comprise 12 - 14 amino acids and show sequence similarity towards the hemolytic peptides A1 and B9 previously isolated from the skin of Rana esculenta. Of the peptides purified, ranatuerin 1 (SMLSVLKNLGKVGLG FVACKINKQC) showed the broadest spectrum of antimicrobial action with inhibitory activity against S. aureus, Escherichia coli and Candida albicans.     

Characterization of natural isolate Lactobacillus acidophilus BGRA43 useful for acidophilus milk production

The adherence of P. aeruginosa to collagen membrane, sponge, and to a new anti-infective COLL dressing and the susceptibility of the organisms attached to the biomaterials to amikacin were investigated in vitro. After 17 h of attachment, the bacteria demonstrated an increased resistance to amikacin compared with their free-floating counterparts. Amikacin, even at a concentration exceeding 150 times the minimal bactericidal concentration (MBC) for the strain tested, did not eradicate the attached bacteria from the surface of collagen membrane. However, when the drug at a high concentration (over 16 times the minimal inhibitory concentration, MIC) was present in the incubation medium before it had been inoculated with P. aeruginosa, a reduction of 2 log10 units in the organisms adherent to the surface of collagen membrane was observed. We conclude that slow release of the antibiotic from the COLL dressing could control the bacterial colonization on the surface. In fact, the released amikacin at the final concentration of 32 times the MBC reduced the number of adherent bacteria by 6 log10 units. In contrast, ciprofloxacin at the same final bactericidal concentration completely eradicated the bacteria from the surface of COLL dressing. However, as ciprofloxacin is not recommended for use as a topical antimicrobial agent, a further search is needed to find an agent with a similar anticolonization activity.     

Optimization of exopolysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus RR grown in a semidefined medium

The role of K+ channels in the nitric oxide-independent renal vasodilator effect of acetylcholine (Ach) was examined to address the hypothesis that the mechanism underlying this response was different from that of bradykinin, because an earlier study indicated the possibility of different mediators. We used the rat isolated, perfused kidney that was constricted with phenylephrine and treated with nitroarginine and indomethacin to inhibit nitric oxide synthase and cyclooxygenase, respectively. The nonspecific K+ channel inhibitors, procaine and tetraethylammonium (TEA), reduced vasodilator responses to Ach and cromakalim, but not those to nitroprusside. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to Ach or nitroprusside. Charybdotoxin, an inhibitor of Ca(++)-activated K+ channels, reduced vasodilator responses to Ach without affecting those to cromakalim or nitroprusside. Iberiotoxin and apamin, inhibitors of large- and small-conductance Ca(++)-activated K+ channels, respectively, did not reduce vasodilation induced by Ach, cromakalim or nitroprusside. The inhibitor of cytochrome P450, clotrimazole, reduced the renal vasodilator effects of Ach and bradykinin but not those of nitroprusside or SCA 40, an agonist for Ca(++)-activated K+ channels. These results suggest that in the rat kidney, Ach, like bradykinin, utilizes a charybdotoxin-sensitive Ca(++)-activated K+ channel of intermediate conductance to elicit vasodilation and that this effect may be dependent on cytochrome P450 activity.     

Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis

We have isolated from the blood of immune-challenged and untreated mussels (Mytilus edulis) antibacterial and antifungal peptides. We have characterized two isoforms of a novel 34-residue, cysteine-rich, peptide with potent bactericidal activity and partially characterized a novel 6.2-kDa antifungal peptide containing 12 cysteines. We report the presence of two members of the insect defensin family of antibacterial peptides and provide a phylogenetic analysis that indicates that mollusc and arthropod defensins have a common ancestry. Our data argue that circulating antimicrobial peptides represent an ancient host defense mechanism that predated the separation between molluscs and arthropods at the root of the Cambrian, about 545 million years ago.     

Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia

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