Correlations of cationic charges with salt sensitivity and microbial specificity of cystine-stabilized beta -strand antimicrobial peptides

The electrostatic interaction of the charge cluster of an amphipathic peptide antibiotic with microbial membranes is a salt-sensitive step that often determines organism specificity. We have examined the correlation between charge clusters and salt insensitivity and microbial specificity in linear, cyclic, and retro-isomeric cystine-stabilized beta-strand (CSbeta) tachyplesin (TP) in a panel of 10 test organisms. Cyclic tachyplesins consisting of 14 and 18 amino acids are constrained by an end-to-end peptide backbone and two or three disulfide bonds to cross-brace the anti-parallel beta-strand that approximates a "beta-tile" structure. Circular dichroism measurements of beta-tile TPs showed that they displayed ordered structures. Control peptides containing the same number of basic amino acids as TP but lacking disulfide constraints were highly salt sensitive. Cyclic TP analogues with six cationic charges were more broadly active and salt-insensitive than those with fewer cationic charges. Reducing their proximity or number of cationic charges, particularly those with three or fewer basic amino acids, led to a significant decrease in potency and salt insensitivity, but an increased selectivity to certain Gram-positive bacteria. An end-group effect of the dibasic N-terminal Lys of TP in the open-chain TP and its retroisomer was observed in certain Gram-negative bacteria under high-salt conditions, an effect that was not found in the cyclic analogs. These results suggest that a stable folded structure together with three or more basic amino acids closely packed in a charged region in CSbeta peptides is important for salt insensitivity and organism specificity.     

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, delta-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be 'special cases' within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.     

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, δ-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be ‘special cases’ within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.     

The Bacillus subtilis extracytoplasmic-function sigmaX factor regulates modification of the cell envelope and resistance to cationic antimicrobial peptides

Bacillus subtilis contains seven extracytoplasmic-function sigma factors that activate partially overlapping regulons. We here identify four additional members of the sigma(X) regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (D-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with D-alanine and incorporation of phosphatidylethanolamine into the cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the cell envelope. The resulting reduction in the net negative charge of the cell envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type cells. We conclude that activation of the sigma(X) regulon serves to alter cell surface properties to provide protection against antimicrobial peptides.     

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.     

Temperature-regulated efflux pump/potassium antiporter system mediates resistance to cationic antimicrobial peptides in Yersinia

Most bacterial pathogens are resistant to cationic antimicrobial peptides (CAMPs) that are key components of the innate immunity of both vertebrates and invertebrates. In Gram-negative bacteria, the known CAMPs resistance mechanisms involve outer membrane (OM) modifications and specifically those in the lipopolysaccharide (LPS) molecule. Here we report, the characterization of a novel CAMPs resistance mechanism present in Yersinia that is dependent on an efflux pump/potassium antiporter system formed by the RosA and RosB proteins. The RosA/RosB system is activated by a temperature shift to 37 degrees C, but is also induced by the presence of the CAMPs, such as polymyxin B. This is the first report of a CAMPs resistance system that is induced by the presence of CAMPs. It is proposed that the RosA/RosB system protects the bacteria by both acidifying the cytoplasm to prevent the CAMPs action and pumping the CAMPs out of the cell.     

Probiotics manipulate host cytokine response and induce antimicrobial peptides

Probiotics modulate production of both cytokine and antimicrobial peptides. This effect can be regarded as a part of complex interplay between them and the host.     

Epithelial antimicrobial peptides in host defense against infection

One component of host defense at mucosal surfaces seems to be epithelium-derived antimicrobial peptides. Antimicrobial peptides are classified on the basis of their structure and amino acid motifs. Peptides of the defensin, cathelicidin, and histatin classes are found in humans. In the airways, α-defensins and the cathelicidin LL-37/hCAP-18 originate from neutrophils. β-Defensins and LL-37/hCAP-18 are produced by the respiratory epithelium and the alveolar macrophage and secreted into the airway surface fluid. Beside their direct antimicrobial function, antimicrobial peptides have multiple roles as mediators of inflammation with effects on epithelial and inflammatory cells, influencing such diverse processes as proliferation, immune induction, wound healing, cytokine release, chemotaxis, protease-antiprotease balance, and redox homeostasis. Further, antimicrobial peptides qualify as prototypes of innovative drugs that might be used as antibiotics, anti-lipopolysaccharide drugs, or modifiers of inflammation.     

Interaction of cationic antimicrobial peptides with Mycoplasma pulmonis

We investigated the mode of action underlying the anti-mycoplasma activity of cationic antimicrobial peptides (AMPs) using four known AMPs and Mycoplasma pulmonis as a model mycoplasma. Scanning electron microscopy revealed that the integrity of the M. pulmonis membrane was significantly damaged within 30 min of AMPs exposure, which was confirmed by measuring the uptake of propidium iodine into the mycoplasma cells. The anti-mycoplasma activity of AMPs was found to depend on the binding affinity for phosphatidylcholine, which was incorporated into the mycoplasma membrane from the growth medium and preferentially distributed in the outer leaflet of the lipid bilayer.     

The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B

A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein’s C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.     

The role of Paneth cells and their antimicrobial peptides in innate host defense

The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.     

肠道微生态系统及其与宿主的协同进化

肠道微生态系统是寄生在宿主肠道内的微生物的总和。微生物进入肠道后,通过一个复杂的过程形成群落,与宿主之间相互作用,形成共生关系。宿主客观上为微生物提供生存和进化场所,微生态系统为宿主提供营养物质、刺激肠道组织的发育、刺激宿主肠道免疫系统的发育、影响宿主能量代谢、协助宿主降解有毒物质、影响宿主生殖活动和寿命等功能。作为一个进化的系统,微生态系统的物种多样性和丰富度对维持宿主正常生理功能具有重要作用,但同时又受宿主的影响,物种间相互作用和宿主-微生物间的相互作用是微生态系统进化的动力。进化主要表现在微生物和宿主基因组上发生适应性变化。因此,系统生态学的理论对理解肠道微生态系统的运行机制和临床应用具有重要指导作用。     

Membrane interactions of designed cationic antimicrobial peptides: the two thresholds

Novel cationic antimicrobial peptides (CAPs) designed in our lab-typified by sequences such as KKKKKKAAX-AAXAAXAA-NH(2), where X = Phe/Trp-display high antibacterial activity but exhibit little or no hemolytic activity towards human red blood cells even at high doses. To clarify the mechanism of their selectivity for bacterial versus mammalian membranes and to increase our understanding of the relationships between primary sequence and bioactivity, a library of derivatives was prepared by increasing segmental hydrophobicity, in which systematic substitutions of Ala for two, three, or four Leu residues were made. Conformationally constrained dimeric and cyclic derivatives were also synthesized. The peptides were examined for activity against pathogenic bacteria (Pseudomonas aeruginosa), hemolytic activity on human red blood cells, and insertion into models of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol). Results were compared with corresponding properties of the natural CAPs magainin and cecropin. Using circular dichroism and fluorescence spectroscopy, we found that peptide conformation and membrane insertion were sequence dependent, both upon the number of Leu residues, and upon their positions along the hydrophobic core. Membrane disruption was likely enhanced by the fact that the peptides contain potent dimerization-promoting sequence motifs, as assessed by SDS-PAGE gel analysis. The overall results led us to identify distinctions in the mechanism of actions of these CAPs for disruption of bacterial versus mammalian membranes, the latter dependent on surpassing a "second hydrophobicity threshold" for insertion into zwitterionic membranes.     

Role of antimicrobial peptides in host defense against mycobacterial infections

Worldwide, tuberculosis remains the most important infectious disease causing morbidity and death. Currently, at least one-third of the world's population is infected with Mycobacterium tuberculosis. In addition, the World Health Organization estimates that about 8-10 million new tuberculosis cases occur annually worldwide and this incidence is currently increasing. Moreover, multidrug-resistant tuberculosis has been increasing in incidence in many areas during the past decade. These situations underscore the importance of the development of new therapeutic agents against mycobacterial infectious diseases. In this article, it is review current progress in the understanding of antimicrobial peptides as potential candidates to develop an alternative/adjunct therapeutic strategy against tuberculosis. This immunoadjunctive therapy might be evaluated in the context of possible drug resistance. This review also summarizes the knowledge about the functions of antimicrobial peptides in the pulmonary innate host defense system and their role in mycobacterial infection, and at the same time outlines recent advances in our understanding of the combined effect of antimicrobial peptides and anti-tuberculosis drugs against intracellular mycobacteria. A concerted effort should now focus on the clinical application of antimicrobial peptides for their practical use.     

Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions

Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH(2)) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH(2)). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μM, but the four-Leu peptides induced 40-80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4-32 μM). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes.     

Conformation and interaction of the cyclic cationic antimicrobial peptides in lipid bilayers.

To investigate the role of peptide-membrane interactions in the biological activity of cyclic cationic peptides, the conformations and interactions of four membrane-active antimicrobial peptides [based on Gramicidin S (GS)] were examined in neutral and negatively charged micelles and phospholipid vesicles, using CD and fluorescence spectroscopy and ultracentrifugation techniques. Moreover, the effects of these peptides on the release of entrapped fluorescent dye from unilamellar vesicles of phosphatidylcholine (PC) and phosphatidylethanolamine/phosphatidylglycerol (PE/PG) were studied. The cyclic peptides include GS10 [Cyclo(VKLdYP)2], GS12 [Cyclo(VKLKdYPKVKLdYP)], GS14 [Cyclo(VKLKVdYPLKVKLdYP)] and [d-Lys]4GS14 [Cyclo(VKLdKVdYPLKVKLdYP)] (underlined residues are d-amino acids), were different in their ring size, structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. Interaction of the peptides with the zwitterionic PC and negatively charged PE/PG vesicles were distinct from each other. The hydrophobic interaction seems to be the dominant factor in the hemolytic activity of the peptides, as well as their interaction with the PC vesicles. A combination of electrostatic and hydrophobic interactions of the peptides induces aggregation and fusion in PE/PG vesicles with different propensities in the order: [d-Lys]4GS14 > GS14 > GS12 > GS10. GS10 and GS14 are apparently located in the deeper levels of the membrane interfaces and closer to the hydrophobic core of the bilayers, whereas GS12 and [d-Lys]4GS14 reside closer to the outer boundary of the interface. Because of differing modes of interaction of the cyclic cationic peptides with lipid bilayers, the mechanism of their biological activity (and its relation to peptide-lipid interaction) proved to be versatile and complex, and dependent on the biophysical properties of both the peptides and membranes.     

Influence of tryptophan on lipid binding of linear amphipathic cationic antimicrobial peptides

We recently demonstrated that a linear 18-residue peptide, (KIGAKI)(3)-NH(2), designed to form amphipathic beta-sheet structure when bound to lipid bilayers, possessed potent antimicrobial activity and low hemolytic activity. The ability of (KIGAKI)(3)-NH(2) to induce leakage from lipid vesicles was compared to that of the amphipathic alpha-helical peptide, (KIAGKIA)(3)-NH(2), which had equivalent antimicrobial activity. Significantly, the lytic properties of (KIGAKI)(3)-NH(2) were enhanced for mixed acidic-neutral lipid vesicles containing phosphatidylethanolamine instead of phosphatidylcholine as the neutral component, while the potency of (KIAGKIA)(3)-NH(2) was significantly reduced [Blazyk, J., et al. (2001) J. Biol. Chem. 276, 27899-27906]. In this paper, we measured the lytic properties of these peptides, as well as several fluorescent analogues containing a single tryptophan residue, by monitoring permeability changes in large unilamellar vesicles with varying lipid compositions and in Escherichia coli cells. The binding of these peptides to lipid bilayers with defined compositions was compared using surface plasmon resonance, circular dichroism, and fluorescence spectroscopy. Surprisingly large differences were observed in membrane binding properties, particularly in the case of KIGAKIKWGAKIKIGAKI-NH(2). Since all of these peptides possess the same charge and very similar mean hydrophobicities, the binding data cannot be explained merely in terms of electrostatic and/or hydrophobic interactions. In light of their equivalent antimicrobial and hemolytic potencies, some of these peptides may employ mechanisms beyond simply increasing plasma membrane permeability to exert their lethal effects.     

Biotic stress resistance in agriculture through antimicrobial peptides

Antimicrobial peptides (AMPs) are the hosts' defense molecules against microbial pathogens and gaining extensive research attention worldwide. These have been reported to play vital role of host innate immunity in response to microbial challenges. AMPs can be used as a natural antibiotic as an alternative of their chemical counterpart for protection of plants/animals against diseases. There are a number of sources of AMPs including prokaryotic and eukaryotic organisms and are present, both in vertebrates and invertebrates. AMPs can be classified as cationic or anionic, based on net charges. Large number of databases and tools are available in the public domain which can be used for development of new genetically modified disease resistant varieties/breeds for agricultural production. The results of the biotechnological research as well as genetic engineering related to AMPs have shown high potential for reduction of economic losses of agricultural produce due to pathogens. In this article, an attempt has been made to introduce the role of AMPs in relation to plants and animals. Their functional and structural characteristics have been described in terms of its role in agriculture. Different sources of AMPs and importance of these sources has been reviewed in terms of its availability. This article also reviews the bioinformatics resources including different database tools and algorithms available in public domain. References of promising biotechnology research in relation to AMPs, prospects of AMPs for further development of genetically modified varieties/breeds are highlighted. AMPs are valuable resource for students, researchers, educators and medical and industrial personnel.     

Rational design of cationic antimicrobial peptides by the tandem of leucine-rich repeat

Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane–peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.     

Staphylococcal resistance to antimicrobial peptides of mammalian and bacterial origin

Antimicrobial host defense peptides, such as defensins, protegrins, and platelet microbicidal proteins are deployed by mammalian skin, epithelia, phagocytes, and platelets in response to Staphylococcus aureus infection. In addition, staphylococcal products with similar structures and activities, called bacteriocins, inhibit competing microorganisms. Staphylococci have developed resistance mechanisms, which are either highly specific for certain host defense peptides or bacteriocins or which broadly protect against a range of cationic antimicrobial peptides. Experimental infection models can be used to study the molecular mechanisms of antimicrobial peptides, the peptide resistance strategies of S. aureus, and the therapeutic potential of peptides in staphylococcal diseases.