阳离子抗菌肽的研究进展

阳离子抗菌肽(Cationic antibacterial peptides)是生物体抵御外源性病原微生物的入侵而产生的一类小分子阳离子多肽,与传统的抗生素相比具有分子量小、抗菌谱广、热稳定性好、抗菌机理独特等优点。本文结合当今阳离子抗菌肽的研究现状和发展前景,从阳离子抗菌肽的理化性质、作用机理及其设计合成等方面进行了综述。     

Rational design of bifunctional chimeric peptides that combine antimicrobial and titanium binding activity

Bacterial biofilm formation remains a serious problem for clinical materials and often leads to implant failure. To counteract bacterial adhesion, which initiates biofilm formation, the development of antibiotic surface coating strategies is of high demand and warrants further investigations. In this study, we have created bifunctional chimeric peptides by fusing the recently developed antimicrobial peptide MGD2 (GLRKRLRKFFNKIKF) with different titanium-binding sequences. The novel peptides were investigated regarding their antibacterial potential against a set of different bacterial strains including drug-resistant Staphylococcus aureus. All peptides showed high antimicrobial activities both when in solution and when immobilized on titanium surfaces. Owing to the ease of synthesis and handling, the herein described peptides might be a true alternative to prevent bacterial biofilm formation.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

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.     

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.     

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.     

Role of membranes in the activities of antimicrobial cationic peptides

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.     

分子串联重复策略用于制备超短肽

超短肽具有更好的稳定性、组织渗透性、生物相容性以及更低的免疫原性。GHK(glycyl-l-histidyl-l-lysine)和GQPR(glycyl-l-glutamyl-l-prolyl-l-arginine)具有刺激胶原蛋白产生、减缓胶原蛋白降解的作用,作为抗皱成分广泛应用于化妆品。超短肽一般都是通过固相合成方法制备,其缺陷是制备过程中大量使用有机化学试剂而造成环境负担,故本文探讨了一种设计和制备超短肽的新方法。因序列短而无法直接重组表达,文中首先构建了适用于融合表达的载体骨架pET28a-Trxm。以GHK和GQPR串联重复基因作为滚环扩增的基本单元(tandem repeat of short peptides,TRSP),反应时随机掺入5-甲基胞嘧啶获得长基因片段,然后经Acc65 Ⅰ和Apa Ⅰ消化产生随机长度的基因。胶回收500 bp到1500bp的DNA片段,克隆得到表达载体pET28a-Trxm-(TRSP)_n并转化获得重组菌。双酶切及测序结果表明,成功构建获得串联重复数n=1、2、3、4、6、7、8、9的阳性克隆。蛋白表达结果显示,当串联重复数n=1、2、3、4、8、9时均有相应融合蛋白表达,表达水平随着重复数增加而降低。Trxm-(TRSP)₁表达水平最高,达总蛋白的50%,而Trxm-(TRSP)₂表达水平为总蛋白的30%。进一步地,含Trxm-(TRSP)₁的清液先后经肠激酶和胰蛋白酶切割后,HPLC分析结果表明,成功获得超短肽GHK和GQPR。该结果对于超短肽重组制备的工业化应用具有重要价值。     

Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.     

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.     

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.     

Generation of novel cationic antimicrobial peptides from natural non-antimicrobial sequences by acid-amide substitution

Background

The accessory gene regulator (agr) is a quorum sensing cluster of genes which control colonization and virulence in Staphylococcus aureus. We evaluated agr function in community- (CA) and healthcare-associated (HA) MRSA, to compare the pharmacodynamics and bactericidal activity of vancomycin against agr functional and dysfunctional HA-MRSA and CA-MRSA.

Methods

40 clinical isolates of MRSA from the Canadian Nosocomial Infection Surveillance Program were evaluated for delta-haemolysin production, as a surrogate marker of agr function. Time kill experiments were performed for vancomycin at 0 to 64 times the MIC against an initial inoculum of 10⁶ and 10⁸ cfu/ml of agr functional and dysfunctional CA-MRSA and HA-MRSA and these data were fit to a hill-type pharmacodynamic model.

Results

15% isolates were agr dysfunctional, which was higher among HA-MRSA (26.3%) versus CA-MRSA (4.76%). Against a low initial inoculum of 10⁶ cfu/ml of CA-MRSA, vancomycin pharmacodynamics were similar among agr functional and dysfunctional strains. However, against a high initial inoculum of 10⁸ cfu/ml, killing activity was notably attenuated against agr dysfunctional CA-MRSA (USA400) and HA-MRSA (USA100). CA-MRSA displayed a 20.0 fold decrease in the maximal reduction in bacterial counts (Emax) which was 3.71 log₁₀ CFU/ml for agr functional vs. 2.41 log₁₀ CFU/ml for agr dysfunctional MRSA (p = 0.0007).

Conclusions

Dysfunction in agr was less common among CA-MRSA vs. HA-MRSA. agr dysfunction demonstrated an impact on vancomycin bactericidal activity and pharmacodynamics against a high initial inoculum of CA-MRSA and HA-MRSA, which may have implications for optimal antimicrobial therapy against persistent, difficult to treat MRSA infections.     

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

Antimicrobial peptides (AMPs), with their extraordinary properties, such as broad-spectrum activity, rapid action and difficult development of resistance, have become promising molecules as new antibiotics. Despite their various mechanisms of action, the interaction of AMPs with the bacterial cell membrane is the key step for their mode of action. Moreover, it is generally accepted that the membrane is the primary target of most AMPs, and the interaction between AMPs and eukaryotic cell membranes (causing toxicity to host cells) limits their clinical application. Therefore, researchers are engaged in reforming or de novo designing AMPs as a ‘single-edged sword’ that contains high antimicrobial activity yet low cytotoxicity against eukaryotic cells. To improve the antimicrobial activity of AMPs, the relationship between the structure and function of AMPs has been rigorously pursued. In this review, we focus on the current knowledge of α-helical cationic antimicrobial peptides, one of the most common types of AMPs in nature.     

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 co-evolution of host cationic antimicrobial peptides and microbial resistance

Endogenous cationic antimicrobial peptides (CAMPs) are among the most ancient and efficient components of host defence. It is somewhat of an enigma that bacteria have not developed highly effective CAMP-resistance mechanisms, such as those that inhibit many therapeutic antibiotics. Here, we propose that CAMPs and CAMP-resistance mechanisms have co-evolved, leading to a transient host-pathogen balance that has shaped the existing CAMP repertoire. Elucidating the underlying principles of this process could help in the development of more sustainable antibiotics.     

The role of cationic antimicrobial peptides in innate host defences

Cationic antimicrobial peptides are found in all living species. A single animal can contain >24 different antimicrobial peptides, which fall into four structural classes. These peptides are produced in large quantities at sites of infection and/or inflammation and can have broad-spectrum antibacterial, antifungal, antiviral, antiprotozoan and antisepsis properties. In addition, they interact directly with host cells to modulate the inflammatory process and innate defences.     

Rational design of alpha-helical antifreeze peptides.

The alanine-rich alpha-helical antifreeze protein from the winter flounder Pseudopleuronectes americanus adsorbs to specific planes of ice guided by an ice lattice match to threonine residues regularly spaced 16.6 A apart. We report here that by redesigning the winter flounder antifreeze peptide to incorporate a 27.1-A spacing between putative 'ice-binding' threonines, the deduced binding alignment of the helical molecule on the ice lattice is changed from the Miller indices directional vector [1102 ] to [2203 ]. Subsequent ice-binding characteristics are altered, including changes in adsorption specificity, decreases in thermal hysteresis activity and the formation of rotated hexagonal bipyramid ice crystal morphology.     

Basolateral targeting by leucine-rich repeat domains in epithelial cells

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure–function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.