iTRAQ-coupled 2-D LC-MS/MS analysis of membrane protein profile in Escherichia coli incubated with apidaecin IB

Apidaecins are a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. They are predominantly active against the gram-negative bacteria. Previous studies mainly focused on the identification of their internal macromolecular targets, few addressed on the action of apidaecins on the molecules, especially proteins, of bacterial cell membrane. In this study, iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered membrane proteins of Escherichia coli cells incubated with one isoform of apidaecins--apidaecin IB. Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB. Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids. Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.     

iTRAQ-coupled 2-D LC-MS/MS analysis of cytoplasmic protein profile in Escherichia coli incubated with apidaecin IB

Apidaecins refer to a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. Accumulating evidence that proline-rich antimicrobial peptides are not-toxic to human and animal cells makes them potential candidates for the development of novel antibiotic drugs. However, the mechanism of action was not fully understood. In this study, antibacterial mechanism of apidaecins was investigated. iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered cytoplasmic proteins of Escherichia coli incubated with one isoform of apidaecins--apidaecin IB. The production of the chaperonin GroEL and its cofactor GroES, which together form the only essential chaperone system in E. coli cytoplasm under all growth conditions, was decreased in cells incubated with apidaecin IB. The decreasing of the GroEL-GroES chaperone team was further found to be involved in a new antibacterial mechanism of apidaecins. Our findings therefore provide important new insights into the antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.     

Experimental and simulation studies reveal mechanism of action of human defensin derivatives

Antimicrobial peptides (AMPs) are naturally occurring promising candidates which can be used as antibiotics against a wide variety of bacteria. The key component for using them as a potent antibiotic is that their mechanism of action is less prone to bacterial resistance. However, the molecular details of their mechanism of action is not yet fully understood. In this study, we try to shed light on the mode of action of AMPs, possible reason behind it, and their interaction with lipid bilayers through experimental as well as molecular dynamics (MD) simulation studies. The focal of our study was Human beta defensin 3 (hBD-3) which is a naturally occurring AMP. We chose three derivatives of hBD-3, namely CHRG01, KSR, and KLR for the detailed analysis presented in this study. These three peptides are evaluated for their antibacterial potency, secondary structure analysis and mechanism of action. The experimental results reveal that these peptides are active against gram positive as well as gram negative bacteria and kill bacteria by forming membrane pores. The MD simulation results correlate well with the antibacterial activity and shed light into the early membrane insertion dynamics. Moreover, the specific amino acids responsible for membrane disruptions are also identified from the MD simulations. Understanding the molecular level interaction of individual amino acids with the lipid bilayer will greatly help in the design of more efficient antimicrobial peptides.     

Cathelicidin-derived Trp/Pro-rich antimicrobial peptides with lysine peptoid residue (Nlys): therapeutic index and plausible mode of action.

Recently, we designed a novel cell-selective antimicrobial peptide (TPk) with intracellular mode of action from Pro --> Nlys (Lys peptoid residue) substitution in a noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptide, tritrpticin-amide (TP; VRRFPWWWPFLRR-NH(2)) (Biochemistry 2006; 45: 13007-13017). In this study, to elucidate the effect of Pro --> Nlys substitution on therapeutic index and mode of action of other noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptides and develop novel short antimicrobial peptides with high cell selectivity/therapeutic index, we synthesized Nlys-substituted antimicrobial peptides, TPk, STPk and INk, in which all proline residues of TP, symmetric TP-analogue (STP; KKFPWWWPFKK-NH(2)) and indolicidin (IN; ILPWKWPWWPWRR-NH(2)) were replaced by Nlys, respectively. Compared to parent Pro-containing peptides (TP, STP and IN), Nlys substituted peptides (TPk, STPk and Ink) had 4- to 26-fold higher cell selectivity/therapeutic index. Parent Pro-containing peptides induced a significant depolarization of the cytoplasmic membrane of intact Staphylococcus aureus at their MIC, whereas Nlys-substituted antimicrobial peptides did not cause visible membrane depolarization at their MIC. These results suggest that the antibacterial action of Nlys-substituted peptides is probably not due to the disruption of bacterial cytoplasmic membranes but the inhibition of intracellular components. Taken together, our results showed that Pro --> Nlys substitution in other noncell-selective Trp/Pro-rich antimicrobial peptides such as STP and IN as well as TP can improve the cell selectivity/therapeutic index and change the mode of antibacterial action from membrane-disrupting to intracellular targeting. In conclusion, our findings suggested that Pro --> Nlys substitution in noncell-selective Trp/Pro-rich antimicrobial peptides is a promising method to develop cell-selective antimicrobial peptides with intracellular target mechanism.     

Apidaecins: antibacterial peptides from honeybees.

Although insects lack the basic entities of the vertebrate immune system, such as lymphocytes and immunoglobulins, they have developed alternative defence mechanisms against infections. Different types of peptide factors, exhibiting bactericidal activity, have been detected in some insect species. These humoral factors are induced upon infection. The present report describes the discovery of the apidaecins, isolated from lymph fluid of the honeybee (Apis mellifera). The apidaecins represent a new family of inducible peptide antibiotics with the following basic structure: GNNRP(V/I)YIPQPRPPHPR(L/I). These heat-stable, non-helical peptides are active against a wide range of plant-associated bacteria and some human pathogens, through a bacteriostatic rather than a lytic process. Chemically synthesized apidaecins display the same bactericidal activity as their natural counterparts. While only active antibacterial peptides are detectable in adult honeybee lymph, bee larvae contain considerable amounts of inactive precursor molecules.     

Cell Penetrating Apidaecin Peptide Interactions with Biomimetic Phospholipid Membranes

Apidaecin peptides from Apis mellifera hemolymph are believed to attack intracellular bacterial targets. Our in vivo results for apidaecins 1a and 1b confirm that bacterial activity is non-lytic, however, the manner in which these peptides pass through the cell membrane to exert this activity is unknown. These data are combined with fluorescence (dye leakage) and quartz crystal microbalance studies to investigate the membrane interaction for these two wildtype peptides. It was found that the peptides penetrate the membrane in a trans-membrane manner. The amount of peptide uptake by the membrane is proportional to the concentration of the peptide, however, this appears to be a dynamic equilibrium which can be almost completely reversed by addition of buffer medium. Interestingly, a small residual mass remains within the membrane and the amount of peptide remaining in the membrane is a function of the buffer-salt concentration viz. in high salt, the residual peptide mass remaining is small whereas at low salt concentration, a larger mass of peptide remains bound. These results support a direct membrane penetration mechanism by the wild type apidaecins 1a and 1b. In both cases the peptide–membrane interaction has a negligible effect on the membrane, although, in high salt a permanent change in the membrane does occur at the highest peptide concentration which does not recover following peptide removal. Stefania Piantavigna and Patricia Czihal contributed equally to this article.     

Mapping of Apidaecin Regions Relevant for Antimicrobial Activity and Bacterial Internalization

Apidaecins are 18–20-residue long proline-rich peptides expressed in insects as part of the innate immune system. They are very active against Gram-negative bacteria, especially Enterobacteriaceae. The C-terminal sequence PRPPHPRL is highly conserved, whereas the N-terminal region is variable. By replacing all 18 residues of apidaecin 1a and apidaecin 1b individually by alanine (Ala-scan), we have shown that single mutations in the C-terminal half of the peptides drastically reduced and mostly abolished the antibacterial activity against Escherichia coli. Conversely, substitutions in the N-terminal eight residues produced no, or only minor effects. The activity loss was correlated to the ability of apidaecin 1b and its mutants to enter Gram-negative bacteria, most likely because they no longer bind to a protein transporter. This assumed binding, however, was not inhibited by truncated apidaecin peptides added at tenfold higher concentrations. Interestingly, the antibacterial activity of full length apidaecin 1b was enhanced about four times by addition of a N-terminally truncated apidaecin peptide [11–18]-apidaecin 1b, as indicated by lower MIC-values against E. coli, although the short 5(6)-carboxyfluorescein-labeled peptide did not enter the bacteria. In contrast, the activity against the Gram-positive bacterium Micrococcus luteus was not located in the C-terminal sequence of apidaecins 1a and b, but depended mostly on the presence of all four basic residues.     

Dual mode of action of Bac7, a proline-rich antibacterial peptide

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.     

Membrane active antimicrobial activity and molecular dynamics study of a novel cationic antimicrobial peptide polybia-MPI,from the venom of Polybia paulista

As the frequent emergence of the resistant bacteria, the development of new agents with a new action mode attracts a great deal of interest. It is now widely accepted that antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics. In this study, antimicrobial peptide polybia-MPI and its analogs were synthesized and their antibacterial activity was studied. Our results revealed that polybia-MPI has potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Its ability to make PI permeate into bacteria and lead to the leakage of calcein from model membrane LUVs, suggests a killing mechanism involving membrane perturbation. SEM and TEM microscopy experiments verified that the morphology of bacteria was changed greatly under the treatment of polybia-MPI. Compared with the conventional chemotherapy, polybia-MPI targets the cell membrane rather than entering into the cell to exert its antibacterial activity. Furthermore, molecular dynamics (MD) simulations were employed to investigate the mechanism of membrane perturbation. The results indicated that the α-helical conformation in the membrane is required for the exhibition of antibacterial activity and the membrane disturbance by polybia-MPI is a cooperative process. In conclusion, with the increasing resistance to conventional antibiotics, there is no doubt that polybia-MPI could offer a new strategy to defend the resistant bacteria.     

Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes

Antimicrobial peptides have been extensively studied in order to elucidate their mode of action. Most of these peptides have been shown to exert a bactericidal effect on the cytoplasmic membrane of bacteria. Lactoferricin is an antimicrobial peptide with a net positive charge and an amphipatic structure. In this study we examine the effect of bovine lactoferricin (lactoferricin B; Lfcin B) on bacterial membranes. We show that Lfcin B neither lyses bacteria, nor causes a major leakage from liposomes. Lfcin B depolarizes the membrane of susceptible bacteria, and induces fusion of negatively charged liposomes. Hence, Lfcin B may have additional targets responsible for the antibacterial effect.     

Structure–activity studies on magainins and other host defense peptides

Host defense peptides are widely distributed in nature, being found in species from bacteria to humans. The structures of these peptides from insects, horseshoe crabs, frogs, and mammals are known to have the common features of a net cationic charge due to the presence of multiple Arg and Lys residues and in most cases the ability to form amphipathic structures. These properties are important for the mechanism of action that is thougln to be a nonreceptor-mediated interaction with the anionic phospholipids of the target cell followed by incorporation into the membrane and disruption of the membrane structure. Host defense peptides have been shown to have broad spectrum antimicrobial activity, able to kill most strains of bacteria as well as some fungi, protozoa, and in addition, many types of tumor cells. Specificity for pathogenic cells over host cells is thought to be due to the composition of the cell membranes, with an increased proportion of anionic phospholipids making the pathogen more susceptible and the presence of cholesterol making the host membranes more resistant. Structure–activity relationship studies have been performed on insect cecropins and apidaecins. horseshoe crab tachyplesins and polyphemusins. and the frog magainins. CPFs (caerulein precursor fragments) and PGLa. In general, changes that increased the basicity and stabilized the amphipathic structure have increased the antimicrobial activity: however, as the peptides become more hydrophobic the degree of specificity decreases. One magainin-2 analogue. MSI-78. has been developed by Magainin Pharmaceuticals as a topical antiinefective and is presently in clinical trials for the treatment of infected diabetic foot ulcers. © 1994 John Wiley & Sons, Inc.     

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1-3 with model membranes investigated by isothermal titration calorimetry

Human-β-defensins HBD-1-3 are important components of the innate immune system. Synthetic peptides Phd-1-3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1-3, have antimicrobial activity. The interaction of Phd-1-3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1-3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH~-1.3 to -2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy-Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1-3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1-3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.     

Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides

The initial stages leading to the binding and functioning of membrane-active polypeptides including hormones, signal sequences, and lytic peptides are mainly governed by electrostatic attraction and hydrophobic partitioning between water and lipid bilayers. Antimicrobial peptides serve as an important model for studying the details of these initial steps. However, a systematic analysis of the contribution of multiple hydrophobic amino acids to these steps have been hindered by the propensity of many peptides to aggregate and become inactivated in solution. To this end, we synthesized a series of model amphipathic all L-amino acid peptides and their diastereomers with the sequence KX(3)KWX(2)KX(2)K, where X = Gly, Ala, Val, Ile, or Leu. The effect of the aliphatic amino acids on the biological activity, binding, structure, membrane localization, and mode of action of these peptides was investigated. Most of the L-amino acid peptides oligomerized and adopted distinct structures in solution and in a membrane mimetic environment. Among this group only the Leu containing peptide was hemolytic and highly active on most bacteria tested. The Val- and Leu-containing peptides were hemolytic but inactive toward most bacteria tested. In contrast, the diastereomeric peptides were monomeric and unstructured in solution, but they adopted distinct structures upon membrane binding. While hemolytic activity was drastically reduced, the spectrum of antibacterial activity was preserved or increased. Importantly, we found a direct correlation with the diastereomers between hydrophobicity and propensity to form a helical/distorted-helix and activity (induced membrane leakage and antibacterial activity), despite the fact that they contained 30% D-amino acids. Furthermore, efficient increase in membrane permeability can proceed through different mechanisms. Specifically, the Leu-containing diastereomeric peptide micellized vesicles and possibly bacterial membranes while the Ile-containing diastereomeric peptide fused model membranes and irregularly disrupted bacterial membranes.     

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides' mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties.     

Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.     

Antibacterial activity of peptides derived from the C-terminal region of a hemolytic lectin, CEL-III, from the marine invertebrate Cucumaria echinata

Several synthetic peptides derived from the C-terminal domain sequence of a hemolytic lectin, CEL-III, were examined as to their action on bacteria and artificial lipid membranes. Peptide P332 (KGVIFAKASVSVKVTASLSK-NH(2)), corresponding to the sequence from residue 332, exhibited strong antibacterial activity toward Gram-positive bacteria. Replacement of each Lys in P332 by Ala markedly decreased the activity. However, when all Lys were replaced by Arg, the antibacterial activity increased, indicating the importance of positively charged residues at these positions. Replacement of Val by Leu also led to higher antibacterial activity, especially toward Gram-negative bacteria. The antibacterial activity of these peptides was correlated with their membrane-permeabilizing activity toward the bacterial inner membrane and artificial lipid vesicles, indicating that the antibacterial action is due to perturbation of bacterial cell membranes, leading to enhancement of their permeability. These results also suggest that the hydrophobic region of CEL-III, from which P332 and its analogs were derived, may play some role in the interaction with target cell membranes to trigger hemolysis.     

家蝇抗菌肽的抑菌动力学研究及其机理初探

利用鼠伤寒沙门氏菌针刺诱导家蝇幼虫表达抗菌肽,对抗菌肽的抑菌动力学进行研究,并通过抗菌肽样品对不同细菌动力学特性的研究出发对抗菌肽抑菌作用机制进行探讨。研究发现抗菌肽样品的活性与作用时间有关,24h内出现一到两个活性峰,同一抗菌肽样品对不同细菌的抑菌动力学有差异,抗菌肽的抑菌动力学机制应该与它的的抑菌作用机制有关。通过电镜观测、细胞磷代谢、紫外吸收物测定以及抗菌肽与细菌DNA相互作用结果可知,微生物诱导家蝇表达的抗菌肽样品不仅能够造成细菌细胞的快速坍塌破裂而且能够破坏细胞核心,与DNA结合作用。抗菌肽抑菌动力学的解释：微生物诱导产物中含有两类抗菌肽,一类抗菌肽能造成细胞膜的快速坍塌破裂形成第一个活性峰;另一类抗菌肽可进入细胞,破坏细胞核心,造成紫外吸收物大量外泄形成第二个活性峰。     

抗菌肽及其临床应用研究进展

抗菌肽是生物体在抵抗病原微生物的防御反应过程中产生的一类具有抗微生物活性的小分子多肽。抗菌肽是机体天然免疫系统的重要组成部分,具有广谱的抗革兰氏阳性、阴性菌活性,对真菌、某些有包膜的病毒、寄生虫以及肿瘤细胞也有抑制活性。抗菌肽具有不同于传统抗生素的独特抗菌机制,病原菌不宜对其产生耐药性,有可能成为一种新的抗生素替代品。介绍了抗菌肽的来源与分类、理化特性与生物学活性,并重点阐述其最新的临床应用进展。     

家蝇幼虫抗菌肽MDL-2的电泳制备及其抗菌作用机理

通过体壁损伤和感染大肠杆菌同时诱导家蝇Musca domestica幼虫产生免疫血淋巴,经沸水浴热变性,透析浓缩处理,然后经Tricine-SDS-PAGE得到诱导前后家蝇幼虫血淋巴中蛋白差异表达条带,将该条带电泳回收,复性,抗菌活性检测等步骤,分离纯化得到抗菌肽MDL-2,其分子中富含Pro,Gly和碱性氨基酸,分子量为11 kD,对革兰氏阴性菌Escherichia coli和革兰氏阳性菌Staphylococcus aureus均有较强抗性,因此电泳制备抗菌肽的方法为此类生物微量活性物质的分离纯化提供一种行之有效的途径。通过MDL-2对大肠杆菌和金黄色葡萄球菌通透性和透射电镜超微结构的图谱分析,MDL-2首先与细菌的外膜结合,然后抗菌肽形成柔性的两亲空间构象与细胞内膜作用,扰乱了膜脂分子的排列,改变了细胞膜的通透性,影响细胞膜的结构和功能,细胞膜上形成了许多孔道,同时造成细胞内的原生质扩散,并从孔道向胞外渗漏,影响了细菌的代谢系统,最终引起细胞膜破碎,细胞完全解体,从而起到抑菌杀菌作用。     

Membrane-active peptides and the clustering of anionic lipids

There is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids.