Resistance to antimicrobial peptides in Gram-negative bacteria

Antimicrobial peptides (AMPs) are present in virtually all organisms and are an ancient and critical component of innate immunity. In mammals, AMPs are present in phagocytic cells, on body surfaces such as skin and mucosa, and in secretions and body fluids such as sweat, saliva, urine, and breast milk, consistent with their role as part of the first line of defense against a wide range of pathogenic microorganisms including bacteria, viruses, and fungi. AMPs are microbicidal and have also been shown to act as immunomodulators with chemoattractant and signaling activities. During the co-evolution of hosts and bacterial pathogens, bacteria have developed the ability to sense and initiate an adaptive response to AMPs to resist their bactericidal activity. Here, we review the various mechanisms used by Gram-negative bacteria to sense and resist AMP-mediated killing. These mechanisms play an important role in bacterial resistance to host-derived AMPs that are encountered during the course of infection. Bacterial resistance to AMPs should also be taken into consideration in the development and use of AMPs as anti-infective agents, for which there is currently a great deal of academic and commercial interest.     

A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic alpha-helical antimicrobial peptide (K(5)L(7)) and its diastereomer (4D-K(5)L(7)) (containing four d-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K(5)L(7) binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K(5)L(7) is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.     

Highly selective end-tagged antimicrobial peptides derived from PRELP

Background

Antimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens.

Methodology and Principal Findings

Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH₂ was effective against various “superbugs” including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection.

Conclusions/Significance

Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise “tuning” of toxicity and proteolytic stability may be achieved by changing tag-length and adding W- or F-amino acid tags.     

Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

Antimicrobial peptides (AMPs) and proteins are important components of innateimmunity against pathogens in insects. The production of AMPs is costly owing toresource-based trade-offs, and strategies maximizing the efficacy of AMPs at lowconcentrations are therefore likely to be advantageous. Here, we show thepotentiating functional interaction of co-occurring insect AMPs (the bumblebeelinear peptides hymenoptaecin and abaecin) resulting in more potentantimicrobial effects at low concentrations. Abaecin displayed no detectableactivity against Escherichia coli when tested alone atconcentrations of up to 200 μM, whereas hymenoptaecin affected bacterialcell growth and viability but only at concentrations greater than 2 μM.In combination, as little as 1.25 μM abaecin enhanced the bactericidaleffects of hymenoptaecin. To understand these potentiating functionalinteractions, we investigated their mechanisms of action using atomic forcemicroscopy and fluorescence resonance energy transfer-based quenching assays.Abaecin was found to reduce the minimal inhibitory concentration ofhymenoptaecin and to interact with the bacterial chaperone DnaK (anevolutionarily conserved central organizer of the bacterial chaperone network)when the membrane was compromised by hymenoptaecin. These naturally occurringpotentiating interactions suggest that combinations of AMPs could be usedtherapeutically against Gram-negative bacterial pathogens that have acquiredresistance to common antibiotics.     

Outer membrane lipoprotein Lpp is Gram-negative bacterial cell surface receptor for cationic antimicrobial peptides

Antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms. Here we demonstrate that the outer membrane lipoprotein, Lpp, of Enterobacteriaceae interacts with and promotes susceptibility to the bactericidal activities of AMPs. The oligomeric Lpp was specifically recognized by several cationic α-helical AMPs, including SMAP-29, CAP-18, and LL-37; AMP-mediated bactericidal activities were blocked by anti-Lpp antibody blocking. Blebbing of the outer membrane and increase in membrane permeability occurred in association with the coordinate internalization of Lpp and AMP. Interestingly, the specific binding of AMP to Lpp was resistant to divalent cations and salts, which were able to inhibit the bactericidal activities of some AMPs. Furthermore, using His-tagged Lpp as a ligand, we retrieved several characterized AMPs, including SMAP-29 and hRNase 7, from a peptide library containing crude mammalian cell lysates. Overall, this study explores a new mechanism and target of antimicrobial activity and provides a novel method for screening of antimicrobials for use against drug-resistant bacteria.     

Design of self-processing antimicrobial peptides for plant protection

Small antimicrobial peptides are excellent candidates for inclusion in self-processing proteins that could be used to confer pathogen resistance in transgenic plants. Antimicrobial peptides as small as 22 amino acids in length have been designed to incorporate the residual amino acids left from protein processing by the tobacco etch virus'(TEVs') NIa protease. Also, by minimizing the length of these peptides and the number of highly hydrophobic residues, haemolytic activity was reduced without affecting the peptide's antimicrobial activity.     

Plant antimicrobial peptides

Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.     

Branched antimicrobial peptides

Branched peptides E(RLAR)2, E[E(RLAR)2]2, E(KLAR)2, and E[E(KLAR)2]2 were synthesized on the basis of tetrapeptides RLAR and KLAR and glutamic acid bis(pentafluorophenyl) ester. Their minimal antimicrobial concentrations were shown to decrease along with increase in branching, achieving 12 microM for Escherichia coli cells, which is comparable to antimicrobial activities of temporin, magainin, and dermaseptin. The branched peptides were found not to act on human erythrocytes.     

Branched antimicrobial peptides

Branched peptides E(RLAR)₂, E[E(RLAR)₂]₂, and E(KLAR)₂, E[E(KLAR)₂]₂ were synthesized on the basis of tetrapeptides RLAR and KLAR and glutamic acid bis(pentafluorophenyl) ester. Their minimal antimicrobial concentrations were shown to decrease along with increase in branching, achieving 12 μM for Escherichia coli cells, which is comparable to antimicrobial activities of temporin, magainin, and dermaseptin. The branched peptides were found not to act on human erythrocytes.     

抗菌肽活性及天然抗菌肽的改造

抗菌肽具有抗菌谱广、热稳定性强、分子量小及免疫原性小等特点,其杀菌机制独特,病原菌不易产生耐药性,有望开发成新一代肽类抗生素。本文主要综述了影响抗菌肽生物活性的生化性质,即螺旋度、疏水性、两亲性、正电荷数等,并从结构的角度论述了其对抗菌肽抑菌活性的影响。部分抗菌肽具有空间结构不稳定、溶血活性等缺点,限制了其临床应用。因此,对天然抗菌肽的改造也成为目前抗菌肽的研究热点,本文还综述了天然抗菌肽的改造方法。     

Design of salt-insensitive glycine-rich antimicrobial peptides with cyclic tricystine structures

Cyclic peptide backbone and cystine constraints were used to develop a broadly active salt-insensitive antimicrobial peptide [Gly(6)]ccTP 1a with eight Gly residues in an 18-residue sequence. The importance of rigidity and amphipathicity imparted by the cyclic and cystine constraints was examined in two peptide series based on tachyplesin, a known beta-stranded antimicrobial peptide. The first series, which retained the charge and hydrophobic amino acids of tachyplesin, but contained zero to four covalent constraints, included a cyclic tricystine tachyplesin (ccTP 1). Corresponding [Gly(6)] analogues were prepared in a parallel series with all six bulky hydrophobic amino acids in their sequences replaced with Gly. Circular dichroism measurements showed that ccTP 1 and [Gly(6)]ccTP 1a exhibited well-ordered beta-sheet structures, while the less constrained [Gly(6)] analogues were disordered. Except for linear peptides assayed under high-salt conditions, peptides with increased or decreased conformational constraints retained broad activity spectra with small variations in potency of 2-10-fold compared to that of tachyplesin. In contrast, Gly replacement analogues resulted in large variations in activity spectra and significant decreases in potency that roughly correlated with the decreases in conformational constraints. Except against Escherichia coli, the Gly-rich analogues with two or fewer covalent constraints were largely inactive under high-salt conditions. Remarkably, the most constrained [Gly(6)]ccTP 1a retained a broad activity spectrum against all 10 test microbes in both low- and high-salt assays. Collectively, our results show that [Gly(6)]ccTP 1acould serve as a template for further analogue study to improve potency and specificity through single or multiple replacements of hydrophobic or unnatural amino acids.     

Design of model amphipathic peptides having potent antimicrobial activities.

Induced amphipathic alpha-helical conformations play an important role in the biological activity of peptides. By using reversed-phase high-performance liquid chromatography (RP-HPLC) as a means to study the secondary structure of peptides at aqueous/lipid interfaces, a sequence (Ac-LKLLKKLLKKLKKLLKKL-NH2) was found to readily adopt an amphipathic alpha-helical conformation upon interacting with the lipid groups of the stationary phase during RP-HPLC. This peptide exhibited potent antimicrobial activities against both Gram-positive and Gram-negative bacteria. We have prepared a complete set of omission, as well as of leucine and lysine substitution, analogs of this sequence. These analogs were used to investigate the effects of such alterations on the parent sequence's antimicrobial and hemolytic activities relative to each analog's behavior during RP-HPLC. The potential for the formation of ion channels through cell membranes by this amphipathic model peptide was also evaluated through preparation of analogs which varied in length from 8 to 22 residues, while maintaining their amphipathicity.     

Prediction of lipoprotein signal peptides in Gram-negative bacteria

A method to predict lipoprotein signal peptides in Gram-negative Eubacteria, LipoP, has been developed. The hidden Markov model (HMM) was able to distinguish between lipoproteins (SPaseII-cleaved proteins), SPaseI-cleaved proteins, cytoplasmic proteins, and transmembrane proteins. This predictor was able to predict 96.8% of the lipoproteins correctly with only 0.3% false positives in a set of SPaseI-cleaved, cytoplasmic, and transmembrane proteins. The results obtained were significantly better than those of previously developed methods. Even though Gram-positive lipoprotein signal peptides differ from Gram-negatives, the HMM was able to identify 92.9% of the lipoproteins included in a Gram-positive test set. A genome search was carried out for 12 Gram-negative genomes and one Gram-positive genome. The results for Escherichia coli K12 were compared with new experimental data, and the predictions by the HMM agree well with the experimentally verified lipoproteins. A neural network-based predictor was developed for comparison, and it gave very similar results. LipoP is available as a Web server at www.cbs.dtu.dk/services/LipoP/.     

Lactococcal membrane-permeabilizing antimicrobial peptides

A number of lactococcal antimicrobial peptides, bacteriocins have been discovered and characterized. Since Lactococcus spp. are generally regarded as safe bacteria, their bacteriocins are expected for various application uses. Most of lactococcal bacteriocins exert antimicrobial activity via membrane permeabilization. The most studied and prominent bacteriocin, nisin A is characterized in the high activity and has been utilized as food preservatives for more than half a century. Recently, other lactococcal bacteriocins such as lacticin Q were found to have distinguished features for further applications as the next generation to nisin.     

抗菌肽的研究进展

近年来,由于细菌耐药性问题日趋严峻,开发新型抗菌制剂已迫在眉睫。抗菌肽具有相对分,子质量小、对热稳定、抗菌谱广及不同于抗生素的抗菌机制,不产生耐药性,因而具有重要的临床应用价值。本文对天然来源、蛋白质酶解、化学合成及基因工程方法产生的抗菌肽及其研究进展进行了综述。     

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

Hevein-like antimicrobial peptides of plants

Plant antimicrobial peptides represent one of the evolutionarily oldest innate immunity components providing the first line of host defense to pathogen attacks. This review is dedicated to a small, currently actively studied family of hevein-like peptides that can be found in various monocot and dicot plants. The review thoroughly describes all known pep- tides belonging to this family including data on their structures, functions, and antimicrobial activity. The main features allowing to assign these peptides to a separate family are given, and the specific characteristics of each peptide are described. Further, the mode of action for hevein-like peptides, their role in plant immune system, and the applications of these mol- ecules in biotechnology and medicine are considered.     

Synthetic mimics of antimicrobial peptides

Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.     

Novel properties of antimicrobial peptides

Endogenous peptide antibiotics are known as evolutionarily old components of innate immunity. Due to interaction with cell membrane these peptides cause permeabilization of the membrane and lysis of invading microbes. However, some studies proved that antimicrobial peptides are universal multifunctional molecules and their functions extend far beyond simple antibiotics. In this review we present an overview of the general mechanism of action of antimicrobial peptides and discuss some of their additional properties, like antitumour activity, mitogenic activity, role in signal transduction pathways and adaptive immune response.