抗菌肽的基因工程研究进展

近年来细菌耐药性问题日趋严峻,寻找新型抗生素已迫在眉睫。抗菌肽是生物体产生的一种阳离子短肽,具有天然的抗菌活性。由于抗菌肽具有与传统抗生素不同的作用机制,不产生耐药性,因而具有重要的临床应用价值。但实践表明,抗菌肽的开发并非易事。针对近年来抗菌肽开发的基因工程策略和实践,尤其是大肠杆菌表达系统和酵母表达系统,进行了简要综述。     

Single molecule resolution of the antimicrobial action of quantum dot-labeled sushi peptide on live bacteria

Background  

Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial.     

Gone gene fishing: how to catch novel marine antimicrobials

Medical or health-promoting products of marine origin are often regarded with skepticism--some, such as shark fins and cod liver oil, are frequently perceived as low-tech "alternative treatments" largely because they have not been exploited to their full potential. The marine environment is an enormous source of biodiversity--80% of all life is found under the oceans' surfaces--yet very little of this rich resource has been utilized. Furthermore, most marine organisms rely heavily on antimicrobial components of their innate immune defenses to combat pathogens. The past three years has seen a revolution in the methods used to identify novel antimicrobials from marine sources; among the most promising are marine cationic antimicrobial peptides (CAPs).     

细菌抗菌肽耐药机制研究进展

细菌对传统抗生素的耐药程度十分严重,寻找克服耐药性的新型抗菌药物已成为当务之急。抗菌肽(antimicrobial peptides,AMPs)是当下较有前景的抗菌药物之一。虽然通常认为,AMPs优先攻击细胞膜的特点使其不会引起广泛的耐药性,但其对特定靶标的识别能力仍为基因突变和细菌耐药性的产生提供了可能。此外,一些细菌还显示出了抵御宿主AMPs的杀伤作用并与宿主细胞共存的能力,相应的细菌防御机制也使其对治疗性AMPs产生抗性,这种交叉抗性近年来也备受关注。这些耐药现象的发现均对AMPs的开发提出了新挑战。本综述就细菌对AMPs耐药的分子机制进行了研究进展的总结,并且对治疗性AMPs与宿主防御肽交叉抗性的相关机制研究进行了归纳,以期寻求新的对抗耐药性的策略。     

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.     

Prediction of Antimicrobial Activity of Synthetic Peptides by a Decision Tree Model

Antimicrobial resistance is a persistent problem in the public health sphere. However, recent attempts to find effective substitutes to combat infections have been directed at identifying natural antimicrobial peptides in order to circumvent resistance to commercial antibiotics. This study describes the development of synthetic peptides with antimicrobial activity, created in silico by site-directed mutation modeling using wild-type peptides as scaffolds for these mutations. Fragments of antimicrobial peptides were used for modeling with molecular modeling computational tools. To analyze these peptides, a decision tree model, which indicated the action range of peptides on the types of microorganisms on which they can exercise biological activity, was created. The decision tree model was processed using physicochemistry properties from known antimicrobial peptides available at the Antimicrobial Peptide Database (APD). The two most promising peptides were synthesized, and antimicrobial assays showed inhibitory activity against Gram-positive and Gram-negative bacteria. Colossomin C and colossomin D were the most inhibitory peptides at 5 μg/ml against Staphylococcus aureus and Escherichia coli. The methods described in this work and the results obtained are useful for the identification and development of new compounds with antimicrobial activity through the use of computational tools.     

Draft genome sequence of the marine Streptomyces sp. strain PP-C42, isolated from the Baltic Sea

Streptomyces, a branch of aerobic Gram-positive bacteria, represents the largest genus of actinobacteria. The streptomycetes are characterized by a complex secondary metabolism and produce over two-thirds of the clinically used natural antibiotics today. Here we report the draft genome sequence of a Streptomyces strain, PP-C42, isolated from the marine environment. A subset of unique genes and gene clusters for diverse secondary metabolites as well as antimicrobial peptides could be identified from the genome, showing great promise as a source for novel bioactive compounds.     

Application of antimicrobial peptides in agriculture and food industry

Antimicrobial peptides have captured the attention of researchers in recent years because of their efficiency in fighting against pathogens. These peptides are found in nature and have been isolated from a wide range of organisms. Furthermore, analogs or synthetic derivatives have successfully been developed on the basis of natural peptide patterns. Long use of pesticides and antibiotics has led to development of resistance among pathogens and other pests as well as increase of environmental and health risks. Antimicrobial peptides are under consideration as new substitutes for conventional pesticides and antibiotics. Many plants and animals have been manipulated with antimicrobial peptide-encoding genes and several pesticides and drugs have been produced based on these peptides. Such strategies and products may still have a long way to go before being confirmed by regulatory bodies and others need to surmount technical problems before being accepted as applicable ones. In spite of these facts, several cases of successful use of antimicrobial peptides in agriculture and food industry indicate a promising future for extensive application of these peptides. In this review, we consider the developing field of antimicrobial peptide applications in various agricultural activities.     

Bacteriocin nanoconjugates: boon to medical and food industry

Resistance to antibiotics is an ongoing problem in the biomedical industry. Developing active, alternative drug therapies would reduce our reliance on antibiotics that induce resistance in micro-organisms. To date, bacteriocins and antimicrobial peptides have shown a positive outcome as antibiotic substitutes and synergists apart from phage therapy, antibodies and probiotics. Bacteriocins are proteinaceous antimicrobial peptides synthesized by lactic acid bacteria extensively used as bio-preservatives and alternative to traditional antibiotics to overcome the problem of drug-resistant pathogens. Nonetheless, the use of bacteriocins has several limitations such as limited antimicrobial spectrum, requiring high dose, sensitivity to proteolytic enzymes, etc. Nanoparticles are one of the promising area of research explored to improve antimicrobial spectrum of bacteriocins. This review therefore highlights the recent developments and research pertaining to use of nanoparticles and bacteriocin conjugates to tackle the resistance crisis as well as its applications in food industry.     

Bioactive peptides from marine organisms: a short overview

Marine organisms are an immense source of new biologically active compounds. These compounds are unique because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides with a wide range of biological activities have been discovered, including antimicrobial, antitumoral, and antiviral activities and toxins amongst others. These proteins have been isolated from different phyla such as Porifera, Cnidaria, Nemertina, Crustacea, Mollusca, Echinodermata and Craniata. Purification techniques used to isolate these peptides include classical chromatographic methods such as gel filtration, ionic exchange and reverse-phase HPLC. Multiple in vivo and in vitro bioassays are coupled to the purification process to search for the biological activity of interest. The growing interest to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs now in clinical trials. This review presents examples of interesting peptides obtained from different marine organisms that have medical relevance. It also presents some of the common methods used to isolate and characterize them.     

Amphipathic, alpha-helical antimicrobial peptides

Gene-encoded antimicrobial peptides are an important component of host defense in animals ranging from insects to mammals. They do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. They are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. A particularly abundant and widespread class of antimicrobial peptides are those with amphipathic, alpha-helical domains. Due to their relatively small size and synthetic accessibility, these peptides have been extensively studied and have generated a substantial amount of structure-activity relationship (SAR) data. In this review, alpha-helical antimicrobial peptides are considered from the point of view of six interrelated structural and physicochemical parameters that modulate their activity and specificity: sequence, size, structuring, charge, amphipathicity, and hydrophobicity. It begins by providing an overview of how these vary in peptides from different natural sources. It then analyzes how they relate to the currently accepted model for the mode of action of alpha-helical peptides, and discusses what the numerous SAR studies that have been carried out on these compounds and their analogues can tell us. A comparative analysis of the many alpha-helical, antimicrobial peptide sequences that are now available then provides further information on how these parameters are distributed and interrelated. Finally, the systematic variation of parameters in short model peptides is used to throw light on their role in antimicrobial potency and specificity. The review concludes with some considerations on the potentials and limitations for the development of alpha-helical, antimicrobial peptides as antiinfective agents.     

Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa

The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions. We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides. This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation. Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated. Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides. The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers. Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system. The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P. aeruginosa infections.     

Antimicrobials,Stress and Mutagenesis

Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs) mostly target the cell wall, a microbial ‘Achilles heel’, it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient ‘weapons’ of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs) do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the ‘Achilles heel’ has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.     

Experimental evolution of resistance to an antimicrobial peptide

A novel class of antibiotics based on the antimicrobial properties of immune peptides of multicellular organisms is attracting increasing interest as a major weapon against resistant microbes. It has been claimed that cationic antimicrobial peptides exploit fundamental features of the bacterial cell so that resistance is much less likely to evolve than in the case of conventional antibiotics. Population models of the evolutionary genetics of resistance have cast doubt on this claim. We document the experimental evolution of resistance to a cationic antimicrobial peptide through continued selection in the laboratory. In this selection experiment, 22/24 lineages of Escherichia coli and Pseudomonas fluorescens independently evolved heritable mechanisms of resistance to pexiganan, an analogue of magainin, when propagated in medium supplemented with this antimicrobial peptide for 600-700 generations.     

Antimicrobial peptides from anurans skin secretions

This article is an overview of antimicrobial peptides found in anurans skin secretions. These molecules constitute an initial barrier against microbial infections because of their activity against a large array of microorganisms. These peptides hold remarkable pharmaceutical and technological interest since they selectively kill microorganisms and are unlikely to induce resistance in pathogens. Also, outstanding synergism occurs when these peptides are combined with classic antibiotics and other antimicrobial peptides.     

Control of cell selectivity of antimicrobial peptides

Antimicrobial peptides (AMPs) are promising novel antibiotics, because they exhibit broad antimicrobial spectra and do not easily induce resistance. For clinical applications, it is important to develop potent AMPs with less toxicity against host cells. This review article summarizes the molecular basis for the cell selectivity (bacteria versus host cells) of AMPs and various attempts to control it, including the optimization of physicochemical parameters of peptides, the introduction of d-, fluorinated, and unusual amino acids into peptides, the constraining of peptide conformations, and the modification of peptides by polymers. Pros and cons of these approaches are discussed.     

Targeting cellular apoptotic pathway with peptides from marine organisms

Apoptosis is a critical defense mechanism against the formation and progression of cancer and exhibits distinct morphological and biochemical traits. Targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. Peptides from marine organisms have become important sources in the discovery of antitumor drugs, especially when modern technology makes it more and more feasible to collect organisms from seas. This primer summarizes several marine peptides, based on their effects on apoptotic signaling pathways, although most of these peptides have not yet been studied in depth for their mechanisms of action. Novel peptides that induce an apoptosis signal pathway are presented in association with their pharmacological properties.     

Antimicrobial activity of marine sponge Clathria indica (Dendy, 1889)

Sponges are sessile filter feeders that have developed efficient defense mechanisms against foreign invaders such as viruses, bacteria or eukaryotic organisms. Antimicrobial peptides are known as major components of the innate immune defense system in marine invertebrates. The aim of the present work was to study the antimicrobial properties of the Indian sponge Clathria indica with special reference to the identification of antimicrobial peptides. Crude methanolic extract and its chloroform, n-butanol and aqueous fractions were tested against 16 human pathogens which include eleven bacteria with four of them being multidrug resistant and five pathogenic fungi. All fractions showed effective antibacterial activity against common and multidrug-resistant Salmonella typhi and antifungal activity against C. albicans and C. neoformans. However, they were ineffective against Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. Chloroform fraction being the most potent among the fractions tested on chemical investigation was indicative of the presence of peptides as evidenced by ninhydrin positive spots on TLC and presence of peptide bonds by NMR. Its ESI-MS showed presence of several peptides in the range of m/z 850 to 980. Structure of three peptides has been tentatively assigned by ESI-MS/MS or tandem mass analysis, on the basis of the amino acid sequence established. The results clearly show that the sponge C. indica represent an interesting source of marine invertebrates-derived antimicrobial peptides in the development of new strategies to treat various infectious diseases.     

抗菌肽的抗菌机制及其临床应用

抗菌肽是广泛存在于生物体内的一种小分子肽, 具有广谱性、高效性、稳定性等特点, 其本身不易产生耐药性。不仅具有杀菌作用, 还能抑杀真菌、寄生虫、病毒以及肿瘤细胞且对正常细胞毒性较小。新颖抗生素发现的缺乏, 导致了大量耐药菌株的出现, 抗菌肽有可能成为一种新的抗生素替代品。本文介绍了抗菌肽的结构特点、生物活性, 并重点阐述了其抗菌机制及最新临床应用进展。