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.     

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.     

Cooperative interaction of antimicrobial peptides with the interrelated immune pathways in plants

Plants express a diverse repertoire of functionally and structurally distinct antimicrobial peptides (AMPs) which provide innate immunity by acting directly against a wide range of pathogens. AMPs are expressed in nearly all plant organs, either constitutively or in response to microbial infections. In addition to their direct activity, they also contribute to plant immunity by modulating defence responses resulting from pathogen‐associated molecular pattern/effector‐triggered immunity, and also interact with other AMPs and pathways involving mitogen‐activated protein kinases, reactive oxygen species, hormonal cross‐talk and sugar signalling. Such links among AMPs and defence signalling pathways are poorly understood and there is no clear model for their interactions. This article provides a critical review of the empirical data to shed light on the wider role of AMPs in the robust and resource‐effective defence responses of plants.     

Adaptive evolution of crustin antimicrobial peptides in decapods

Antimicrobial peptides (AMPs) are present in a wide range of taxonomic groups and played a crucial role in host adaptation to a diverse array of ever-changing pathogens. Crustin, a cysteine-rich cationic AMP, is known to exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria in decapods. Given their important role in host-immune defense, a large proportion of amino acid substitutions in crustin AMPs are expected to be fixed by natural selection. Utilizing the complete coding nucleotide sequence data of crustin, the present study revealed the pervasive role of positive Darwinian selection in the evolution and divergence of crustin AMPs in decapods. Approximately, 20–35?% of codons in two phylogenetically distinct groups of closely related crustins in Penaeid shrimps are shown to have evolved under positive selection. Interestingly, several of these positively selected sites are located at the carboxyl-terminal region, the region that directly interacts with the invading pathogens. Pathogen-mediated selection pressure could be the likely cause for such an accelerated rate of amino acid substitutions and could have contributed to the structural and functional diversification of crustin AMPs in several taxa.     

Antimicrobial peptides and proteins, exercise and innate mucosal immunity

This review examines the question of whether exercise can be used as an experimental model to further our understanding of innate antimicrobial peptides and proteins (AMPs) and their role in susceptibility to infection at mucosal surfaces. There is strong evidence to suggest that AMPs, in combination with cellular and physical factors, play an important role in preventing infection. Although AMPs act directly on microorganisms, there is increasing recognition that they also exert their protective effect via immunomodulatory mechanisms, especially in noninflammatory conditions. Further studies that manipulate physiologically relevant concentrations of AMPs are required to shed light on the role they play in reducing susceptibility to infection. Evidence shows that in various form prolonged and/or exhaustive exercise is a potent modulator of the immune system, which can either sharpen or blunt the immune response to pathogens. The intensity and duration of exercise can be readily controlled in experimental settings to manipulate the degree of physical stress. This would allow for an investigation into a potential dose-response effect between exercise and AMPs. In addition, the use of controlled exercise could provide an experimental model by which to examine whether changes in the concentration of AMPs alters susceptibility to illness.     

The interplay between the clustering of transmembrane proteins and coupling of anchored membrane proteins

Clustering of membrane proteins plays an important role in many cellular activities such as protein sorting and signal transduction. In this study, we used dissipative particle dynamics simulation method to investigate the clustering of anchored membrane proteins (AMPs) in the presence of transmembrane proteins (TMPs). First, our simulation results show that clustering of AMPs and that of TMPs are in fact interdependent, and depending on their hydrophobic length, both protein mixing and protein demixing are observed. Especially, the protein demixing occurs only when the hydrophobic mismatch of TMPs is negative while that of AMPs is positive. Second, our simulation results indicate that the clustering of TMPs also modulates the coupling of the clustering of AMPs in both leaflets. On the one hand, the coupling between AMPs in different leaflets will be strongly restrained if TMPs form protein mixing with AMPs in one leaflet and protein demixing with AMPs in the other leaflet. On the other hand, the coupling between AMPs can be enhanced or mediated by TMPs when TMPs mix with AMPs in both leaflets. Our results may have some implications on our understanding of how different types of membrane proteins cluster and provide a possible explanation of how TMPs participate in signal transduction across cellular membranes.     

The role of natural antimicrobial peptides during infection and chronic inflammation

Natural antimicrobial peptides (AMPs), a family of small polypeptides that are produced by constitutive or inducible expression in organisms, are integral components of the host innate immune system. In addition to their broad-spectrum antibacterial activity, natural AMPs also have many biological activities against fungi, viruses and parasites. Natural AMPs exert multiple immunomodulatory roles that may predominate under physiological conditions where they lose their microbicidal properties in serum and tissue environments. Increased drug resistance among microorganisms is occurring far more quickly than the discovery of new antibiotics. Natural AMPs have shown promise as ‘next generation antibiotics’ due to their broad-spectrum curative effects, low toxicity, the fact that they are not residual in animals, and the low rates of resistance exhibited by many pathogens. Many types of synthetic AMPs are currently being tested in clinical trials for the prevention and treatment of various diseases such as chemotherapy-associated infections, diabetic foot ulcers, catheter-related infections, and other conditions. Here, we provide an overview of the types and functions of natural AMPs and their role in combating microorganisms and different infectious and inflammatory diseases.     

Role of Antimicrobial Peptides in Amphibian Defense Against Trematode Infection

Antimicrobial peptides (AMPs) contribute to the immune defenses of many vertebrates, including amphibians. As larvae, amphibians are often exposed to the infectious stages of trematode parasites, many of which must penetrate the host’s skin, potentially interacting with host AMPs. We tested the effects of the natural AMPs repertoires on both the survival of trematode infectious stages as well as their ability to infect larval amphibians. All five trematode species exhibited decreased survival of cercariae in response to higher concentrations of adult bullfrog AMPs, but no effect when exposed to AMPs from larval bullfrogs. Similarly, the use of norepinephrine to remove AMPs from larval bullfrogs, Pacific chorus frogs, and gray treefrogs had only weak (gray treefrogs) or non-significant (other tested species) effects on infection success by Ribeiroia ondatrae. We nonetheless observed strong differences in parasite infection as a function of both host stage (first- versus second-year bullfrogs) and host species (Pacific chorus frogs versus gray treefrogs) that were apparently unrelated to AMPs. Taken together, our results suggest that AMPs do not play a significant role in defending larval amphibians against trematode cercariae, but that they could be one mechanism helping to prevent infection of post-metamorphic amphibians, particularly for highly aquatic species.     

Variation in Streptococcus pneumoniae susceptibility to human antimicrobial peptides may mediate intraspecific competition

Streptococcus pneumoniae is a facultative pathogen inhabiting the nasopharynx of humans where it is exposed to a range of antimicrobial peptides (AMPs) of the innate immune response. It is possible therefore that the susceptibility of strains to AMPs plays a role in determining their ability to colonize, and furthermore, that AMPs could mediate competitive interactions between co-colonizing genotypes. However, little is known about patterns of natural variation in AMP susceptibility of S. pneumoniae, and it is unclear whether the susceptibilities of an isolate to multiple human AMPs are correlated. We tested this by characterizing the susceptibility of 31 S. pneumoniae natural isolates to human neutrophil peptide (HNP-1) (α-defensin) and LL-37 (cathelicidin). We observed significant variation in susceptibility between isolates to both AMPs, and in the majority of isolates, susceptibilities to HNP-1 and LL-37 were uncorrelated. Clinical isolates were more susceptible to AMPs than were carriage isolates. The polysaccharide capsule of S. pneumoniae is thought to protect cells against AMPs. However, serotype alone could not explain the observed variation in susceptibility suggesting that genetic background plays an equally important role. We tested directly whether AMPs could mediate competition between isolates using competition experiments in the presence and absence of AMPs. These experiments demonstrated that AMPs could indeed reverse the outcome of competition between selected isolates. AMP-mediated competition could therefore contribute to the maintenance of intraspecific genetic diversity in S. pneumoniae.     

抗菌肽融合表达研究进展

抗菌肽抗菌谱广、活性稳定,且具有与抗生素不同的抗菌机制,在抑杀病原微生物的同时不易产生耐药性,因而在食品、饲料、医药等领域具有重要的应用价值。基因工程技术是降低抗菌肽生产成本的主要方式,其中融合表达在提高抗菌肽产量方面起到了重要作用。文中综述了抗菌肽融合表达的国内外研究进展,探讨了部分融合标签用于抗菌肽表达的策略,并对今后的发展提出了自己的看法。     

Specification and behavior of AMPs, muscle-committed transient Drosophila stem cells

During development, transient stem cells play critical roles in the formation of specific tissues. Adult Muscle Precursors (AMPs) are at the origin of all adult Drosophila muscles and as we report here represent a novel population of muscle-committed transient stem cells. Similar to vertebrate muscle stem cells, AMPs keep Notch signaling active and express Enhancer of split m6 (E(spl)m6) gene, a read-out of Notch pathway. To get insights into AMP cell specification we performed a gain-of-function screen and found that the rhomboid-triggered Epidermal Growth Factor (EGF) signaling pathway controls both the specification and the subsequent maintenance of AMPs. Our findings are supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Interestingly, by targeting GFP to the AMP cell membranes we also demonstrated that AMPs send long cellular processes and form a network of interconnected cells. As revealed by laser ablation experiments, the main role of AMP cell connections is to maintain their correct spatial positioning.     

Antimicrobial peptides of invertebrates. Part 1. structure,biosynthesis, and evolution

Antimicrobial peptides and proteins (AMPs) are among the most important components of the immune system of multicellular organisms. The role of AMPs is of particular importance for invertebrates which constitute the vast majority of species diversity of the living world, because these animals lack acquired immunity. The AMPs of animal origin are ribosomally-synthesized molecules that have, as a rule, a positive net charge and amphiphilic properties. They can act against bacteria, yeast and filamentous fungi, protozoa, and enveloped viruses. AMPs can also play a role of mediators of the immune system. Search and investigation for protective factors of the invertebrate host defense provide a better understanding of mechanisms of the innate immunity of humans and other mammals and give a key to a development of new medicines. The first part of this review focuses on special features of a structure, biosynthesis, regulation of gene expression, and molecular evolution of AMPs of invertebrates.     

Molecular mechanisms of antitumor effect of natural antimicrobial peptides

In spite of all the advances in cancer treatment made in recent years, one of the main problems in this field that remains extremely urgent is the development of drug resistance to the chemotherapeutic agents currently in use due to clonal microevolution of tumor tissue. Numerous publications devoted to the study of cationic antimicrobial peptides (AMPs) as molecular factors of the innate immune system suggest that these compounds possess significant therapeutic potential and can be considered as candidates for the role of not only antimicrobial, but also next-generation anticancer drugs. AMPs are characterized by a variety of mechanisms of cytotoxic action that can lead to either necrosis or apoptosis of the target cells. These effects are based on the selective interaction with the membranes of tumor cells, which have a number of similarities, in structural and physiological aspects, with the microbial membranes. AMPs were found to be able to inhibit tumor growth by interrupting the formation of its vascular network. The antitumor effect of AMPs may also be enhanced by the modulation of host immune system, as previously observed for their antimicrobial effects. The described properties of AMPs give hope for the development of new drugs that will be able to overcome the resistance of tumor cells.     

Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria

Analysis of a Selected Set of Antimicrobial Peptides

The rapid emergence of resistance to classical antibiotics has increased the interest in novel antimicrobial compounds. Antimicrobial peptides (AMPs) represent an attractive alternative to classical antibiotics and a number of different studies have reported antimicrobial activity data of various AMPs, but there is only limited comparative data available. The mode of action for many AMPs is largely unknown even though several models have suggested that the lipopolysaccharides (LPS) play a crucial role in the attraction and attachment of the AMP to the bacterial membrane in Gram-negative bacteria. We compared the potency of Cap18, Cap11, Cap11-1-18m², Cecropin P1, Cecropin B, Bac2A, Bac2A-NH₂, Sub5-NH₂, Indolicidin, Melittin, Myxinidin, Myxinidin-NH₂, Pyrrhocoricin, Apidaecin and Metalnikowin I towards Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas salmonicida, Listeria monocytogenes, Campylobacter jejuni, Flavobacterium psychrophilum, Salmonella typhimurium and Yersinia ruckeri by minimal inhibitory concentration (MIC) determinations. Additional characteristics such as cytotoxicity, thermo and protease stability were measured and compared among the different peptides. Further, the antimicrobial activity of a selection of cationic AMPs was investigated in various E. coli LPS mutants.

Cap18 Shows a High Broad Spectrum Antimicrobial Activity

Of all the tested AMPs, Cap18 showed the most efficient antimicrobial activity, in particular against Gram-negative bacteria. In addition, Cap18 is highly thermostable and showed no cytotoxic effect in a hemolytic assay, measured at the concentration used. However, Cap18 is, as most of the tested AMPs, sensitive to proteolytic digestion in vitro. Thus, Cap18 is an excellent candidate for further development into practical use; however, modifications that should reduce the protease sensitivity would be needed. In addition, our findings from analyzing LPS mutant strains suggest that the core oligosaccharide of the LPS molecule is not essential for the antimicrobial activity of cationic AMPs, but in fact has a protective role against AMPs.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

LAMP: A Database Linking Antimicrobial Peptides

The frequent emergence of drug-resistant bacteria has created an urgent demand for new antimicrobial agents. Traditional methods of novel antibiotic development are almost obsolete. Antimicrobial peptides (AMPs) are now regarded as a potential solution to revive the traditional methods of antibiotic development, although, until now, many AMPs have failed in clinical trials. A comprehensive database of AMPs with information about their antimicrobial activity and cytotoxicity will help promote the process of finding novel AMPs with improved antimicrobial activity and reduced cytotoxicity and eventually accelerate the speed of translating the discovery of new AMPs into clinical or preclinical trials. LAMP, a database linking AMPs, serves as a tool to aid the discovery and design of AMPs as new antimicrobial agents. The current version of LAMP has 5,547 entries, comprising 3,904 natural AMPs and 1,643 synthetic peptides. The database can be queried using either simply keywords or combinatorial conditions searches. Equipped with the detailed antimicrobial activity and cytotoxicity data, the cross-linking and top similar AMPs functions implemented in LAMP will help enhance our current understanding of AMPs and this may speed up the development of new AMPs for medical applications. LAMP is freely available at: http://biotechlab.fudan.edu.cn/database/lamp.     

In vivo Toxicity Assessment of Antimicrobial Peptides (AMPs LR14) Derived from Lactobacillus plantarum Strain LR/14 in Drosophila melanogaster

Lactic acid bacteria are known to produce antimicrobial peptides (AMPs) such as bacteriocins which can be employed to control pathogens and food spoilage microorganisms. However, their possible role as toxic agents against a eukaryotic system still remains unexplored. The present study deals with the in vivo evaluation of acute toxic effect of AMPs LR14, a mixture of AMPs isolated from Lactobacillus plantarum LR/14 on Drosophila melanogaster. The fly was used as a model system to measure the extent of toxicity of these peptides. The results showed that concentrations below 10 mg/ml are not significantly effective. When exposed to 10 mg/ml of AMPs LR14, acute toxic effect and a significant delay in the developmental cycle of the fly could be observed. Also, the weight and size of the flies were significantly reduced upon ingestion of these peptides. Higher concentrations (beyond 15 mg/ml) exerted a strong larvicidal effect. Detailed analysis on larval tissues and adult germ cells of the insect revealed deformity in cellular architecture, DNA fragmentation, and premature apoptosis, confirming that the peptides have a dose-dependent toxic property. Our studies provide the first information on the role of AMPs LR14 as an insecticidal agent.