Antimicrobial peptide resistance mechanisms of human bacterial pathogens

The critical role played by antimicrobial peptides (AMPs) in mammalian innate immunity is increasingly recognized. Bacteria differ in their intrinsic susceptibility to AMPs, and the relative resistance of some important human pathogens to these defense molecules is now appreciated as an important virulence phenotype. Experimental analysis has identified diverse mechanisms of bacterial AMP resistance including altered cell surface charge, active efflux, production of proteases or trapping proteins, and modification of host cellular processes. The contribution of these resistance mechanisms to pathogenesis is confirmed through direct comparison of wild-type bacteria and AMP-sensitive mutants using in vivo infection models. Knowledge of the molecular basis of bacterial AMP resistance may provide new targets for antimicrobial therapy of human infectious diseases.     

Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.     

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.     

Identification of novel host defense peptides and the absence of alpha-defensins in the bovine genome

Host defense peptides (historically called antimicrobial peptides, AMPs) are key components in the mammalian innate immune system, and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. In order to identify novel host defense peptides by sequence analysis, we constructed the AMPer resource (http://www.cnbi2.com/cgi-bin/amp.pl) that utilizes hidden Markov models to recognize sequences of antimicrobial peptides. In the current work, we utilized the AMPer resource to search bovine expressed sequence tags from the NCBI dbEST project and the bovine genome sequence for novel host defense peptides. Of the 34 known bovine AMPs, 27 were identified with high confidence in the AMPs predicted from ESTs. A further potential 68 AMPs predicted from the EST data were found that appear to be novel giving a total estimate of 102 AMPs present in the genome. Two of these were cathelicidins and selected for experimental verification in RNA derived from bovine tissue. One predicted AMP, most similar to rabbit '15 kDa protein' AMP, was confirmed to be present in infected bovine intestinal tissue using PCR. These findings demonstrated the practical applicability of the developed bioinformatics approach and laid a foundation for future discoveries of gene-coded AMPs. No members of the alpha-defensin family were found in the bovine sequences. Since we could find no technical reasons these would be missed and no references to bovine alpha-defensins in the literature, this suggests that cattle lack this important family of host defense peptides.     

Computational resources and tools for antimicrobial peptides

Antimicrobial peptides (AMPs), as evolutionarily conserved components of innate immune system, protect against pathogens including bacteria, fungi, viruses, and parasites. In general, AMPs are relatively small peptides (<10 kDa) with cationic nature and amphipathic structure and have modes of action different from traditional antibiotics. Up to now, there are more than 19 000 AMPs that have been reported, including those isolated from nature sources or by synthesis. They have been considered to be promising substitutes of conventional antibiotics in the quest to address the increasing occurrence of antibiotic resistance. However, most AMPs have modest direct antimicrobial activity, and their mechanisms of action, as well as their structure–activity relationships, are still poorly understood. Computational strategies are invaluable assets to provide insight into the activity of AMPs and thus exploit their potential as a new generation of antimicrobials. This article reviews the advances of AMP databases and computational tools for the prediction and design of new active AMPs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Tethering antimicrobial peptides: Current status and potential challenges

Antimicrobial peptides (AMPs) are next generation antibiotics which will make excellent coating agents for a myriad of devices because they are far less susceptible to the development of pathogen resistance compared to conventional antibiotics, exhibit rapid and broad-spectrum killing profiles, and are effective at low concentrations. These advantages, however, are compromised upon AMP tethering to solid supports. The effects of peptide-tethering strategies in governing AMP orientation, surface density, flexibility, and activity are reviewed. Understanding AMP structure-function relationship in the tethered conformation will enable rational improvements of immobilisation parameters. Foreseeable challenges in the development of AMP-coated devices such as microbial accumulation on implant surface and the lack of direct biomolecular structure and orientation data of peptides on surfaces are also discussed, and solutions to address these roadblocks are also proposed.     

Expression systems for heterologous production of antimicrobial peptides

Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.     

Antimicrobial peptides (AMPs): peptide structure and mode of action

Antimicrobial peptides (AMPs) have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum. Their amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of AMP activity, their relevance to resolving how peptides damage and kill microorganisms still needs to be clarified. Moreover, many AMPs employ sophisticated and dynamic mechanisms of action to carry out their likely roles in antimicrobial host defense. Recently, it has been speculated that transmembrane pore formation is not the only mechanism of microbial killing by AMPs. In fact, several observations suggest that translocated AMPs can alter cytoplasmic membrane septum formation, reduce cell-wall, nucleic acid, and protein synthesis, and inhibit enzymatic activity. In this review, we present the structures of several AMPs as well as models of how AMPs induce pore formation. AMPs have received special attention as a possible alternative way to combat antibiotic-resistant bacterial strains. It may be possible to design synthetic AMPs with enhanced activity for microbial cells, especially those with antibiotic resistance, as well as synergistic effects with conventional antibiotic agents that lack cytotoxic or hemolytic activity.     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Interplay between Candida albicans and the Antimicrobial Peptide Armory

Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral, and fungal pathogens. The medically important fungus Candida albicans colonizes different host niches as part of the normal human microbiota. Proliferation of C. albicans is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host, C. albicans has developed a three-phase AMP evasion strategy, including secretion of peptide effectors, AMP efflux pumps, and regulation of signaling pathways. These mechanisms prevent C. albicans from the antifungal activity of the major AMP classes, including cathelicidins, histatins, and defensins leading to a basal resistance. This minireview summarizes human AMP attack and C. albicans resistance mechanisms and current developments in the use of AMPs as antifungal agents.     

Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.     

Effect of positive charges in the structural interaction of crabrolin isoforms with lipopolysaccharide

Antimicrobial peptides (AMPs) appear as chemical compounds of increasing interest for their role in killing bacteria and, more recently, for their ability to bind endotoxin (lipopolysaccharide, LPS) that is released during bacterial infection and that may lead to septic shock. This dual role in the mechanism of action can further be enhanced in a synergistic way when two or more AMPs are combined together. Not all AMPs are able to bind LPS, suggesting that several modes of binding to the bacterial surface may exist. Here we analyze a natural AMP, crabrolin, and two mutated forms, one with increased positive charge (Crabrolin Plus) and the other with null charge (Crabrolin Minus), and compare their binding abilities to LPS. While Crabrolin WT as well Crabrolin Minus do not show binding to LPS, the mutated Crabrolin Plus exhibits binding and forms a well defined structure in the presence of LPS. The results strengthen the importance of positive charges for the binding to LPS and suggest the mutated form with increased positive charge as a promising candidate for antimicrobial and antiseptic activity.     

D-alanylation of teichoic acids promotes group a streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion

Group A streptococcus (GAS) is a leading cause of severe, invasive human infections, including necrotizing fasciitis and toxic shock syndrome. An important element of the mammalian innate defense system against invasive bacterial infections such as GAS is the production of antimicrobial peptides (AMPs) such as cathelicidins. In this study, we identify a specific GAS phenotype that confers resistance to host AMPs. Allelic replacement of the dltA gene encoding d-alanine-d-alanyl carrier protein ligase in an invasive serotype M1 GAS isolate led to loss of teichoic acid d-alanylation and an increase in net negative charge on the bacterial surface. Compared to the wild-type (WT) parent strain, the GAS DeltadltA mutant exhibited increased susceptibility to AMP and lysozyme killing and to acidic pH. While phagocytic uptake of WT and DeltadltA mutants by human neutrophils was equivalent, neutrophil-mediated killing of the DeltadltA strain was greatly accelerated. Furthermore, we observed the DeltadltA mutant to be diminished in its ability to adhere to and invade cultured human pharyngeal epithelial cells, a likely proximal step in the pathogenesis of invasive infection. Thus, teichoic acid d-alanylation may contribute in multiple ways to the propensity of invasive GAS to bypass mucosal defenses and produce systemic infection.     

宿主防御肽抗病毒感染机制研究进展

宿主防御肽是一类广泛存在于脊椎动物的小分子多肽,具有广谱的抗菌活性以及抗炎、细胞趋化、促进血管生成和修复损伤等免疫调节功能。以往的研究多集中在宿主防御肽抗细菌和真菌感染的研究上。近年来大量研究发现,宿主防御肽也具有广泛的抗病毒活性,在临床各类病毒病的预防和治疗上具有潜在的应用前景。本文围绕宿主防御肽直接杀伤病毒、调节病毒感染过程和参与宿主抗病毒天然免疫调节这3个方面的作用机制进行综述,为宿主防御肽抗病毒相关研究和相关抗病毒生物药物的研发提供参考和借鉴。     

How are the expression patterns of gut antimicrobial peptides modulated by human gastrointestinal diseases? A bridge between infectious,inflammatory, and malignant diseases

The human gut barrier is the tissue exposed to the highest load of microorganisms, harbouring 100 trillion bacteria. In addition, the gut's renewal rate outruns that of any other human tissue. Antimicrobial peptides (AMPs) are highly optimized defense molecules in the intestinal barrier optimized to maintain gastrointestinal homeostasis. Alterations in AMPs activity can lead to or result from human gastrointestinal diseases. In this review, unique, conserved, or otherwise regular alterations in the expression patterns of human AMPs across gastrointestinal inflammatory and infectious diseases were analyzed for pattern elucidation. Human gastrointestinal diseases are associated with alterations in gut AMPs' expression patterns in a peptide‐specific, disease‐specific, and pathogen‐specific way, modulating human gastrointestinal functioning. Across diseases, there is a (i) marked reduction in otherwise constitutively expressed AMPs, leading to increased disease susceptibility, and a (ii) significant increase in the expression of inducible AMPs, leading to tissue damage and disease severity. Infections and inflammatory conditions are associated with altered gene expression in the gut, whose patterns may favour cellular metaplasia, mucosal dysfunction, and disease states. Altered expression of AMPs can thus thrive disease severity and evolution since its early stages. Nevertheless, the modulation of AMP expression patterns unveils promising therapeutic targets.     

Peptide Design Principles for Antimicrobial Applications

The increased incidence of bacterial resistance to available antibiotics represents a major global health problem and highlights the need for novel anti-infective therapies. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. AMPs are versatile, have almost unlimited sequence space, and can be tuned for broad-spectrum or specific activity against microorganisms. However, several obstacles remain to be overcome in order to develop AMPs for medical use, such as toxicity, stability, and bacterial resistance. We lack standard experimental procedures for quantifying AMP activity and do not yet have a clear picture of the mechanisms of action of AMPs. The rational design of AMPs can help solve these issues and enable their use as new antimicrobials. Here we provide an overview of the main physicochemical features that can be engineered to achieve enhanced bioactivity and describe current strategies being used to design AMPs.     

Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.     

Truncated antimicrobial peptides from marine organisms retain anticancer activity and antibacterial activity against multidrug-resistant Staphylococcus aureus

Antimicrobial peptides (AMPs) were recently determined to be potential candidates for treating drug-resistant bacterial infections. The aim of this study was to develop shorter AMP fragments that combine maximal bactericidal effect with minimal synthesis cost. We first synthesized a series of truncated forms of AMPs (anti-lipopolysaccharide factor from shrimp, epinecidin from grouper, and pardaxin from Pardachirus marmoratus). The minimum inhibitory concentrations (MICs) of modified AMPs against ten bacterial species were determined. We also examined the synergy between peptide and non-peptide antibiotics. In addition, we measured the inhibitory rate of cancer cells treated with AMPs by MTS assay. We found that two modified antibacterial peptides (epinecidin-8 and pardaxin-6) had a broad range of action against both gram-positive and gram-negative bacteria. Furthermore, epinecidin and pardaxin were demonstrated to have high antibacterial and anticancer activities, and both AMPs resulted in a significant synergistic improvement in the potencies of streptomycin and kanamycin against methicillin-resistant Staphylococcus aureus. Neither AMP induced significant hemolysis at their MICs. In addition, both AMPs inhibited human epithelial carcinoma (HeLa) and fibrosarcoma (HT-1080) cell growth. The functions of these truncated AMPs were similar to those of their full-length equivalents. In conclusion, we have successfully identified shorter, inexpensive fragments with maximal bactericidal activity. This study also provides an excellent basis for the investigation of potential synergies between peptide and non-peptide antibiotics, for a broad range of antimicrobial and anticancer activities.     

Escherichia coli Cell Surface Perturbation and Disruption Induced by Antimicrobial Peptides BP100 and pepR

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the Gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic Gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.     

Meningococcal resistance to antimicrobial peptides is mediated by bacterial adhesion and host cell RhoA and Cdc42 signalling

Antimicrobial peptides (AMPs) constitute an essential part of the innate immune defence. Pathogenic bacteria have evolved numerous strategies to withstand AMP‐mediated killing. The influence of host epithelia on bacterial AMP resistance is, however, still largely unknown. We found that adhesion to pharyngeal epithelial cells protected Neisseria meningitidis, a leading cause of meningitis and sepsis, from the human cathelicidin LL‐37, the cationic model amphipathic peptide (MAP) and the peptaibol alamethicin, but not from polymyxin B. Adhesion to primary airway epithelia resulted in a similar increase in LL‐37 resistance. The inhibition of selective host cell signalling mediated by RhoA and Cdc42 was found to abolish the adhesion‐induced LL‐37 resistance by a mechanism unrelated to the actin cytoskeleton. Moreover, N. meningitidis triggered the formation of cholesterol‐rich membrane microdomains in pharyngeal epithelial cells, and host cell cholesterol proved to be essential for adhesion‐induced resistance. Our data highlight the importance of Rho GTPase‐dependent host cell signalling for meningococcal AMP resistance. These results indicate that N. meningitidis selectively exploits the epithelial microenvironment in order to protect itself from LL‐37.