Mechanism Of Antimicrobial Peptides Research Articles

Mechanisms of Antimicrobial Peptides from Bagasse against Human Pathogenic Bacteria

Nonedible agricultural wastes (agricultural wastes, agro-industrial wastes, and fishery wastes) were chosen as potential sources of antimicrobial peptides and evaluated for antibacterial efficiency against human pathogens. Specifically, protein hydrolysates were first obtained by hydrolysis with pepsin. Filtrated peptides smaller than 3 kDa were then purified by C18 reversed-phase chromatography, cation exchange chromatography, and off-gel fractionation. NanoLC-MS/MS was used to investigate the amino acid sequences of active peptide candidates. Five candidate peptides were finally chosen for chemical synthesis and evaluation of growth inhibition against human pathogenic bacteria. Two synthetic peptides from bagasse, NLWSNEINQDMAEF (Asn-Leu-Trp-Ser-Asn-Glu-Ile-Asn-Gln-Asp-Met-Ala-Glu-Phe) and VSNCL (Val-Ser-Asn-Cys-Leu), showed the most potent antibacterial activity against three pathogens: Pseudomonas aeruginosa, Bacillus subtilis, and Burkholderia cepacia. The antibacterial mechanisms of these peptides were then examined using shotgun proteomics, which revealed their effects to involve both intracellular-active and membrane-active mechanisms. Further investigation and modification of peptides are needed to increase the efficiency of these peptides against human pathogens.

The Prepropalustrin-2CE2 and Preprobrevinin-2CE3 Gene from Rana chensinensis: Gene Expression, Genomic Organization and Functional Analysis of the Promoter Activity.

For amphibians, antimicrobial peptides are innate immune molecules that resist adverse external environmental stimuli. However, the regulation mechanism of antimicrobial peptide gene expression in frogs is still unclear. The two antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, are produced under external stimulation in Rana chensinensis. Using this model, we analyzed the gene structure and regulatory elements of the two antimicrobial peptide genes and explored the regulatory effects of related transcription factors on the two genes. Different stimuli such as E. coli, S. aureus, and chemical substance lipopolysaccharide (LPS) were applied to Rana chensinensis tadpoles at different developmental stages, and antimicrobial peptide expression levels were detected by RT-PCR. Bioinformatics analysis and 5'-RACE and genome walking technologies were employed to analyze the genome structure and promoter region of the antimicrobial peptide genes. With dual-luciferase reporter gene assays, yeast one-hybrid experiment and EMSA assays, we assessed the regulatory effect of the endogenous regulators of the cell on the antimicrobial peptide promoter. The transcription levels of prepropalustrin-2CE2 and preprobrevinin-2CE3 were significantly upregulated after different stimulations. Genomic structure analysis showed that both genes contained three exons and two introns. Promoter analysis indicated that there are binding sites for regulatory factors of the NF-κB family in the promoter region, and experiments showed that endogenous NF-κB family regulatory factors in frog cells activate the promoters of the antimicrobial peptide genes. Yeast one-hybrid experiment and EMSA assay demonstrated that RelA and NF-κB1 might interact with specific motifs in the prepropalustrin-2CE2 promoter. In this paper, we found that the gene expression levels of the antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, in R. chensinensis will increase under environmental stimuli, and we verified that the changes in gene expression levels are affected by the transcription factors RelA and NF-κB1. The yeast one-hybrid experiment and EMSA assay confirmed that RelA and NF-κB1 could directly interact with the frog antimicrobial peptide gene promoter, providing new data for the regulatory mechanism of antimicrobial peptides in response to environmental stimuli.

Antimicrobial peptides (AMP) in biofilm induced orthopaedic device-related infections

Orthopaedic device-related infection (ODRI) represent is one of the most challenging complications to manage in orthopaedic and trauma surgery. Biofilm formation is one of the crucial steps in the development of implant related orthopaedic infections due to the surface-adherent bacteria. Bacterial biofilms have several innate antimicrobial resistance mechanisms and hence difficult to eliminate with conventional antibiotics. Chronic, indolent, unresponsive infection can lead to clinical disability affecting quality of life with socio-economic consequences and compromised patient related health care outcomes. Although there is a basic understanding of the mechanism of biofilm associated antimicrobial resistance, enhanced knowledge, innovative treatment strategies and new therapeutic modalities is the need of the hour to manage biofilm associated Orthopaedic device-related infection (ODRI). Antimicrobial peptides (AMPs) represent an exciting opportunity to treat biofilm infections due to their diverse mechanisms of action. This article highlights the current role and mechanism of Antimicrobial peptides (AMPs) in preventing and eradicating Orthopaedic device-related infection (ODRI).

Systematic comparison of activity and mechanism of antimicrobial peptides against nosocomial pathogens

The World Health Organisation has deemed several multi-drug resistant (MDR) nosocomial bacterial pathogens to be of significant threat to human health. A stark increase in morbidity, mortality and the burden to healthcare systems around the world can be attributed to the development of resistance in these bacteria. Accordingly, alternative antimicrobial agents have been sought as an attractive means to combat MDR pathogens, with one such example being antimicrobial peptides (AMPs). Given the reported activity of AMPs, including Pardaxin, MSI-78, dermaseptin-PC (DMPC) and Cecropin B, it is important to understand their activities and modes of action against bacteria for further AMP design. In this study, we compared these AMPs against a panel of nosocomial bacterial pathogens, followed by detailed mechanistic studies. It was found that Pardaxin (1–22) and MSI-78 (4–20) displayed the most pronounced antimicrobial activity against the tested bacteria. The mechanistic studies by membrane permeability and molecular dynamics simulation further confirmed the strong membrane interaction and structure of Pardaxin (1–22) and MSI-78 (4–20), which contributed to their potent activity. This study demonstrated a structure and activity guidance for further design of Pardaxin (1–22) and MSI-78 (4–20) as therapeutics against MDR pathogens. The different effects of DMPC (1–19) and Cecropin B (1–21) on membrane integrity and phospholipid membrane interactions provided critical information for the rational design of next-generation analogues with specificity against either Gram-negative or Gram-positive bacteria.

Mechanism of Antimicrobial Peptides: Antimicrobial, Anti-Inflammatory and Antibiofilm Activities.

Antimicrobial peptides (AMPs) are regarded as a new generation of antibiotics. Besides antimicrobial activity, AMPs also have antibiofilm, immune-regulatory, and other activities. Exploring the mechanism of action of AMPs may help in the modification and development of AMPs. Many studies were conducted on the mechanism of AMPs. The present review mainly summarizes the research status on the antimicrobial, anti-inflammatory, and antibiofilm properties of AMPs. This study not only describes the mechanism of cell wall action and membrane-targeting action but also includes the transmembrane mechanism of intracellular action and intracellular action targets. It also discusses the dual mechanism of action reported by a large number of investigations. Antibiofilm and anti-inflammatory mechanisms were described based on the formation of biofilms and inflammation. This study aims to provide a comprehensive review of the multiple activities and coordination of AMPs in vivo, and to fully understand AMPs to realize their therapeutic prospect.

Influence of Different Aromatic Hydrophobic Residues on the Antimicrobial Activity and Membrane Selectivity of BRBR-NH2 Tetrapeptide.

The ultrashort linear antimicrobial tetrapeptide BRBR-NH2 with an unnatural residue biphenylalanine (B) has potent and rapid antimethicillin-resistant Staphylococcus aureus (MRSA) activity but lacks hemolytic activity. The anti-MRSA activity of BRBR-NH2 is 8-fold more potent than that of WRWR-NH2 and 16-fold more potent than that of FRFR-NH2. However, how to influence their antimicrobial activities and mechanisms through the substitution of different aromatic hydrophobic residues is still unclear. In this work, to study the effects of varying hydrophobic interactions and membrane selectivities of BRBR-NH2, we performed multiple long-time (1000 ns) molecular dynamics (MD) simulations to investigate the interactions of a red blood cell (RBC) membrane and a Gram-positive bacterial cell membrane with three different tetrapeptides (BRBR-NH2, WRWR-NH2, and FRFR-NH2) under different ratios of peptides and lipids and also explored the changes in the membrane and structural characteristics of peptides. The binding energy results show that BRBR-NH2 interacts weakly with the RBC membrane, while not all BRBR-NH2 can be adsorbed to the RBC membrane surface. The MD simulation results produced significant local membrane thinning of multiBRBR-NH2 peptides in the Gram-positive bacterial cell membrane. An in-depth analysis of structural features and peptide-membrane interactions suggests that the aggregation of BRBR-NH2 on the membrane surface plays a crucial role in the destruction of the cell membrane. Taken together with the observed local membrane thinning, the in-depth analysis demonstrated that the interactions between the lipid bilayer and the BRBR-NH2 aggregation surface result in a local disturbance of the membrane structure. It can be concluded that the high anti-MRSA activity of BRBR-NH2 is attributed to the aggregation of BRBR-NH2 on the membrane surface. On the other hand, WRWR-NH2 and FRFR-NH2 peptides tend to bind with the membrane surface in a monomeric form and cover the membrane surface in a carpet-like manner. Therefore, these results provide an advanced microscopic understanding of how hydrophobic interactions or hydrophobic residues affect the antimicrobial activity and mechanism of antimicrobial peptides (AMPs).

Using fluorescence microscopy to shed light on the mechanisms of antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising in the fight against increasing bacterial resistance, but the development of AMPs with enhanced activity requires a thorough understanding of their mechanisms of action. Fluorescence microscopy is one of the most flexible and effective tools to characterize AMPs, particularly in its ability to measure the membrane interactions and cellular localization of peptides. Recent advances have increased the scope of research questions that can be addressed via microscopy through improving spatial and temporal resolution. Unique combinations of fluorescent labels and dyes can simultaneously consider different aspects of peptide-membrane interaction mechanisms. This review emphasizes the central role that fluorescence microscopy will continue to play in the interrogation of AMP structure-function relationships and the engineering of more potent peptides.

Research on the effect and mechanism of antimicrobial peptides HPRP-A1/A2 work against Toxoplasma gondii infection.

With increasing antibiotic resistance and drug safety concerns, novel therapeutics are urgently needed. Antimicrobial peptides are promising candidates that could address the spread of multidrug-resistant pathogens. HPRP-A1/A2 are known to display antimicrobial activity against gram-negative bacteria, gram-positive bacteria and some pathogenic fungi, but whether HPRP-A1/A2 work on Toxoplasma gondii (Tgondii) is unknown. In this study, we found that the viability of tachyzoites that received HPRP-A1/A2 treatment was significantly decreased, and there was a reduction in the adhesion to and invasion of macrophages by tachyzoites after HPRP-A1/A2 treatment. HPRP-A1/A2 damaged the integrity of tachyzoite membranes, as characterized by membrane disorganization in and cytoplasm outflow from tachyzoites. In addition, in vivo injection with HPRP-A1/A2 resulted in a significantly decreased number of tachyzoites and an accelerated Th1/Tc1 response, and elicited pro-inflammatory cytokines in Tgondii-infected mice. Furthermore, HPRP-A1/A2-treated splenocytes exhibited a significantly increased Tc1/Th1 response, and HPRP-A1/A2-stimulated macrophages inhibited the growth of carboxyfluorescein succinimidyl amino ester (CFSE)-labelled tachyzoites, which had higher TNF-α/IL-12 mRNA levels. Altogether, these results imply that HPRP-A1/A2 are effective against Tgondii through damaging the structure of tachyzoites and inducing a protective immune response, which could offer an alternative approach against Tgondii infection.

Role of calcium in reactive oxygen species-induced apoptosis in Candida albicans: an antifungal mechanism of antimicrobial peptide, PMAP-23

PMAP-23 (RIIDLLWRVRRPQKPKFVTVWVR-NH2) is an antimicrobial peptide (AMP) derived from porcine myeloid. Membrane disruption is thought to underpin the anticandidal activity of PMAP-23. However, many AMPs act via mechanisms other than simple membrane permeabilisation. Here, we investigated the anticandidal mechanism of PMAP-23 at low concentrations. Membrane disruption and depolarisation and rapid K+ efflux were observed in Candida albicans cells treated with 5 µM PMAP-23. In contrast, 2.5 µM PMAP-23 caused membrane depolarisation and K+ efflux without membrane disruption. The lower PMAP-23 concentration increased cytosolic and mitochondrial Ca2+ levels. Disruption of Ca2+ homeostasis altered the NAD+/NADH ratio and resulted in reactive oxygen species (ROS) accumulation and glutathione oxidation. PMAP-23 treatment also stimulated apoptosis, as evidenced by metacaspase activation, DNA fragmentation, and phosphatidylserine externalisation. Pretreatment with the mitochondrial Ca2+ uptake inhibitor (ruthenium red) or ROS scavenger (N-acetylcysteine) attenuated these apoptotic events. Our results suggest that PMAP-23 induces apoptosis as antifungal mechanism, and mitochondrial Ca2+-induced ROS is major factor to trigger the apoptosis. Thus, the anticandidal activity of PMAP-23 is not based solely on disruption of biological membranes but also involves induction of apoptosis via mitochondrial Ca2+-dependent ROS. PMAP-23 mode of action sheds new light on the antifungal mechanism of antimicrobial peptides, supporting the role of Ca2+ and ROS in apoptosis regulation.

Studies on the Antibacterial Activity and Mechanism of Antimicrobial Peptides Against Drug-Resistant Bacteria.

Antibiotic resistance is a growing threat to human health. More than anything else, multidrug-resistant gram-negative bacteria pose the biggest challenge to healthcare, which is predominantly due to the lack of effective therapeutic options. In the present study, we investigated the activities of peptides HX-12A, HX-12B and HX-12C against MDRAB and MDRKP isolates in vitro. Those three peptides displayed high antibacterial activities and rapid bactericidal effects against MDRAB and MDRKP isolates. Additionally, the peptides retained their activity even being present in pH 6.8 and 8.0, salt (NaCl 100 mM, CaCl2 1 mM, MgCl2 1 mM) or human serum (5%), especially peptide HX-12C. Moreover, the killing kinetics and transmission electron microscopy results suggested that the possible sterilization mechanism of peptides is to destroy the cell membrane, which makes it more difficult for bacteria to develop resistance. Thus, these three peptides may be developed into new antimicrobial agents.

Disruption and Proteome Alterations of Escherichia coli Induced by a Novel Antimicrobial Peptide from Tibetan Kefir

Our previous study has demonstrated the broad spectrum of antibacterial activities of a novel peptide F1 against fungi, gram‐positive and gram‐negative bacteria. F1 was produced by Lactobacillus paracasei subsp. Tolerans FX‐6 isolated from Tibetan kefir. This study was aimed to reveal the mechanisms for the antibacterial effects of F1 on Escherichia coli using transmission electron microscope and iTRAQ‐based quantitative proteomic analysis. The ultrastructural observation showed that the Escherichia coli cells shrunk after being treated with F1 for 0.5 h or 1 h, suggesting that there was a large extent of cytoplasmic leak. After 2 h, the cell was completely deformed and lysed. The results from iTRAQ‐based quantitative proteomic analysis showed that among 280 identified proteins in Escherichia coli, there were 31 proteins with significantly altered expression levels in response to the treatment of antimicrobial peptide F1, including 10 down‐regulated proteins and 21 up‐regulated proteins. Each of these proteins has different molecular functions according to the Kyoto Encyclopedia of Genes and Genomes pathways. Specifically, the tricarboxylic acid cycle, oxidative phosphorylation, glycerophospholipid metabolism, and cell cycle‐caulobacter pathways, which are associated with Escherichia coli cell death, were affected to the largest extent by the antimicrobial peptide F1. Overall, our study demonstrated a global response of Escherichia coli to the antimicrobial peptide F1, which provides novel insights on the mechanisms of antimicrobial peptides.

Connecting Peptide Physicochemical and Antimicrobial Properties by a Rational Prediction Model

The increasing rate in antibiotic-resistant bacterial strains has become an imperative health issue. Thus, pharmaceutical industries have focussed their efforts to find new potent, non-toxic compounds to treat bacterial infections. Antimicrobial peptides (AMPs) are promising candidates in the fight against antibiotic-resistant pathogens due to their low toxicity, broad range of activity and unspecific mechanism of action. In this context, bioinformatics' strategies can inspire the design of new peptide leads with enhanced activity. Here, we describe an artificial neural network approach, based on the AMP's physicochemical characteristics, that is able not only to identify active peptides but also to assess its antimicrobial potency. The physicochemical properties considered are directly derived from the peptide sequence and comprise a complete set of parameters that accurately describe AMPs. Most interesting, the results obtained dovetail with a model for the AMP's mechanism of action that takes into account new concepts such as peptide aggregation. Moreover, this classification system displays high accuracy and is well correlated with the experimentally reported data. All together, these results suggest that the physicochemical properties of AMPs determine its action. In addition, we conclude that sequence derived parameters are enough to characterize antimicrobial peptides.

Interactions of α-helices with lipid bilayers: a review of simulation studies

Membrane proteins, of which the majority seem to contain one or more α-helix, constitute approx. 30% of most genomes. A complete understanding of the nature of helix/bilayer interactions is necessary for an understanding of the structural principles underlying membrane proteins. This review describes computer simulation studies of helix/bilayer interactions. Key experimental studies of the interactions of α-helices and lipid bilayers are briefly reviewed. Surface associated helices are found in some membrane-bound enzymes (e.g. prostaglandin synthase), and as stages in the mechanisms of antimicrobial peptides and of pore-forming bacterial toxins. Transmembrane α-helices are found in most integral membrane proteins, and also in channels formed by amphipathic peptides or by bacterial toxins. Mean field simulations, in which the lipid bilayer is approximated as a hydrophobic continuum, have been used in studies of membrane-active peptides (e.g. alamethicin, melittin, magainin and dermaseptin) and of simple membrane proteins (e.g. phage Pf1 coat protein). All atom molecular dynamics simulations of fully solvated bilayers with transmembrane helices been applied to: the constituent helices of bacteriorhodopsin; peptide-16 (a simple model TM helix); and a number of pore-lining helices from ion channels. Surface associated helices (e.g. melittin and dermaseptin) have been simulated, as have α-helical bundles such as bacteriorhodopsin and alamethicin. From comparison of the results from the two classes of simulation, it emerges that a major theoretical challenge is to exploit the results of all atom simulations in order to improve the mean field approach.