Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis.     

Role of charge properties of bacterial envelope in bactericidal action of human group IIA phospholipase A2 against Staphylococcus aureus

Mammalian Group IIA phospholipases A(2) (PLA(2)) potently kill Staphylococcus aureus. Highly cationic properties of these PLA(2) are important for Ca(2+)-independent binding and cell wall penetration, prerequisites for Ca(2+)-dependent degradation of membrane phospholipids and bacterial killing. To further delineate charge properties of the bacterial envelope important in Group IIA PLA(2) action against S. aureus, we examined the effects of mutations that prevent specific modifications of cell wall (dltA) and cell membrane (mprF) polyanions. In comparison to the parent strain, isogenic dltA(-) bacteria are approximately 30-100x more sensitive to PLA(2), whereas mprF(-) bacteria are <3-fold more sensitive. Differences in PLA(2) sensitivity of intact bacteria reflect differences in cell wall, not cell membrane, properties since protoplasts from all three strains are equally sensitive to PLA(2). A diminished positive charge in PLA(2) reduces PLA(2) binding and antibacterial activity. In contrast, diminished cell wall negative charge by substitution of (lipo)teichoic acids with d-alanine reduces antibacterial activity of bound PLA(2), but not initial PLA(2) binding. Therefore, the potent antistaphylococcal activity of Group IIA PLA(2) depends on cationic properties of the enzyme that promote binding to the cell wall, and polyanionic properties of cell wall (lipo)teichoic acids that promote attack of membrane phospholipids by bound PLA(2).     

Interaction between heat shock proteins and antimicrobial peptides

Drosocin, pyrrhocoricin, and apidaecin, representing the short (18-20 amino acid residues) proline-rich antibacterial peptide family, originally isolated from insects, were shown to act on a target bacterial protein in a stereospecific manner. Native pyrrhocoricin and one of its analogues designed for this purpose protect mice from bacterial challenge and, therefore, may represent alternatives to existing antimicrobial drugs. Furthermore, this mode of action can be a basis for the design of a completely novel set of antibacterial compounds, peptidic or peptidomimetic, if the interacting bacterial biopolymers are known. Recently, apidaecin was shown to enter Escherichia coli and subsequently kill bacteria through sequential interactions with diverse target macromolecules. In this paper report, we used biotin- and fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin analogues to identify biopolymers that bind to these peptides and are potentially involved in the above-mentioned multistep killing process. Through use of a biotin-labeled pyrrhocoricin analogue, we isolated two interacting proteins from E. coli. According to mass spectrometry, Western blot, and fluorescence polarization, the short, proline-rich peptides bound to DnaK, the 70-kDa bacterial heat shock protein, both in solution and on the solid-phase. GroEL, the 60-kDa chaperonin, also bound in solution. Control experiments with an unrelated labeled peptide showed that while binding to DnaK was specific for the antibacterial peptides, binding to GroEL was not specific for these insect sequences. The killing of bacteria and DnaK binding are related events, as an inactive pyrrhocoricin analogue made of all-D-amino acids failed to bind. The pharmaceutical potential of the insect antibacterial peptides is underscored by the fact that pyrrhocoricin did not bind to Hsp70, the human equivalent of DnaK. Competition assay with unlabeled pyrrhocoricin indicated differences in GroEL and DnaK binding and a probable two-site interaction with DnaK. In addition, all three antibacterial peptides strongly interacted with two bacterial lipopolysaccharide (LPS) preparations in solution, indicating that the initial step of the bacterial killing cascade proceeds through LPS-mediated cell entry.     

Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 mug of MC 540 per ml and a light dose of 600 J/cm2 or to 20 mug of MC 540 per ml and a light dose of 450 J/cm2. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

Action of antimicrobial peptides: two-state model

The argument and experimental evidence are presented for a two-state model that explains the action of both helical and beta-sheet antimicrobial peptides after they bind to the plasma membranes of cells. Each peptide has two distinct physical states of binding to lipid bilayers. At low peptide-to-lipid ratios (P/L), the peptide tends to adsorb in the lipid headgroup region in a functionally inactive state. At a P/L above a threshold value P/L, the peptide forms a multiple-pore state that is lethal to a cell. The susceptibility of a cell to an antimicrobial peptide depends on the value of P/L that is determined by the lipid composition of the cell membrane. This model provides plausible explanations for the experimental findings that the susceptibility of different bacteria to a peptide is not directly correlated to its binding affinity, different peptides preferentially kill different pathogens, and peptides exhibit varying levels of lytic activity against different eukaryotic cells.     

Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides

Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Resistance mechanisms of bacteria to antimicrobial compounds

A range of antimicrobial compounds (bactericides) commonly termed biocides, microbicides, sanitizers, antiseptics and disinfectants are available, all of which are claimed by their producers to kill bacteria. Resistance has been defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they are not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tolerance is a genetic change, and where the biochemical basis is known. Antimicrobial substances target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes and the genetic material. However, different bacteria react differently to bactericides, either due to inherent differences such as unique cell envelope composition and non-susceptible proteins, or to the development of resistance, either by adaptation or by genetic exchange. At low concentrations bactericides often act bacteriostatically, and are only bacteriocidal at higher concentrations. For bactericides to be effective, they must attain a sufficiently high concentration at the target site in order to exert their antibacterial action. In order to reach their target site(s), they must traverse the outer membrane of the gram negative bacteria. Bacteria with effective penetration barriers to biocides generally display a higher inherent resistance than those bacteria which are readily penetrated. The rate of penetration is linked to concentration, so that a sufficiently high bactericide concentration will kill bacteria with enhanced penetration barriers. It has been indicated that susceptible bacterial isolates acquire increased tolerance to bactericides following serial transfer in sub-inhibitory concentrations. Whereas the basis of bacterial resistance to antibiotics is well know, that of resistance to antiseptics, disinfectants and food preservatives is less well understood.Three mechanisms of resistance that have been reported include:

• limited diffusion of antimicrobial agents through the biofilm matrix,

• interaction of the antimicrobial agents with the biofilm matrix (cells and polymer),

• enzyme mediated resistance,

• level of metabolic activity within the biofilm

• genetic adaptation

• efflux pumps and

• outer membrane structure.

     

Surface kinetic test method for determining rate of kill by an antimicrobial solid.

An antimicrobial-surface kinetic test which maximizes probability of cell-to-surface contact has been developed. The measurement of rate of kill by a nonleaching antimicrobial surface is based on the number of surviving bacterial cells at specific times of exposure to various amounts of total treated surface area of test substrate. This method gives information for direct comparison of rate of kill for a variety of antimicrobial surfaces in terms of rate of kill per square centimeter of surface area. Data obtained by this method can also give valuable dose response application information as an indication of the exponential efficiency of concentration in terms of treated surface area.     

Surface kinetic test method for determining rate of kill by an antimicrobial solid.

An antimicrobial-surface kinetic test which maximizes probability of cell-to-surface contact has been developed. The measurement of rate of kill by a nonleaching antimicrobial surface is based on the number of surviving bacterial cells at specific times of exposure to various amounts of total treated surface area of test substrate. This method gives information for direct comparison of rate of kill for a variety of antimicrobial surfaces in terms of rate of kill per square centimeter of surface area. Data obtained by this method can also give valuable dose response application information as an indication of the exponential efficiency of concentration in terms of treated surface area.     

Use of Merocyanine 540 for Photodynamic Inactivation of Staphylococcus aureus Planktonic and Biofilm Cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm² or to 20 μg of MC 540 per ml and a light dose of 450 J/cm². A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

The human immune system can directly lyse invading micro‐organisms and aberrant host cells by generating pores in the cell envelope, called membrane attack complexes (MACs). Recent studies using single‐particle cryoelectron microscopy have revealed that the MAC is an asymmetric, flexible pore and have provided a structural basis on how the MAC ruptures single lipid membranes. Despite these insights, it remains unclear how the MAC ruptures the composite cell envelope of Gram‐negative bacteria. Recent functional studies on Gram‐negative bacteria elucidate that local assembly of MAC pores by surface‐bound C5 convertase enzymes is essential to stably insert these pores into the bacterial outer membrane (OM). These convertase‐generated MAC pores can subsequently efficiently damage the bacterial inner membrane (IM), which is essential for bacterial killing. This review summarizes these recent insights of MAC assembly and discusses how MAC pores kill Gram‐negative bacteria. Furthermore, this review elaborates on how MAC‐dependent OM damage could lead to IM destabilization, which is currently not well understood. A better understanding on how MAC pores kill bacteria could facilitate the future development of novel strategies to treat infections with Gram‐negative bacteria.     

甘氨酸脂肪醇酯盐酸盐抗菌作用的测试

本文用滤纸片琼脂弥散试验和活菌平板计数法比较了五种甘氨酸脂肪醇酯盐酸盐对不同细菌，霉菌的抗菌活性，认为甘氨酸癸酯盐酸盐表现出最高的抗菌活性，测试出杀死细菌繁殖体的较适浓度和时间，并讨论了抗菌能力不同的原因。     

From "carpet" mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides

Living organisms of all types produce a large repertoire of gene-encoded, net positively charged, antimicrobial peptides as part of their innate immunity to microbial invasion. Despite significant variations in composition, length and secondary structure most antimicrobial peptides are active in micromolar concentrations, suggesting a common general mechanism for their mode of action. Many antimicrobial peptides bind bacterial phospholipid membranes up to a threshold concentration, followed by membrane permeation/disintegration (the "carpet" mechanism). Recent data suggest that the details of the permeation pathways may vary for different peptides and are assigned to different modes of action. Accumulating data reveal that the molecular basis for cell selectivity is the ability of peptides to specifically bind the negatively charged bacterial membrane, as well as their oligomeric state in solution and in the membrane. Based on the "carpet" mechanism and the role of the peptide oligomeric state, a novel group of diastereomeric (containing D- and L-amino acids) antimicrobial peptides were developed. These peptides may serve as promising templates for the future designs of antimicrobial peptides.     

Phagocytosis of bacterial pathogens

Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general.     

Phagocytosis of bacterial pathogens

Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general.     

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.     

Nusbiarylins,a new class of antimicrobial agents: Rational design of bacterial transcription inhibitors targeting the interaction between the NusB and NusE proteins

Discovery of antibiotics of a novel mode of action is highly required in the fierce battlefield with multi-drug resistant bacterial infections. Previously we have validated the protein-protein interaction between bacterial NusB and NusE proteins as an unprecedented antimicrobial target and reported the identification of a first-in-class inhibitor of bacterial ribosomal RNA synthesis with antimicrobial activities. In this paper, derivatives of the hit compound were rationally designed based on the pharmacophore model for chemical synthesis, followed by biological evaluations. Some of the derivatives demonstrated the improved antimicrobial activity with the minimum inhibitory concentration (MIC) at 1–2 μg/mL against clinically significant bacterial pathogens. Time-kill kinetics, confocal microscope, ATP production, cytotoxicity, hemolytic property and cell permeability using Caco-2 cells of a representative compound were also measured. This series of compounds were named “nusbiarylins” based on their target protein NusB and the biaryl structure and were expected to be further developed towards novel antimicrobial drug candidates in the near future.     

A rapid microplate method for quantifying inhibition of bacterial adhesion to eukaryotic cells

Adhesion of bacteria to the mucosal epithelial cell surface is the first step in infection, and studies have shown that inhibition of this step may be useful therapeutically. To test compounds that may prevent bacterial binding to a number of epithelial cell lines, we have developed a high-throughput adhesion assay using a microtitre plate system and bacteria that have been modified to express firefly luciferase. This method has proved to be a sensitive, rapid, and reproducible system for screening antiadhesive agents for their effects on bacterial adhesion.     

Antimicrobial activity profiles of Amphiphilic Xanthone derivatives are a function of their molecular Oligomerization

Currently, membrane-targeting small antimicrobial peptidomimetics (SAP) are important in antibiotic development because bacteria appear to develop resistance to these surface-active compounds less readily. However, the molecular membrane-targeting action of SAPs has received little attention. In this study, we investigated the effect of oligomerization of amphiphilic xanthone, a model SAP, on its antimicrobial properties against both Gram-positive and Gram-negative bacteria. First, oligomer formation by an amphiphilic xanthone, compound 2 (also coded as AM052), was investigated via solution-state nuclear magnetic resonance (NMR) spectroscopy. Then, the effects of oligomerization on membrane disruption were further studied via biophysical approaches. The results showed that the antimicrobial activities of SAPs develop in several stages: oligomer formation in aqueous solution, initial binding of oligomers to the membrane-water interface followed by insertion into the membrane bilayer, aggregation of antimicrobial oligomers in the membrane, and induced membrane leakage. Ultimately, the presence of the oligomers in the bacterial membrane leads to decreased membrane fluidity and bacterial cell death. Interestingly, the early formation of large oligomers leads to stronger membrane disruption and more rapid bacterial killing. However, reduced antimicrobial activities against Gram-negative bacteria were observed for compounds that formed larger oligomers because the LPS layer acts as a barrier to large complexes. Taken together, our results suggest that oligomerization of SAPs has a strong impact on their antimicrobial properties.