Binding Of Antimicrobial Peptides Research Articles

Free Energy Analysis of Peptide-Induced Pore Formation in Lipid Membranes by Bridging Atomistic and Coarse-Grained Simulations.

Antimicrobial peptides (AMPs) are attractive materials for combating the antimicrobial resistance crisis because they can kill target microbes by directly disrupting cell membranes. Although thousands of AMPs have been discovered, their molecular mechanisms of action are still poorly understood. One broad mechanism for membrane disruption is the formation of membrane-spanning hydrophilic pores which can be stabilized by AMPs. In this study, we use molecular dynamics simulations to investigate the thermodynamics of pore formation in model single-component lipid membranes in the presence of one of three AMPs: aurein 1.2, melittin and magainin 2. To overcome the general challenge of modeling long time scale membrane-related behaviors, including AMP binding, clustering, and pore formation, we develop a generalizable methodology for sampling AMP-induced pore formation. This approach involves the long equilibration of peptides around a pore created with a nucleation collective variable by performing coarse-grained simulations, then backmapping equilibrated AMP-membrane configurations to all-atom resolution. We then perform all-atom simulations to resolve free energy profiles for pore formation while accurately modeling the interplay of lipid-peptide-solvent interactions that dictate pore formation free energies. Using this approach, we quantify free energy barriers for pore formation without direct biases on peptides or whole lipids, allowing us to investigate mechanisms of pore formation for these 3 AMPs that are a consequence of unbiased peptide diffusion and clustering. Further analysis of simulation trajectories then relates variations in pore lining by AMPs, AMP-induced lipid disruptions, and salt bridges between AMPs to the observed pore formation free energies and corresponding mechanisms. This methodology and mechanistic analysis have the potential to generalize beyond the AMPs in this study to improve our understanding of pore formation by AMPs and related antimicrobial materials.

Electrostatics-Based Computational Design and Experimental Analysis of Buforin II Antimicrobial Peptide Variants with Increased DNA Affinities.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics in the treatment of bacterial infections in part due to their targeting of generic bacterial structures that make it more difficult to develop drug resistance. In this study, we introduce and implement a design workflow to develop more potent AMPs by improving their electrostatic interactions with DNA, which is a putative intracellular target. Using the existing membrane-translocating AMP buforin II (BF2) as a starting point, we use a computational workflow that integrates electrostatic charge optimization, continuum electrostatics, and molecular dynamics simulations to suggest peptide positions at which a neutral BF2 residue could be substituted with arginine to increase DNA-binding affinity either significantly or minimally, with the latter choice done to determine whether AMP binding affinity depends on charge distribution and not just overall monopole. Our analyses predicted that T1R and L8R BF2 variants would yield substantial and minimal increases in DNA-binding affinity, respectively. These predictions were validated with experimental peptide-DNA binding assays with additional computational analyses providing structural insights. Additionally, experimental measurements of antimicrobial potency showed that a design to increase DNA binding can also yield greater potency. As a whole, this study takes initial steps to support the idea that (i) a design strategy aimed to increase AMP binding affinity to DNA by focusing only on electrostatic interactions can improve AMP potency and (ii) the effect on DNA binding of increasing the overall peptide monopole via arginine substitution depends on the position of the substitution. More broadly, this design strategy is a novel way to increase the potency of other membrane-translocating AMPs that target nucleic acids.

Antimicrobial peptides alter lipid bilayer properties before disrupting membrane barrier property.

Antimicrobial peptides (AMPs) have been the subject of inquiry since the initial search for antibiotics in the ­first half of 20th century; the first antibiotic used in humans was the linear pentadecapeptide gramicidin. Much of this interest comes from the modest frequency of bacterial resistance against AMPs. Though AMPs differ in sequence and structure, the majority share three core physicochemical properties; they are short, cationic and amphipathic. Moreover, enantiomers of AMPs tend to have similar potencies. These common features have led to a focus on the bacterial membrane as a target for AMPs. High concentrations of AMPs cause gross bilayer defects, through pore formation or micellar breakdown. At lower concentrations, AMPs have effects on bacterial physiology that appear unrelated to membrane permeabilization, suggesting that they may act through bilayer-mediated regulation (BMR) of membrane protein function. If correct, this offers a mechanism of action that integrates the known membrane-modifying effects of AMPs with their ability to broadly alter bacterial physiology at sub-pore forming concentrations. We tested this hypothesis using melittin, gramicidin S, and indolicidin, three AMPs with distinct structures, that indeed alter bilayer properties at concentrations below those required for pore formation. Contrary to expectation, AMP induced BMR is attenuated in anionic bilayers despite increased AMP binding, suggesting an organizing effect of membrane-bound counterions. We further found that amphiphilic bilayer modifiers promote membrane permeabilization, suggesting that BMR may be responsible for the cooperativity of AMP-induced permeabilization. Two membrane-impermeant detergents, CHAPS and zwittergent, promote pore formation despite inhibiting AMP binding, suggesting a role for leaflet-specific lateral pressure differences. These results suggest a new mechanism by which AMPs exert their bactericidal effect, which may guide antibiotic design and provide a path for circumventing bacterial resistance.

How do antimicrobial peptides disrupt the lipopolysaccharide membrane leaflet of Gram-negative bacteria?

HypothesisIt is widely regarded that antimicrobial peptides (AMPs) kill bacteria by physically disrupting microbial membranes and causing cytoplasmic leakage, but it remains unclear how AMPs disrupt the outer membrane (OM) of Gram-negative bacteria (GNB) and then compromise the inner membrane. We hypothesise that different AMPs impose different structural disruptions, with direct implications to their antimicrobial efficacies. ExperimentsThe antimicrobial activities of three typical AMPs, including the designed short AMP, G3, and two natural AMPs, melittin and LL37, against E. coli and their haemolytic activities were studied. Lipopolysaccharide (LPS) and anionic di-palmitoyl phosphatidyl glycerol (DPPG) monolayer models were constructed to mimic the outer membrane and inner membrane leaflets of Gram-negative bacteria. The binding and penetration of AMPs to the model lipid monolayers were systematically studied by neutron reflection via multiple H/D contrast variations. FindingG3 has relatively high antimicrobial activity, low cytotoxicity, and high proteolytic stability, whilst melittin has significant haemolysis and LL37 has weaker antimicrobial activity. G3 could rapidly lyse LPS and DPPG monolayers within 10–20 min. In contrast, melittin was highly active against the LPS membrane, but the dynamic process lasted up to 80 min, with excessive stacking in the OM. LL37 caused rather weak destruction to LPS and DPPG monolayers, leading to massive adsorption on the membrane surface without penetrating the lipid tail region. These findings demonstrate that the rationally designed AMP G3 was well optimised to impose most effective destruction to bacterial membranes, consistent with its highest bactericidal activity. These different interfacial structural features associated with AMP binding shed light on the future development of active and biocompatible AMPs for infection and wound treatments.

The Remarkable Innate Resistance of Burkholderia bacteria to Cationic Antimicrobial Peptides: Insights into the Mechanism of AMP Resistance.

Gram-negative bacteria belonging to the genus Burkholderia are remarkably resistant to broad-spectrum, cationic, antimicrobial peptides (AMPs). It has been proposed that this innate resistance is related to changes in the outer membrane lipopolysaccharide (OM LPS), including the constitutive, essential modification of outer membrane Lipid A phosphate groups with cationic 4-amino-4-deoxy-arabinose. This modification reduces the overall negative charge on the OM LPS which may change the OM structure and reduce the binding, accumulation, and permeation of cationic AMPs. Similarly, the Gram-negative pathogen Pseudomonas aeruginosa can quickly become resistant to many AMPs by multiple mechanisms, frequently, including activation of the arn operon, which leads, transiently, to the same modification of Lipid A. We recently discovered a set of synthetically evolved AMPs that do not invoke any resistance in P. aeruginosa over multiple passages and thus are apparently not inhibited by aminorabinosylation of Lipid A in P. aeruginosa. Here we test these resistance-avoiding peptides, within a set of 18 potent AMPs, against Burkholderia thailandensis. We find that none of the AMPs tested have measurable activity against B. thailandensis. Some were inactive at concentrations as high as 150μM, despite all having sterilizing activity at ≤ 10μM against a panel of common, human bacterial pathogens, including P. aeruginosa. We speculate that the constitutive modification of Lipid A in members of the Burkholderia genus is only part of a broader set of modifications that change the architecture of the OM to provide such remarkable levels of resistance to cationic AMPs.

Mechanical rigidity of bacterial membrane models: effects of lipids and peptides probed by MD

Antimicrobial peptides (AMPs) are membrane-targeting short sequence peptides that have potential for the development of new antibiotics. AMPs are generally cationic, and target negatively charged bacterial membranes, resulting in their disruption. The multiple peptide resistance factor (MprF) protein reduces AMP binding to bacterial membranes via the conversion of anionic phosphatidylglycerol (PG) lipids into either zwitterionic Alanyl-phosphatidylglycerol (Ala-PG) or cationic Lysyl-phosphatidylglycerol (Lys-PG) lipids, reducing the negative surface charge present on the membrane.

A rapid one-step process for the isolation of antibacterial peptides by silica-decorated Fe3O4nanoparticles

Antimicrobial peptides (AMPs), which are widely used as food preservatives, were purified to control the spoilage microorganism Alicyclobacillus acidoterrestris. The AMPs were produced by Pediococcus acidilactici CICC10344 and purified by silica-decorated Fe3O4 nanoparticles (SMNPs). The SMNPs were synthesized and characterized by transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR). The diameter of the SMNPs was 31 ± 7 nm. The VSM measurements indicated that the saturation magnetization value (Ms) of the SMNPs was 30 emu/g. We monitored AMP binding to the surface of Fe3O4 nanoparticles by FT-IR, showing that the amide had been successfully formed. The optimal condition for purification by SMNPs was an AMP concentration of 0.5 mg/mL for 30 min of exposure. The AMP purified by SMNPs retained good antimicrobial activity in pH 4–8 and temperature of 80 °C and 100 °C for 20 min. Scanning electron microscope (SEM) images indicated that a likely antibacterial mechanism is pore formation in the cell membrane. The one-step process proposed in the present work is convenient and effective for the purification of antibacterial peptides that may be widely used for the purification of other peptides.

Role of membrane proteins in bacterial resistance to antimicrobial peptides.

Several natural antimicrobial peptides (AMPs), including the novel semisynthetic lipoglycopeptide antibiotics telavancin, dalbavancin, and oritavancin, have been approved for clinical use to address the growing problem of multiple antibiotic-resistant Gram-positive bacterial infections. Nevertheless, the efficacy of these antibiotics has already been compromised. The SARS-CoV-2 pandemic led to the increased clinical use of all antibiotics, further promoting the development of bacterial resistance. Therefore, it is critical to gain a deeper understanding of the role of resistance mechanisms to minimize the consequential risks of long-term antibiotic use and misuse. Here, we summarize for the first time the current knowledge of resistance mechanisms that have been shown to cause resistance to clinically used AMPs, with particular focus on membrane proteins that have been reported to interfere with the activity of AMPs by affecting the binding of AMPs to bacteria.

Interaction of Human β Defensin Type 3 (hBD-3) with Different PIP2-Containing Membranes, a Molecular Dynamics Simulation Study.

Human β defensin type 3 (hBD-3) is a cysteine-rich small antibacterial peptide. It belongs to the human innate immune system. hBD-3 has six cysteine residues, which form three pairs of disulfide bonds, and those bonds break in the reducing condition. It is known that hBD-3 can interact with bacterial membrane, and even eukaryotic cell membrane, which has a low concentration of phosphatidylinositol 4,5-bisphosphate (PIP2) lipids. PIP2 is a vital component in cell membranes and has been found to play important roles during antimicrobial peptide (AMP) interaction with membranes. To understand the functional mechanism of hBD-3 interacting with PIP2-containing membranes, the binding structures of hBD-3 on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers mixed with 10% of PIP2 were predicted using two kinds of methods. The first one is by placing the hBD-3 monomer in different orientations above the POPC + 10%PIP2 membrane to set up five different initial simulation systems and performing long-term simulations on each to predict the most stable binding structure. It was found that hBD-3 analogue binds on the mixed lipid membrane on the two loop regions. The second method is by running long-term simulations on one or nine hBD-3 dimers binding on POPC mixed with 10%PIP2 lipid bilayer starting from the solid-state NMR (ssNMR)-suggested orientation. The dimer dissociated, and the most stable binding of hBD-3 in wild-type on the mixed membrane is also through the two loop regions, which agrees with the prediction from both the first method and the lipid self-assembly result. The PIP2 lipids can form long-lasting hydrogen bonds with positively charged residues such as Arg and Lys on hBD-3, thus forming clusters with hBD-3. As a comparison, hBD-3 dimers binding with a combined bilayer having 1,2-palmitoyl-oleoyl-sn-glycero-3-phosphoserine (POPS) on the upper and POPC on the lower leaflets and the combined POPS + POPC bilayer mixing with 10%PIP2 were also studied. The long-term simulation result shows that hBD-3 can bind with the heads of negatively charged POPS and PIP2 lipids and form hydrogen bonds. The stable binding sites of hBD-3 on PIP2 or POPS mixed bilayers are still on the two loop regions. On the combined POPS + POPC mixed with 10%PIP2 bilayer, the binding of hBD-3 with PIP2 lipids became less stable and fewer because of the competition of binding with the POPS lipids. Besides that, binding with hBD-3 can decrease the membrane thickness of the POPC + PIP2, POPS + POPC, and POPS + POPC + PIP2 bilayers and make POPS and PIP2 lipids more flexible based on the order parameter calculations. Our results supply molecular insight on AMP binding with different membranes and can help understand the functional mechanism of hBD-3 disrupting PIP2-containing membranes.

Membrane Binding of Antimicrobial Peptides Is Modulated by Lipid Charge Modification.

Peptide interactions with lipid bilayers play a key role in a range of biological processes and depend on electrostatic interactions between charged amino acids and lipid headgroups. Antimicrobial peptides (AMPs) initiate the killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defense mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysyl-phosphatidyl glycerol (Lys-PG). The resulting change in the membrane charge is suggested to reduce the binding of AMPs to membranes, thus impeding downstream AMP activity. Using coarse-grained molecular dynamics to investigate the effects of these modified lipids on AMP binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. More than 5000 simulations in total are used to define the relationship between lipid bilayer composition, peptide sequence (using five different membrane-active peptides), and peptide binding to membranes. The degree of interaction of a peptide with a membrane correlates with the membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that the lipid modifications due to MprF alter the energy barrier to peptide helix penetration of the bilayer. These results will offer a guide to the design of novel peptides, which addresses the issue of resistance via MprF-mediated membrane modification.

A steady-state modeling approach for simulation of antimicrobial peptide-cell membrane interaction

Antimicrobial Peptides (AMPs) are host defense molecules that initiate microbial death by binding to the membrane. On membrane binding, AMPs undergo changes in conformation and aggregation state to enable killing action. Depending on the AMP and cell membrane characteristics, the nature of binding can be aggregating or non-aggregating, with high/low cooperativity, at single or multiple sites with high/low affinity leading to a unique killing action that needs to be studied individually. In the present study, a steady-state model that simulates AMP-membrane interaction was developed and was used to predict the mechanism of AMP binding. The predictions obtained from the model were validated with experimentally deciphered values available in literature. The model was further used to predict the mechanism for a set of designed AMPs with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family. Depending on the predicted mechanism, a unique half saturation constant and steepness of response (Hill coefficient) was obtained which was further validated with available data from literature. The model could reliably predict the mechanism, the half saturation constant and the Hill coefficient values. Further based on the analysis, it was observed that aggregation and oligomerization result in drastic killing action in a short range of peptide concentration owing to high Hill coefficient values. Mechanisms such as monomers binding at multiple sites with/without cooperativity result in antimicrobial activity at low half saturation constant though the killing action may not be steep. Thus, the methodology developed here can be used to develop hypothesis for studying AMP-membrane interaction mechanisms.

Hydrophobic Control of the Bioactivity and Cytotoxicity of de Novo-Designed Antimicrobial Peptides.

Antimicrobial peptides (AMPs) can target bacterial membranes and kill bacteria through membrane structural damage and cytoplasmic leakage. A group of surfactant-like cationic AMPs was developed from substitutions to selective amino acids in the general formula of G(IIKK)3I-NH2, (called G3, a de novo AMP), to explore the correlation between AMP hydrophobicity and bioactivity. A threshold surface pressure over 12 mN/m was required to cause measurable antimicrobial activity and this corresponded to a critical AMP concentration. Greater surface activity exhibited stronger antimicrobial activity but had the drawback of worsening hemolytic activity. Small unilamellar vesicles (SUVs) with specific lipid compositions were used to model bacterial and host mammalian cell membranes by mimicking the main structural determinants of the charge and composition. Leakage from the SUVs of encapsulated carboxyfluorescein measured by fluorescence spectroscopy indicated a negative correlation between hydrophobicity and model membrane selectivity, consistent with measurements of the zeta potential that demonstrated the extent of AMP binding onto model SUV lipid bilayers. Experiments with model lipid membranes thus explained the trend of minimum inhibitory concentrations and selectivity measured from real cell systems and demonstrated the dominant influence of hydrophobicity. This work provides useful guidance for the improvement of the potency of AMPs via structural design, whilst taking due consideration of cytotoxicity.

Charges' interaction in polyelectrolyte (nano)complexing of His6-OPH with peptides: Unpredictable results due to imperfect or useless concept?

Quorum Quenching (QQ) enzymes can be used to prevent bacterial antibiotic resistance by degradation of Quorum Sensing (QS) signaling molecules, for example N-acyl homoserine lactones (AHLs). This paper is aimed at the in silico investigation of the possible combinations of hexahistidine-tagged organophosphorus hydrolase (His6-OPH) with antimicrobial peptides (AMPs) to improve the enzyme activity and, promisingly, stability. This shall help creating a nanosized QQ preparation capable to hydrolyze different AHLs and possessing an antimicrobial activity. To achieve this, binding of AMPs and His6-OPH was simulated by molecular docking, and various interaction parameters (affinity, charge, contact area, etc.) of the generated models were studied. Both anionic and cationic polypeptides were shown to bind to His6-OPH with negligible effect of their charge, that significantly deviates from the charge-to-charge interaction concept. The (nano)complexes of His6-OPH with Indolicidin and Temporin A appear to have the most balanced characteristics which were issued experimentally also.

Understanding Spatiotemporal Aspects of Cecropin a Attack on Single, Live Bacteria using Time-Lapse Fluorescence Microscopy

Antimicrobial peptides (AMPs) halt growth of bacterial cells without causing substantial damage to eukaryotic cells. This specificity arises from charge-mediated electrostatic binding of AMPs with anionic bacterial membranes. Previous studies in our group have explored the membrane permeabilization caused by AMPs in detail. Low micromolar concentrations of AMPs can permeabilize exponentially growing cells within ∼10 minutes. Here we investigate parameters that might influence the spatial and temporal aspects of Cecropin A action, as observed in our microscopy assays. The effects of lipopolysaccharide (LPS) core components, i.e., negative charge from phosphorylation, and number of sugar layers was studied. Single cell, time resolved assays were performed on the K-12 BW25113 wild type strain and its deep rough mutants that have reduced LPS layers in terms of the charge or the number of sugars. A reduction in the length and/or the charge of the core LPS layer resulted in faster permeabilization of the E. coli membranes. This suggests that the sugars and the phosphate groups are both hindrances for the permeabilization action of Cecropin A on E. coli. Additionally, the spatial pattern of permeabilization of any of the membranes did not change as a result of deep rough mutations, suggesting that a different factor may be causing the pattern.

Supported Lipid Bilayers with Phosphatidylethanolamine as the Major Component.

Phosphatidylethanolamine (PE) is notoriously difficult to incorporate into model membrane systems, such as fluid supported lipid bilayers (SLBs), at high concentrations because of its intrinsic negative curvature. Using fluorescence-based techniques, we demonstrate that having fewer sites of unsaturation in the lipid tails leads to high-quality SLBs because these lipids help to minimize the curvature. Moreover, shorter saturated chains can help maintain the membranes in the fluid phase. Using these two guidelines, we find that up to 70 mol % PE can be incorporated into SLBs at room temperature and up to 90 mol % PE can be incorporated at 37 °C. Curiously, conditions under which three-dimensional tubules project outward from the planar surface as well as conditions under which domain formation occurs can be found. We have employed these model membrane systems to explore the ability of Ni2+ to bind to PE. It was found that this transition metal ion binds 1000-fold tighter to PE than to phosphatidylcholine lipids. In the future, this platform could be exploited to monitor the binding of other transition metal ions or the binding of antimicrobial peptides. It could also be employed to explore the physical properties of PE-containing membranes, such as phase domain behavior and intermolecular hydrogen bonding.

Modulating charge-dependent and folding-mediated antimicrobial interactions at peptide\u2013lipid interfaces

Peptide–lipid interactions support a variety of biological functions. Of particular interest are those that underpin fundamental mechanisms of innate immunity that are programmed in host defense or antimicrobial peptide sequences found virtually in all multicellular organisms. Here we synthetically modulate antimicrobial peptide–lipid interactions using an archetypal helical antimicrobial peptide and synthetic membranes mimicking bacterial and mammalian membranes in solution. We probe these interactions as a function of membrane-induced folding, membrane stability and peptide–lipid ratios using a correlative approach encompassing light scattering and spectroscopy measurements such as circular dichroism spectroscopy, fluorescence and nuclear magnetic resonance spectroscopy. The peptide behavior is assessed against that of its anionic counterpart having similar propensities for α-helical folding. The results indicate strong correlations between peptide folding and membrane type, supporting folding-responsive binding of antimicrobial peptides to bacterial membranes. The study provides a straightforward approach for modulating structure–activity relationships in the context of membrane-induced antimicrobial action, thus holding promise for the rational design of potent antimicrobial agents.

Gold-nanoparticles coated with the antimicrobial peptide esculentin-1a(1-21)NH2 as a reliable strategy for antipseudomonal drugs.

Despite conjugation of AMPs to AuNPs represents a worthwhile solution to face some limitations for their development as new therapeutics, only a very limited number of studies is available on peptide-coated AuNPs. Importantly, this is the first report showing that a covalent binding of a linear AMP via a poly(ethylene glycol) linker to AuNPs highly enhances antipseudomonal activity, preserving the same mode of action of the free peptide, without being harmful. Furthermore, AuNPs@Esc(1-21) are expected to accelerate recovery of an injured skin layer. All together, these findings suggest our peptide-coated AuNPs as attractive novel nanoscale formulation to treat bacterial infections and to heal the injured tissue.

Mode of Action of Antimicrobial Peptides on E. coli Spheroplasts

We investigated the phenomena of antimicrobial peptides (AMPs) directly attacking the cytoplasmic membranes of Escherichia coli spheroplasts. We developed a procedure for fluorescence recovery after photobleaching to examine dye leakage through bacterial membranes as AMPs in solution bound to the membranes. We found that the AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the response of a lipid-bilayer vesicle, which always showed a membrane area expansion by AMP binding. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of fluorescence recovery after photobleaching showed that the fluxes of dye molecules into and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. We discovered a new, to our knowledge, experimental parameter called the flux rate that characterizes the AMP-induced permeability of dye molecules through bacterial membranes. The phenomena observed in bacterial membranes are consistent with the pore-forming activities of AMPs previously observed in lipid bilayers. The experimental value of the flux rate per pore is much smaller than a theoretical value that assumes no friction for the dye molecule’s permeation through the pore. We believe that experimental studies of the flux rate will be useful for further analysis of AMPs’ permeabilization mechanisms.

Interactions of Histidine-Rich Antimicrobial Peptides, Gad-1 and Gad-2, with Model Membranes at low and high pH

Antimicrobial peptides (AMPs) are an important component of the innate immune system of many different organisms. Their cationic nature and amphipathic structure make them well suited to interact with and perturb bacterial membranes. A major driving force in the binding of AMPs to bacterial membranes is the electrostatic interaction between positively charged residues of AMPs and anionic lipids of the bacterial membrane. Since histidine has a pKa ∼ 6, histidine-rich AMPs can exhibit greater activity at low pH, when the histidine is positively charged, as compared to at neutral pH, when the histidine is neutral. While Gad-1 and Gad-2 are both histidine-rich AMPs, only Gad-2 exhibits pH-dependent activity against E. coli. Hence, it was surprising to find that neither peptide exhibited pH-dependent membrane disruption in model membranes, when probed by 2H NMR. Thus, we hypothesized the differences in activity might be related to differences in binding affinity. In order to probe the binding, we carried out zeta potential measurements with both Gad-1 and Gad-2 at pH 7.0 and pH 5.0. Additionally, 15N solid state NMR measurements were carried out to establish the topology of the Gad peptides in the bilayer. Together these studies can provide a better understanding of membrane-peptide interactions and the effect of pH on these interactions.

Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985-8994, 2010, http://dx.doi.org/10.1074/jbc.M109.078725; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem 287:418-428, 2012, http://dx.doi.org/10.1074/jbc.M111.290361). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with Escherichia coli signal peptide was expressed in a hexamer, which was anchored on the surface of E. coli, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native P. aeruginosa OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.