Cationic spacer arm design strategy for control of antimicrobial activity and conformation of amphiphilic methacrylate random copolymers

Antimicrobial and hemolytic activities of amphiphilic random copolymers were modulated by the structure of the cationic side chain spacer arms, including 2-aminoethylene, 4-aminobutylene, and 6-aminohexylene groups. Cationic amphiphilic random copolymers with ethyl methacrylate (EMA) comonomer were prepared with a range of comonomer fractions, and the library of copolymers was screened for antimicrobial and hemolytic activities. Copolymers with 4-aminobutylene cationic side chains showed an order of magnitude enhancement in their antimicrobial activity relative to those with 2-aminoethylene spacer arms, without causing adverse hemolysis. When the spacer arms were further elongated to hexylene, the copolymers displayed potent antimicrobial and hemolytic activities. The 4-aminobutylene side chain appears to be the optimal spacer arm length for maximal antimicrobial potency and minimal hemolysis, when combined with hydrophobic ethylmethacrylate in a roughly 70/30 ratio. The copolymers displayed relatively rapid bactericidal kinetics and broad-spectrum activity against a panel of Gram-positive and Gram-negative bacteria. The effect of the spacer arms on the polymer conformation in the membrane-bound state was investigated by molecular dynamics simulations. The polymer backbones adopt an extended chain conformation, parallel to the membrane surface. A facially amphiphilic conformation at the membrane surface was observed, with the primary ammonium groups localized at the lipid phoshophate region and the nonpolar side chains of EMA comonomers buried in the hydrophobic membrane environment. This study demonstrates that the antimicrobial activity and molecular conformation of amphiphilic methacrylate random copolymers can be modulated by adjustment of cationic side chain spacer arms.     

Biological activities of retro and diastereo analogs of a 13-residue peptide with antimicrobial and hemolytic activities.

The biological activities of synthetic retro and diastereo analogs of PKLLKTFLSKWIG (SPFK), a 13-residue peptide with antimicrobial and hemolytic activities, have been investigated. Retro peptides with C-terminal acid and amide exhibited antibacterial activities comparable with those of SPFK. Their hemolytic activities were, however, only marginally lower. The diastereo analog with C-terminal acid was not antibacterial and was weakly hemolytic. Amidation of this analog could restore antibacterial activity. Both retro analogs were unordered in aqueous medium but had a propensity for a helical structure in trifluoroethanol. However, diastereo analogs were unordered in both aqueous medium and trifluoroethanol. Thus, reversing the sequence in a short amphiphilic peptide may not always result in the selective loss of biological activity such as hemolytic activity. Also, introduction of enantiomeric amino acids in a short peptide to generate a diastereomer may result in loss of structure as well as antimicrobial and hemolytic activities, unless compensated by an increase in positive charges.     

Exploring the antibacterial and hemolytic activity of shorter- and longer-chain beta-, alpha,beta-, and gamma-peptides, and of beta-peptides from beta2-3-aza- and beta3-2-methylidene-amino acids bearing proteinogenic side chains--a survey

The antibacterial activities of 31 different beta-, mixed alpha/beta-, and gamma-peptides, as well as of beta-peptides derived from beta2-3-aza- and beta3-2-methylidene-amino acids were assayed against six pathogens (Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), and the results were compared with literature data. The interaction of these peptides with mammalian cells, as modeled by measuring the hemolysis of human erythrocytes, was also investigated. In addition to those peptides designed to fold into amphiphilic helical conformations with positive charges on one face of the helix, one new peptide with hemolytic activity was detected within the sample set. Moreover, it was demonstrated that neither cationic peptides used for membrane translocation (beta3-oligoarginines), nor mixed alpha/beta- or gamma-peptides with somatostatin-mimicking activities display unwanted hemolytic activity.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences, model membrane binding and biological activities.

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt beta-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Structure-activity relationship study of anoplin.

Anoplin is a decapeptide amide, GLLKRIKTLL-NH2 derived from the venom sac of the solitary spider wasp, Anoplius samariensis. It is active against Gram-positive and Gram-negative bacteria and is not hemolytic towards human erythrocytes. The present paper reports a structure-activity study of anoplin based on 37 analogues including an Ala-scan, C- and N-truncations, and single and multiple residue substitutions with various amino acids. The analogues were tested for antibacterial activity against both S. aureus ATCC 25923 and E. coli ATCC 25922, and several potent antibacterial analogues were identified. The cytotoxicity of the analogues against human erythrocytes was assessed in a hemolytic activity assay. The antibacterial activity and selectivity of the analogues against S. aureus and E. coli varied considerably, depending on the hydrophobicity and position of the various substituted amino acids. In certain cases the selectivity for Gram-positive and Gram-negative bacteria was either reversed or altogether eliminated. In addition, it was generally found that antibacterial activity coincided with hemolytic activity.     

Dimeric unnatural polyproline-rich peptides with enhanced antibacterial activity

We report a dimerization strategy to enhance the antibacterial potency of an otherwise weak cationic amphiphilic polyproline helical (CAPH) peptide. Overall, the dimeric CAPHs were more active against Escherichia coli and Staphylococcus aureus than the monomeric counterpart, reaching up to a 60-fold increase in potency. At their minimum inhibitory concentration (MIC), the dimeric peptides demonstrated no hemolytic activity or bacterial membrane disruption as monitored by β-galactosidase release in E. coli. At higher concentrations the dimeric agents were found to induce β-galactosidase release, but maintained negligible hemolytic activity, pointing to a potential shift in the mechanism of action at higher concentrations. Thus, discontinuous dimerization of an unnatural proline-rich peptide was a successful strategy to create potent de novo antibacterial peptides without membrane lysis.     

Homogeneous hybrid droplet interface bilayers assembled from binary mixtures of DPhPC phospholipids and PB-b-PEO diblock copolymers

Hybrid membranes built from phospholipids and amphiphilic block copolymers seek to capitalize on the benefits of both constituents for constructing biomimetic interfaces with improved performance. However, hybrid membranes have not been formed or studied using the droplet interface bilayer (DIB) method, an approach that offers advantages for revealing nanoscale changes in membrane structure and mechanics and offers a path toward assembling higher-order tissues. We report on hybrid droplet interface bilayers (hDIBs) formed in hexadecane from binary mixtures of synthetic diphytanoyl phosphatidylcholine (DPhPC) lipids and low molecular weight 1,2 polybutadiene-b-polyethylene oxide (PBPEO) amphiphilic block copolymers and use electrophysiology measurements and imaging to assess the effects of PBPEO in the membrane. This work reveals that hDIBs containing up to 15 mol% PBPEO plus DPhPC are homogeneously mixtures of lipids and polymers, remain highly resistive to ion transport, and are stable—including under applied voltage. Moreover, they exhibit hydrophobic thicknesses similar to DPhPC-only bilayers, but also have significantly lower values of membrane tension. These characteristics coincide with reduced energy of adhesion between droplets and the formation of alamethicin ion channels at significantly lower threshold voltages, demonstrating that even moderate amounts of amphiphilic block copolymers in a lipid bilayer provide a route for tuning the physical properties of a biomimetic membrane.     

Design and synthesis of self-degradable antibacterial polymers by simultaneous chain- and step-growth radical copolymerization

Self-degradable antimicrobial copolymers bearing cationic side chains and main-chain ester linkages were synthesized using the simultaneous chain- and step-growth radical polymerization of t-butyl acrylate and 3-butenyl 2-chloropropionate, followed by the transformation of t-butyl groups into primary ammonium salts. We prepared a series of copolymers with different structural features in terms of molecular weight, monomer composition, amine functionality, and side chain structures to examine the effect of polymer properties on their antimicrobial and hemolytic activities. The acrylate copolymers containing primary amine side chains displayed moderate antimicrobial activity against E. coli but were relatively hemolytic. The acrylate copolymer with quaternary ammonium groups and the acrylamide copolymers showed low or no antimicrobial and hemolytic activities. An acrylate copolymer with primary amine side chains degraded to lower molecular weight oligomers with lower antimicrobial activity in aqueous solution. This degradation was due to amidation of the ester groups of the polymer chains by the nucleophilic addition of primary amine groups in the side chains resulting in cleavage of the polymer main chain. The degradation mechanism was studied in detail by model reactions between amine compounds and precursor copolymers.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Effect of hydrogen peroxide on antibacterial activities of Canadian honeys

Honey is recognized as an efficacious topical antimicrobial agent in the treatment of burns and wounds. The antimicrobial activity in some honeys depends on the endogenous hydrogen peroxide content. This study was aimed to determine whether honey's hydrogen peroxide level could serve as a honey-specific, activity-associated biomarker that would allow predicting and assessing the therapeutic effects of honey. Using a broth microdilution assay, I analyzed antibacterial activities of 42 Canadian honeys against two bacterial strains: Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633). The MIC90 and MIC50 were established from the dose-response relationship between antibacterial activities and honey concentrations. The impact of H2O2 on antibacterial activity was determined (i) by measuring the levels of H2O2 before and after its removal by catalase and (ii) by correlating the results with levels of antibacterial activities. Canadian honeys demonstrated moderate to high antibacterial activity against both bacterial species. Both MIC90 and MIC50 revealed that the honeys exhibited a selective growth inhibitory activity against E. coli, and this activity was strongly influenced by endogenous H2O2 concentrations. Bacillus subtilis activity was marginally significantly correlated with H2O2 content. The removal of H2O2 by catalase reduced the honeys' antibacterial activity, but the enzyme was unable to completely decompose endogenous H2O2. The 25%-30% H2O2 "leftover" was significantly correlated with the honeys' residual antibacterial activity against E. coli. These data indicate that all Canadian honeys exhibited antibacterial activity, with higher selectivity against E. coli than B. subtilis, and that these antibacterial activities were correlated with hydrogen peroxide production in honeys. Hydrogen peroxide levels in honey, therefore, is a strong predictor of the honey's antibacterial activity.     

Structure‐activity relationship of indolicidin,a Trp‐rich antibacterial peptide

A series of Trp and Arg analogs of antibacterial indolicidin (Ind) was synthesized and the antimicrobial and hemolytic activities were investigated. [L⁹]Ind, [L¹¹]Ind, [K⁸,L⁹]Ind and [K^{6, 8},L⁹]Ind showed desirable characteristics, exhibiting negligible hemolytic activity while keeping strong antibacterial activity. The results indicated that the Trp residue at position 11 essentially contributes to both activities and one can not be exchanged for the other, whereas the Trp residues at positions 4 and 9 play important roles in antimicrobial and hemolytic activities, respectively. The Trp residues at positions 6 and 8 play no important roles in biological activities. We then found that the retro analog of Ind showed higher antibacterial activity than Ind against both Gram‐positive and Gram‐negative bacteria but remarkably lower hemolytic activity than that of Ind. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Structure--activity study of the antibacterial peptide fallaxin

Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure-activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N- and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, (VISA), methicillin-susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 muM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity H, mean hydrophobic moment mu(H), and alpha-helicity. We were able to group the active and inactive analogs according to mean hydrophobicity H and mean hydrophobic moment mu(H). Far-UV CD-spectroscopy experiments on fallaxin and several analogs in buffer, in TFE, and in membrane mimetic environments (small unilamellar vesicles) indicated that a coiled-coil conformation could be an important structural trait for antibacterial activity. This study provides data that support fallaxin analogs as promising lead structures in the development of new antibacterial agents.     

Synthesis,micellisation and interaction of novel quaternary ammonium compounds derived from l-Phenylalanine with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as model membrane in relation to their antibacterial activity,and their selectivity over human red blood cells

A series of quaternary ammonium compounds (QUATS) derived from l-Phenylalanine have been synthesized and their antibacterial efficiencies were determined against various strains of Gram-positive and Gram-negative bacteria. The antibacterial activity increased with increasing chain length, exhibiting a cut-off effect at C₁₄ for Gram-positive and C₁₂ for Gram-negative bacteria. The l-Phenylalanine QUATS displayed enhanced antibacterial properties with a higher cut-off point compared to their corresponding l-Phenylalanine ester hydrochlorides. The CMC was correlated with the MIC, inferring that micellar activity contributes to the cut-off effect in antibacterial activity. The hemolytic activities (HC₅₀) of the QUATS against human red blood cells were also determined to illustrate the selectivity of these QUATS for bacterial over mammalian cells. In general, the MIC was lower than the HC₅₀, and assessment of the micellar contribution to the antibacterial and hemolytic evaluation in TBS as a common medium confirmed that these QUATS can act as antibacterial, yet non-toxic molecules at their monomer concentrations. The interaction of the QUATS with the phospholipid vesicles (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) in the presence of 1-anilino-8-naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probes showed that the presence of the quaternary ammonium moiety causes an increase in hydrophobic interactions, thus causing an increase in antibacterial activity.     

新型载药高分子囊泡的制备与抗菌性能研究

相对于小分子抗菌剂,高分子抗菌材料会对细菌的胞膜产生物理破坏,从而降低病原体产生耐药性的可能;另外,高分子抗菌材料不但具有较高的抗菌性能,抗菌作用时效长,环境污染小,而且对人体伤害小,具有更好的选择性。因此,高分子抗菌材料的研究受到广泛关注。此外,还可以将小分子抗生素负载到高分子纳米载体中,实现小分子抗菌剂的传输与协同抗菌。本文构筑了两亲性高分子聚(N,N-二甲氨基乙酯-嵌段-聚苯氧异丙醇)(PDMAEMA-b-PPOPMA),自组装得到纳米囊泡,被用来物理包埋万古霉素(Vancomycin),对金黄色葡萄球菌(S.aureus)表现出良好的抗菌活性。     

Antimicrobial characteristic of insoluble alkylpyridinium iodide

Insoluble and soluble alkylpyridinium iodides (C8 to C18) were synthesized. The insoluble agents were quaternized 4-vinylpyridine-divinylbenzene copolymers. The insoluble agent [C12(50)] that contained 50% divinylbenzene and had a C12 alkyl chain was selected as the most suitable insoluble agent. C12(50) showed poor durability of the antibacterial activity, but C12(50), which had lost the activity, was refreshed by washing with ethanol. This washing became ineffective after a few cycles of antibacterial treatment and refreshment. Such C12(50) recovered the activity upon 1.0 N NaOH treatment. The antibacterial activity of C12(50) depended on its surface area. It showed high antimicrobial activity against gram-positive bacteria and also showed activity against gram-negative bacteria and yeasts. But the activities of C12(50) and laurylpyridinium iodide solution were different against some microbes. The antibacterial activities of the agents were investigated against Escherichia coli and Micrococcus luteus under various conditions. The activity of C12(50) was higher at a higher temperature or at a lower cell concentration. The activity of C12(50) decreased on addition of NaCl, glucose, or bovine albumin to the cell suspension or in 0.01 M sodium-potassium phosphate buffer. C12(50) showed less activity when cells were mixed with dead cells or the supernatant of dead cells killed in an autoclave. The mode of action of the laurylpyridinium iodide solution against E. coli and M. luteus was similar to that of C12(50) except for the influence of E. coli cell concentration.     

Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide.

The effect of introducing a disulfide bridge between the N- and C-terminal ends on the structure and biological activities of the 13-residue linear peptide PKLLKTFLSKWIG(SPFK), which has both antibacterial and hemolytic activity, have been investigated. The terminal amino acids P and G in SPFK were replaced by cysteines to form a disulfide bridge. The linear peptides C(Acm)KLLKTFLSKWIC(Acm) and C(Acm) KLLKTFLSKWIC(Acm)-amide, where Acm is acetamidomethyl group, showed antibacterial activity but did not possess hemolytic activity unlike SPFK. Introduction of an S-S bridge resulted in enhanced hemolytic activity compared with SPFK. The hemolytic activity was particularly pronounced in the cyclic peptide CKLLKTFLSKWIC-amide. Circular dichroism studies indicate that the cyclic peptides tend to adopt distorted helical structures. The cyclic peptides also have a greater affinity for lipid vesicles, which could be the reason for the effective perturbation of the erythrocyte membrane.     

Antimicrobial characteristic of insoluble alkylpyridinium iodide.

Insoluble and soluble alkylpyridinium iodides (C8 to C18) were synthesized. The insoluble agents were quaternized 4-vinylpyridine-divinylbenzene copolymers. The insoluble agent [C12(50)] that contained 50% divinylbenzene and had a C12 alkyl chain was selected as the most suitable insoluble agent. C12(50) showed poor durability of the antibacterial activity, but C12(50), which had lost the activity, was refreshed by washing with ethanol. This washing became ineffective after a few cycles of antibacterial treatment and refreshment. Such C12(50) recovered the activity upon 1.0 N NaOH treatment. The antibacterial activity of C12(50) depended on its surface area. It showed high antimicrobial activity against gram-positive bacteria and also showed activity against gram-negative bacteria and yeasts. But the activities of C12(50) and laurylpyridinium iodide solution were different against some microbes. The antibacterial activities of the agents were investigated against Escherichia coli and Micrococcus luteus under various conditions. The activity of C12(50) was higher at a higher temperature or at a lower cell concentration. The activity of C12(50) decreased on addition of NaCl, glucose, or bovine albumin to the cell suspension or in 0.01 M sodium-potassium phosphate buffer. C12(50) showed less activity when cells were mixed with dead cells or the supernatant of dead cells killed in an autoclave. The mode of action of the laurylpyridinium iodide solution against E. coli and M. luteus was similar to that of C12(50) except for the influence of E. coli cell concentration.     

Design and synthesis of novel antimicrobial peptides on the basis of alpha helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study

Tenecin 1, a peptide consisting of 43 amino acids, exhibits a potent bactericidal activity against various Gram-positive bacteria and shares a common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. Our previous research indicated that an active fragment was successfully extracted from C-terminal beta sheet domain of Tenecin 1, whereas the fragment corresponding to the alpha helical region of the protein had no antibacterial activity. We chose this inactive fragment corresponding to alpha helical region of Tenecin 1 and synthesized derivatives with a different net positive charge by using rational design. Interestingly, we successfully endowed antibacterial activity as well as antifungal activity to the inactive alpha helical fragment by single or double amino acid replacement(s) without an increase of hemolytic activity. The leakage of dye from vesicles induced by the active peptides suggested that these peptides act on the membranes of pathogen as a primary mode of action. Structure-activity relationship study of a series of the active derivatives revealed that amphiphilic structure and high net positive charge were prerequisite factors for the activity and that there was a relationship between the antibacterial activity and the isoelectric point of the active peptides. In this work, we showed an efficient method to endow the antibacterial activity as well as antifungal activity to the inactive fragment derived from a cyclic insect defensin protein and suggested a facile method to screen for active fragments from cyclic host defense peptides.     

Induction, purification and characterization of an antibacterial peptide scolopendrin I from the venom of centipede Scolopendra subspinipes mutilans

The crude venom of the centipede Scolopendra subspinipes mutilans, injected with Escherichia coli K12D31 for 3-4 days showed broad-spectrum antimicrobial activity against Gram-positive. Gram-negative bacteria and fungi. It showed good antibacterial activity against E. coli K12D31 at different temperatures, pH, and ionic strengths. The crude venom was heated at 100 degrees C for 30 min, centrifuged at 10,000 rpm for 30 min at 4 degrees C and the supernatants were obtained, from which an antibacterial fraction having a molecular mass of 3000-5000 Da, was further separated by ultrafiltration. A homogeneous antibacterial peptide named scolopendrin I, having a molecular mass of 4,498 Da, was isolated using cation-exchange chromatography and two steps of reverse-phase high performance liquid chromatography (RP-HPLC). Scolopendrin I did not show any hemolytic and agglutination activities at the concentration below 30 microM.     

Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.