Membrane-perturbing Activity Research Articles

Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins.

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the "histidine switch" principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes-including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase-into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.

Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop

In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I.

Carboxyl group-modified myoglobin shows membrane-permeabilizing activity

In this study, we investigated whether modification of the carboxyl group with semicarbazide-enabled myoglobin (Mb) exhibits membrane-perturbing activity in physiological solutions. Mass spectrometry analysis showed that semicarbazide molecules were coupled to 19 of the 22 carboxyl groups in semicarbazide-modified Mb (SEM-Mb). Measurements of the absorption and circular dichroism spectra indicated that SEM-Mb lost its heme group and reduced the content of the α-helix structure in Mb. The microenvironment surrounding Trp residues in Mb changes after blocking negatively charged residues, as shown by fluorescence quenching studies. The results of the trifluoroethanol-induced structural transition indicated that SEM-Mb had higher structural flexibility than that of Mb. SEM-Mb, but not Mb, induced the permeability of bilayer membranes. Both proteins showed similar lipid-binding affinities. The conformation of SEM-Mb and Mb changed upon binding to lipid vesicles or a membrane-mimicking environment composed of SDS micelles, suggesting that membrane interaction modes differ. Unlike lipid-bound Mb, Trp residues in lipid-bound SEM-Mb are located at the protein-lipid interface. Altogether, our data indicate that modifying negatively charged groups relieves the structural constraints in Mb, consequently switching Mb structure to an active conformation that exhibits membrane-permeabilizing activity.

SC5005 dissipates the membrane potential to kill Staphylococcus aureus persisters without detectable resistance.

In the past few decades, multiple-antibiotic-resistant Staphylococcus aureus has emerged and quickly spread in hospitals and communities worldwide. Additionally, the formation of antibiotic-tolerant persisters and biofilms further reduces treatment efficacy. Previously, we identified a sorafenib derivative, SC5005, with bactericidal activity against MRSA in vitro and in vivo. Here, we sought to elucidate the resistance status, mode of action and anti-persister activity of this compound. The propensity of S. aureus to develop SC5005 resistance was evaluated by assessment of spontaneous resistance and by multi-passage selection. The mode of action of SC5005 was investigated using macromolecular synthesis, LIVE/DEAD and ATPlite assays and DiOC2(3) staining. The effect of SC5005 on the mammalian cytoplasmic membrane was measured using haemolytic and lactate dehydrogenase (LDH) assays and flow cytometry. SC5005 depolarized and permeabilized the bacterial cytoplasmic membrane, leading to reduced ATP production. Because of this mode of action, no resistance of S. aureus to SC5005 was observed after constant exposure to sub-lethal concentrations for 200 passages. The membrane-perturbing activity of SC5005 was specific to bacteria, as no significant haemolysis or release of LDH from human HT-29 cells was detected. Additionally, compared with other bactericidal antibiotics, SC5005 exhibited superior activity in eradicating both planktonic and biofilm-embedded S. aureus persisters. Because of its low propensity for resistance development and potent persister-eradicating activity, SC5005 is a promising lead compound for developing new therapies for biofilm-related infections caused by S. aureus.

Biophysical Insights into the Spatial Organization and Dynamics of Structurally Complex Mycobacterial Membrane Lipids

The remarkably complex cell wall of mycobacterium tuberculosis (Mtb) forms a formidable barrier to many host stresses and therapeutics. It houses distinct lipids with long hydrocarbon chains, and chemical groups such as trehalose, methyl branches and cyclopropane rings; not found in eukaryotic membranes. The current holistic picture of the Mtb cell envelope from cryo-TEM comprises of peptidoglycan (PG) covalently modified with arabinogalactan polymers that are in turn elaborated with mycolic acids (C60-C90). These mycolic acids form the inner leaflet of the mycomembrane (MM); MM also includes an outer leaflet constituted by a variety of non-covalently associated lipids such as phthiocerol dimycocerosate (PDIM), dimycolyltrehalose (TDM), sulfoglycolipids (SL), phosphatidylinositol mannosides (PIMs), lipoarabinomannan (man-LAM) and DAGs along with phospholipids. The vital role of each Mtb membrane component in forming a spatially complex barrier, and how mycobacteria uses its membrane platforms to impact infection biology remain understudied. in our current work we have characterized the outer membrane (OM) and inner membrane (IM) from Mycobacterium smegmatis by isolating and reconstituting lipids from these regions to form model membranes mimicking the natural cell envelope. Using methods such as atomic force microscopy and spectroscopy, infrared spectroscopy, time-resolved fluorescence spectroscopy, two-photon and confocal microscopy, we provide crucial insights into the lateral organization, fluidity and domain architecture of mycobacterial membrane lipids and its correlation to lipid structure. Our findings may facilitate identification of compounds with membrane perturbing activity against specific membranous regions of mycobacterial envelope. We also foresee our characterized mycobacterial model membrane system to serve as molecularly complete membrane scaffolds for anti-tubercular drug membrane screening in future.

Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.

Peptide Inhibitors of Bacterial Protein Synthesis with Broad Spectrum and SbmA-Independent Bactericidal Activity against Clinical Pathogens.

Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials; however, their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, X-ray crystallography, and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Staphylococcus aureus. Two peptides out of five acquired a weak membrane-perturbing activity while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat antibiotic-resistant pathogens.

Phosphate-dependent aggregation of [KL]n peptides affects their membranolytic activity

In this study, we investigate how the length of amphiphilic β-sheet forming peptides affects their interaction with membranes. Four polycationic model peptides with lengths from 6 to 18 amino acids were constructed from simple Lys-Leu repeats, giving [KL]n=3,5,7,9. We found that (1) they exhibit a pronounced antimicrobial activity with an intriguing length dependent maximum for [KL]5 with 10 amino acids; (2) their hemolytic effect, on the other hand, increases steadily with peptide length. CD analysis (3) and TEM (4) show that all peptides-except for the short [KL]3-aggregate into amyloid-like fibrils in the presence of phosphate ions, which in turn has a critical effect on the results in (1) and (2). In fact, (5) vesicle leakage reveals an intrinsic membrane-perturbing activity (at constant peptide mass) of [KL]5 > [KL]9 > [KL]7 in phosphate buffer, which changes to [KL]5 ≈ [KL]7 ≈ [KL]9 in PIPES. A specific interaction with phosphate ions thus explains the subtle balance between two counteracting effects: phosphate-induced unproductive pre-aggregation in solution versus monomeric membrane binding and vigorous lipid perturbation due to self-assembly of the bound peptides within the bilayer. This knowledge can now be used to control and optimize the peptides in further applications.

Status of Asp29 and Asp40 in the Interaction of Naja atra Cardiotoxins with Lipid Bilayers.

It is widely accepted that snake venom cardiotoxins (CTXs) target the plasma membranes of cells. In the present study, we investigated the role of Asp residues in the interaction of Naja atra cardiotoxin 1 (CTX1) and cardiotoxin 3 (CTX3) with phospholipid bilayers using chemical modification. CTX1 contains three Asp residues at positions 29, 40, and 57; CTX3 contains two Asp residues at positions 40 and 57. Compared to Asp29 and Asp40, Asp57 was sparingly modified with semi-carbazide, as revealed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass and mass/mass analyses. Thus, semi-carbazide-modified CTX1 (SEM-CTX1) mainly contained modified Asp29 and Asp40, while SEM-CTX3 contained modified Asp40. Compared to that of native toxins, trifluoroethanol easily induced structural transition of SEM-CTX1 and SEM-CTX3, suggesting that the structural flexibility of CTXs was constrained by Asp40. Modification of Asp29 and Asp40 markedly promoted the ability of CTX1 to induce permeability of cell membranes and lipid vesicles; CTX3 and SEM-CTX3 showed similar membrane-damaging activity. Modification of Asp residues did not affect the membrane-binding capability of CTXs. Circular dichroism spectra of SEM-CTX3 and CTX3 were similar, while the gross conformation of SEM-CTX1 was distinct from that of CTX1. The interaction of CTX1 with membrane was distinctly changed by Asp modification. Collectively, our data suggest that Asp29 of CTX1 suppresses the optimization of membrane-bound conformation to a fully active state and that the function of Asp40 in the structural constraints of CTX1 and CTX3 is not important for the manifestation of membrane-perturbing activity.

Evaluating the Photodynamic Biocidal Activity and Investigating the Mechanism of Thiazolium Cyanine Dyes

In order to develop ideal photosensitizers for photodynamic therapy (PDT), a thiazolium group was introduced in cyanine dyes to possess the advantages of intense absorption in the visible region and anti-microbial activity. We evaluated the anti-bacterial activity of the three thiazolium cyanine dyes against Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) in vitro. This is the first time that such kinds of cyanine dyes are applied in the photodynamic antibiotic area. Cyanines display excellent anti-bacterial activity to S. aureus and E. coli exposed to white light irradiation, and the corresponding light-induced biocidal efficacy of cyanines increased with irradiation time and concentration. At the same time, there was no observation of dark anti-bacterial abilities. Especially, cyanines show low cell cytotoxicity and excellent biocompatibility. These results suggest that thiazolium cyanine could work as a photosensitizer in PDT with great promise and broad applications for killing bacteria. Mechanistic studies suggest that the reactive oxygen species (ROS) is the vital factor for combating bacteria exposure to white light conditions, whereas it is not the only determining factor of the biocidal activity. The interaction of the cyanine to the cell membrane also plays a critical role in killing bacteria, which has exhibited a synergic effect of electrostatic and hydrophobic interactions. It influences the cell uptake and the membrane perturbation activity of the cyanines, which indirectly affects the biocidal activity.

Naja atra Cardiotoxin 3 Elicits Autophagy and Apoptosis in U937 Human Leukemia Cells through the Ca2+/PP2A/AMPK Axis.

Cardiotoxins (CTXs) are suggested to exert their cytotoxicity through cell membrane damage. Other studies show that penetration of CTXs into cells elicits mitochondrial fragmentation or lysosome disruption, leading to cell death. Considering the role of AMPK-activated protein kinase (AMPK) in mitochondrial biogenesis and lysosomal biogenesis, we aimed to investigate whether the AMPK-mediated pathway modulated Naja atra (Taiwan cobra) CTX3 cytotoxicity in U937 human leukemia cells. Our results showed that CTX3 induced autophagy and apoptosis in U937 cells, whereas autophagic inhibitors suppressed CTX3-induced apoptosis. CTX3 treatment elicited Ca2+-dependent degradation of the protein phosphatase 2A (PP2A) catalytic subunit (PP2Acα) and phosphorylation of AMPKα. Overexpression of PP2Acα mitigated the CTX3-induced AMPKα phosphorylation. CTX3-induced autophagy was via AMPK-mediated suppression of the Akt/mTOR pathway. Removal of Ca2+ or suppression of AMPKα phosphorylation inhibited the CTX3-induced cell death. CTX3 was unable to induce autophagy and apoptosis in U937 cells expressing constitutively active Akt. Met-modified CTX3 retained its membrane-perturbing activity, however, it did not induce AMPK activation and death of U937 cells. These results conclusively indicate that CTX3 induces autophagy and apoptosis in U937 cells via the Ca2+/PP2A/AMPK axis, and suggest that the membrane-perturbing activity of CTX3 is not crucial for the cell death signaling pathway induction.

Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem.

The use of enhanced sampling molecular dynamics simulations to facilitate the folding of proteins is a relatively new approach which has quickly gained momentum in recent years. Accelerated molecular dynamics (aMD) can elucidate the dynamic path from the unfolded state to the near-native state, “flattened” by introducing a non-negative boost to the potential. Alamethicin F30/3 (Alm F30/3), chosen in this study, belongs to the class of peptaibols that are 7–20 residue long, non-ribosomally synthesized, amphipathic molecules that show interesting membrane perturbing activity. The recent studies undertaken on the Alm molecules and their transmembrane channels have been reviewed. Three consecutive simulations of ~900 ns each were carried out where N-terminal folding could be observed within the first 100 ns, while C-terminal folding could only be achieved almost after 800 ns. It took ~1 μs to attain the near-native conformation with stronger potential boost which may take several μs worth of classical MD to produce the same results. The Alm F30/3 hexamer channel was also simulated in an E. coli mimicking membrane under an external electric field that correlates with previous experiments. It can be concluded that aMD simulation techniques are suited to elucidate peptaibol structures and to understand their folding dynamics.

De novo Design of Selective Membrane-Active Peptides by Enzymatic Control of Their Conformational Bias on the Cell Surface.

Selectively targeting the membrane-perturbing potential of peptides towards a distinct cellular phenotype allows one to target distinct populations of cells. We report the de novo design of a new class of peptide whose ability to perturb cellular membranes is coupled to an enzyme-mediated shift in the folding potential of the peptide into its bioactive conformation. Cells rich in negatively charged surface components that also highly express alkaline phosphatase, for example many cancers, are susceptible to the action of the peptide. The unfolded, inactive peptide is dephosphorylated, shifting its conformational bias towards cell-surface-induced folding to form a facially amphiphilic membrane-active conformer. The fate of the peptide can be further tuned by peptide concentration to affect either lytic or cell-penetrating properties, which are useful for selective drug delivery. This is a new design strategy to afford peptides that are selective in their membrane-perturbing activity.

Glycosylation differentially modulates membranolytic and chaperone-like activities of PDC-109, the major protein of bovine seminal plasma

The major bovine seminal plasma protein, PDC-109, is a mixture of glycosylated (BSP-A1) and non-glycosylated (BSP-A2) isoforms of a 109-residue long polypeptide. It binds to spermatozoa by specifically recognizing choline phospholipids on the plasma membrane and destabilizes it by penetrating the hydrophobic interior, resulting in lipid efflux, which is necessary for sperm capacitation and successful fertilization. PDC-109 also acts as a molecular chaperone and protects target proteins from denaturation and aggregation induced by various types of stress. In order to investigate the role of glycosylation in these activities, we have separated BSP-A1 and BSP-A2 from PDC-109, and also cloned and expressed BSP-A2 in E. coli and purified the recombinant BSP-A2 (rBSP-A2) to homogeneity. Employing biophysical and biochemical approaches we have investigated the membrane-perturbing and chaperone-like activities (CLA) of PDC-109, BSP-A1, BSP-A2 and recombinant BSP-A2 (rBSP-A2). The results obtained demonstrate that glycan-lacking wild-type BSP-A2 and rBSP-A2 exhibit higher membrane-perturbing activity but decreased CLA as compared to PDC-109. In contrast, BSP-A1 exhibits significantly higher CLA than PDC-109, but its ability to destabilize membranes is considerably lower. This differential modulation of the membrane-perturbing and chaperone-like activities has been explained on the basis of higher membrane-penetrating ability and lower solubility of glycan-lacking BSP-A2 as compared to the glycosylated BSP-A1.

Generation of Persister Cells of Pseudomonas aeruginosa and Staphylococcus aureus by Chemical Treatment and Evaluation of Their Susceptibility to Membrane-Targeting Agents.

Persister cells (PCs) are a subset of dormant, phenotypic variants of regular bacteria, highly tolerant to antibiotics. Generation of PCs in vivo may account for the recalcitrance of most chronic infections to antimicrobial treatment and demands for the identification of new antimicrobial agents able to target such cells. The present study explored the possibility to obtain in vitro PCs of Pseudomonas aeruginosa and Staphylococcus aureus at high efficiency through chemical treatment, and to test their susceptibility to structurally different antimicrobial peptides (AMPs) and two clinically used peptide-based antibiotics, colistin and daptomycin. The main mechanism of action of these molecules (i.e., membrane-perturbing activity) renders them potential candidates to act against dormant cells. Exposure of stationary-phase cultures to optimized concentrations of the uncoupling agent cyanide m-chlorophenylhydrazone (CCCP) was able to generate at high efficiency PCs exhibiting an antibiotic-tolerant phenotype toward different classes of antibiotics. The metabolic profile of CCCP-treated bacteria was investigated by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry. CCCP-pretreated bacteria of both bacterial species underwent a substantial decrease in heat production and oxidoreductase activity, as compared to the untreated controls. After CCCP removal, induced persisters showed a delay in heat production that correlated with a lag phase before resumption of normal growth. The metabolic reactivation of bacteria coincided with their reversion to an antibiotic-sensitive phenotype. Interestingly, PCs generated by CCCP treatment resulted highly sensitive to three different membrane-targeting AMPs at levels comparable to those of CCCP-untreated bacteria. Colistin was also highly active against PCs of P. aeruginosa, while daptomycin killed PCs of S. aureus only at concentrations 32 to 64-fold higher than those of the tested AMPs. In conclusion, CCCP treatment was demonstrated to be a suitable method to generate in vitro PCs of medically important bacterial species at high efficiency. Importantly, unlike conventional antibiotics, structurally different AMPs were able to eradicate PCs suggesting that such molecules might represent valid templates for the development of new antimicrobials active against persisters.

Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NH2 and its Diastereomer Esc(1-21)-1c: Correlation with their antipseudomonal and cytotoxic activity

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. DLeu14 and DSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.

Haemolytic activity of formyl- and acetyl-derivatives of bile acids and their gramine salts

Bile acids (lithocholic: LCA, deoxycholic: DCA and cholic: CA) and their formyl- and acetyl-derivatives can be used as starting material in chemical synthesis of compounds with different biological activity strongly depended on their chemical structures. Our previous studies showed that biological activity of bile acids salts with gramine toward human erythrocytes was significantly different from the activity of bile acids alone. Moreover, gramine effectively modified the membrane perturbing activity of other steroids. As a continuation of our work, the haemolytic activity of formyl- and acetyl-substituet bile acids as well as their gramine salts was studied in vitro. The structures of new compounds were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. The results shown that the haemolytic activity of formyl- and acetyl-LCA and DCA was significantly higher in comparison with their native forms at the whole concentration range. At high concentration, formyl derivative of CA was as effective as LCA and DCA derivatives whereas at lower concentration its haemolytic activity was at the level of original acid. The acetyl-CA was not active as membrane perturbing agents. Furthermore, gramine significantly decreased the membrane-perturbing activity of hydrophobic bile acids derivatives. The results obtained with the cellular system are in line with physicochemical calculation.

Quercetin 7-O-glutamate sensitizes Escherichia coli to vancomycin

We have reported previously that conjugation of glutamic acid at the 7-O position of quercetin could significantly enhance its biological activity. In this study, as a part of our ongoing efforts to investigate the therapeutic potential of this novel quercetin–glutamate conjugate (1), we evaluated the effect of its combination with vancomycin against Gram-negative Escherichia coli, which are not normally affected by vancomycin alone. Quercetin conjugate 1 was shown to potentiate the antibacterial activity of vancomycin against E. coli, which could be attributed to its membrane perturbation activity, but not to facilitation of the efflux pump-mediated penetration of vancomycin.

Understanding the dark and light-enhanced bactericidal action of cationic conjugated polyelectrolytes and oligomers.

A multiscale investigation was carried out to study the dark and light-enhanced bactericidal mechanisms of poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPEs) and oligo-phenylene ethynylenes (OPEs). On the morphological scale, Gram-negative E. coli cells exposed to CPE and OPE compounds in the dark show damage to the cell envelope, plasma membrane, and in some cases the cytoplasm, while with UV-irradiation, E. coli sustained catastrophic damages to both the cell envelope and cytoplasm. In contrast, the Gram-positive S. epi bacteria appeared intact when exposed to CPE and OPE compounds in the dark but showed damages to the cell envelope with UV-irradiation. To better understand the molecular basis of CPE- and OPE-induced morphological changes and damages to bacteria, we investigated the effect of these compounds on model bacterial plasma membrane and bacterial proteins and plasmid DNA. Measurements of dark membrane perturbation activity of the CPEs and OPEs using model lipid membranes support a carpet or detergent-like mechanism by which the antimicrobial compounds induce membrane collapse and phase transitions. Under UV-irradiation, E. coli bacteria exposed to CPEs and OPEs showed covalent modifications and damages to both cellular protein and plasmid DNA, likely through oxidative pathways mediated by singlet oxygen and subsequent reactive oxygen species sensitized by the CPE and OPE compounds. Our finding thus show that the antimicrobial polymers and oligomers exert toxicity toward Gram-negative bacteria by disrupting the morphology and structures of cell envelope and cytoplasm, including cellular components such as proteins and DNA, while exert toxicity toward Gram-positive bacteria by binding to and disrupting just the cell wall.

Unusual titration of the membrane-bound artificial hemagglutinin fusion peptide

E5 is a 20-residue-long analog of the fusion peptide from influenza hemagglutinin (GLFEAIAEFIEGGWEGLIEG). It has been suggested that two of its five glutamates, Glu11and Glu15, are critical in its pH-dependent membrane perturbation. To reveal their specific involvement, a pair of analogs with substitution of either Glu11 or Glu15 for Ala were synthesized. By analysis of the pH-dependence of the chemical shifts of protons of these peptides bound to dodecylphosphocholine micelles we found: (1) the peptides adopt an amphiphilic alpha-helical structure within residues 2-18, similar to the parent peptide; (2) the helix is significantly more disordered at neutral pH than at acidic pH for E5 peptide only; and (3) in E5 and mutant peptides the Glu11 and 15 residues have similar pK (a) values, higher than those of the other glutamates. This excludes their mutual interaction in E5, being a source of the elevated pK (a) values. We attribute this phenomenon to the presence of minor states caused by deepening of the Glu11 and 15 side-chains in the hydrophobic environment of the membrane. As the mid-pH of membrane-perturbation activity of E5 matches the pK (a) value of these glutamates, we conclude their presence contributes to the plasticity of the peptide and determines the pH-dependence of membrane perturbation caused by E5.