Trichokonins from Trichoderma pseudokoningii SMF2 induce resistance against Gram-negative Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage.

Peptaibols, mainly produced by Trichoderma, play a pivotal role in controlling plant disease caused by fungi, virus, and Gram-positive bacteria. In the current study, we evaluated the control effect of Trichokonins, antimicrobial peptaibols from Trichoderma pseudokoningii SMF2, on soft rot disease of Chinese cabbage caused by a Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum and analyzed the mechanism involved. Trichokonins treatment (0.3 mg L(-1) ) enhanced the resistance of Chinese cabbage against Pcc infection. However, Trichokonins could hardly inhibit the growth of Pcc in vitro, even at high concentration (500 mg L(-1) ). Therefore, the direct effect of Trichokonins on Pcc may not the main reason why Trichokonins could control soft rot of Chinese cabbage. Trichokonin treatment led to an obvious increase in the production of reactive oxygen species hydrogen peroxide and superoxide radical, a significant enhance of the activities of pathogenesis-related enzymes catalase, polyphenoloxidase and peroxidase, and upregulation of the expression of salicylic acid - responsive pathogenesis-related protein gene acidic PR-1a in Chinese cabbage. These results indicate that Trichokonins induce resistance in Chinese cabbage against Pcc infection through the activation of salicylic acid signaling pathway, which imply the potential of Trichoderma and peptaibols in controlling plant disease caused by Gram-negative bacteria.

Lipid charge regulation of non-specific biological ion channels.

Ion channels are specialized proteins that enable the movement of charges through otherwise impermeable lipidic membranes. Their action is essential in living organisms facilitating electric signaling, muscle contraction or osmotic stress response among other effects. The protein and the lipid charges configure a polarized interface that yields local ionic concentrations and electric potentials that are very different from those of the bulk electrolyte. The combined effect of gradients of ionic concentration and electric potential causes the transport of ions through channels. Here we analyze charge regulation effects in different protein-lipid conformations, stressing how important is the role of electrostatic interactions in the ion channel function that traditionally has been rationalized paying attention mainly to changes in pore size. Tuning lipid charge combined with conductance and selectivity measurements is shown to be a complementary method to evidence lipid involvement in the structure of a biological ion channel.

Meningococcal resistance to antimicrobial peptides is mediated by bacterial adhesion and host cell RhoA and Cdc42 signalling.

Antimicrobial peptides (AMPs) constitute an essential part of the innate immune defence. Pathogenic bacteria have evolved numerous strategies to withstand AMP-mediated killing. The influence of host epithelia on bacterial AMP resistance is, however, still largely unknown. We found that adhesion to pharyngeal epithelial cells protected Neisseria meningitidis, a leading cause of meningitis and sepsis, from the human cathelicidin LL-37, the cationic model amphipathic peptide (MAP) and the peptaibol alamethicin, but not from polymyxin B. Adhesion to primary airway epithelia resulted in a similar increase in LL-37 resistance. The inhibition of selective host cell signalling mediated by RhoA and Cdc42 was found to abolish the adhesion-induced LL-37 resistance by a mechanism unrelated to the actin cytoskeleton. Moreover, N. meningitidis triggered the formation of cholesterol-rich membrane microdomains in pharyngeal epithelial cells, and host cell cholesterol proved to be essential for adhesion-induced resistance. Our data highlight the importance of Rho GTPase-dependent host cell signalling for meningococcal AMP resistance. These results indicate that N. meningitidis selectively exploits the epithelial microenvironment in order to protect itself from LL-37.

Toward understanding protocell mechanosensation.

Mechanosensitive (MS) channels can prevent bacterial bursting during hypo-osmotic shocks by responding to increases in lateral tension at the membrane level through an integrated and coordinated opening mechanism. Mechanical regulation in protocells could have been one of the first mechanisms to evolve in order to preserve their integrity against changing environmental conditions. How has the rich functional diversity found in present cells been created throughout evolution, and what did the primordial MS channels look like? This review has been written with the aim of identifying which factors may have been important for the appearance of the first osmotic valve in a prebiotic context, and what this valve may have been like. It highlights the mechanical properties of lipid bilayers, the association of peptides as aggregates in membranes, and the conservation of sequence motifs as central aspects to understand the evolution of proteins that gate below the tension required for spontaneous pore formation and membrane rupture. The arguments developed here apply to both MscL and MscS homologs, but could be valid to mechano-susceptible proteins in general.

Radio frequency rectification on membrane bound pores.

Probing the interaction of biological systems with radio frequencies holds great promise for research and drug screening applications. While a common assumption is that biological systems do not operate at radio frequencies, we find that currents due to ion transport through channels and pores in cell membranes are in the pA to nA range. These values translate via the average current I = ne/tau(d) = nef to frequencies in the range of 1 MHz-1 GHz, where n is the average number of ions transported and tau(d) is the dwell time of the ions in the channel. It is thus desirable to have circuitry available which facilitates radio frequency spectroscopy of ion transport. This will yield real-time in vitro information on ion channel operation. Here we present measurements on the local interaction of a radio frequency signal with single ion channels and pores. We find radio frequency rectification and pumping on the channels and pores embedded in suspended bilipid membranes, recorded in direct current measurements. This electromagnetic modulation can be used to probe the dynamics of ion channel conformational changes.

Orientation and peptide-lipid interactions of alamethicin incorporated in phospholipid membranes: polarized infrared and spin-label EPR spectroscopy.

Alamethicin is a 20-residue peptaibiotic that induces voltage-dependent ion channels in lipid membranes. The mode by which alamethicin inserts into membranes was investigated using measurements of peptide-lipid interactions by spin-label electron paramagnetic resonance (EPR) and of peptide orientation by polarized infrared (IR) spectroscopy. In fluid membranes, spin-labeled stearic acid shows no evidence of a specific motionally restricted population of lipid chains, such as that found at the intramembranous surface of integral membrane proteins or oligomeric assemblies of transmembrane alpha-helices. In agreement with recent results from TOAC-substituted alamethicin analogues, native alamethicin is predominantly monomeric in fluid lipid membranes and presents an intramembrane surface that integrates well with the lipid chains but is insufficiently extensive to induce specific motional restriction. Channel formation takes place by transient association of transmembrane monomers. In aligned fluid membranes, alamethicin exhibits a large tilt in short chain-length lipids that decreases first rapidly with increasing chain-length and then more gradually for the lipids with longer chains. This macroscopically low order contrasts with the high local order, relative to the local membrane normal, that is found by EPR for alamethicins spin-labeled with TOAC. The macroscopic behavior is consistent with predictions for the chain-length dependence of elastic bending fluctuations of the membrane surface, which was invoked recently to explain the spontaneous insertion of beta-barrel proteins in short-chain lipid membranes.

Peptide aggregation and pore formation in a lipid bilayer: a combined coarse-grained and all atom molecular dynamics study.

We present a simulation study where different resolutions, namely coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, are used sequentially to combine the long timescale reachable by CG simulations with the high resolution of AA simulations, to describe the complete processes of peptide aggregation and pore formation by alamethicin peptides in a hydrated lipid bilayer. In the 1-micros CG simulations the peptides spontaneously aggregate in the lipid bilayer and exhibit occasional transitions between the membrane-spanning and the surface-bound configurations. One of the CG systems at t = 1 micros is reverted to an AA representation and subjected to AA simulation for 50 ns, during which water molecules penetrate the lipid bilayer through interactions with the peptide aggregates, and the membrane starts leaking water. During the AA simulation significant deviations from the alpha-helical structure of the peptides are observed, however, the size and arrangement of the clusters are not affected within the studied time frame. Solid-state NMR experiments designed to match closely the setup used in the molecular dynamics simulations provide strong support for our finding that alamethicin peptides adopt a diverse set of configurations in a lipid bilayer, which is in sharp contrast to the prevailing view of alamethicin oligomers formed by perfectly aligned helical alamethicin peptides in a lipid bilayer.

Enzymatic reaction in a vesicular microreactor: peptaibol-facilitated substrate transport.

Catalytic reactions performed with enzymes localized in lipid vesicles or in whole cells represent a new, promising approach in biocatalysis. The delivery of different substrates into these micro- or nano-'reactors' requires a sufficient permeability of lipid membranes. To increase the permeability of lipid bilayers, one may use different membrane-active peptides, including peptaibols. In the present study, the trypsin-catalyzed hydrolysis of N(alpha)-benzoyl-L-arginine-para-nitroanilide (BAPA; 1) was studied in a phospholipid vesicular system made of phosphatidylcholine (POC), in the presence of the peptaibols alamethicin (ALM) or zervamicin IIB (ZER). Two different manners of compartmentalization of substrate and enzyme (enzyme- vs. substrate-containing vesicles) were used. The kinetics parameters of the reaction in homogeneous solution and in the vesicular systems were determined. The rate of the extra- or intravesicular enzymatic reaction was found to be controlled by substrate diffusion through the lipid bilayer. In comparison with untreated vesicular systems, an up to seven-fold increase in reaction rate was observed in the presence of either ALM or ZER.

Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: implications for drug-drug interactions.

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-beta-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC50 of 30 microM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 microM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC50 for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 microM to 1.8 microM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K(I) (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 microM and a k(inact) (maximal rate of inactivation) of 0.21 min(-1). Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.

A high-throughput screen for identifying transmembrane pore-forming peptides.

We have developed a visual microwell plate assay for rapid, high-throughput screening for membrane-disrupting molecules such as de novo designed pore formers, antibiotic peptides, bacterial toxins, and lipases. The detectability is based on the strong fluorescence emission of the lanthanide metal terbium(III) (Tb(3+)) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb(3+) is not strongly fluorescent alone, the binary complex emits bright green fluorescence when irradiated with uv light. For the microwell plate assay, we prepared unilamellar phospholipid vesicles that had either Tb(3+) or DPA entrapped and the opposite molecule in the external solution. Disruption of the membranes allows the Tb(3+)/DPA complex to form, giving rise to a visibly fluorescent solution. In plates with 20-microl wells, the lower limit of visual detectability of the Tb(3+)/DPA complex in solution was about 2.5 microM. The lower limit of detectability using vesicles with entrapped Tb(3+) or DPA was about 50 microM phospholipid. We show that the membrane-disrupting effect of as little as 0.25 microM or 5 pmol of the pore-forming, antibiotic peptide alamethicin can be detected visually with this system. This sensitive, high-throughput assay is readily automatable and makes possible the visual screening of combinatorial peptide libraries for members that permeabilize lipid bilayer membranes.

A universal thermodynamic approach to analyze biomolecular binding experiments.

Binding processes of any kind can be characterized as an association of a given ligand with some binding factor. This includes macromolecules as well as supramolecular aggregates such as micelles or membranes. The underlying molecular binding mechanism may be more or less complicated due to various intermediate steps (involving for instance conformational changes, aggregation, cooperativity, etc.). A sensible discussion of possible binding models naturally calls for a model-independent access to basic thermodynamic properties. The present contribution will demonstrate how this can quite generally be accomplished by a pertinent processing of properly selected experimental data. The method requires a series of titration measurements comprising the use of variable amounts of both the ligand and the binding factor. It leads to a linear mass conservation plot (i.e. amount of the ligand vs. a matching amount of the binding factor) whose slope and ordinate intercept are equal to the binding ratio (i.e. bound ligand per binding factor) and the free ligand concentration, respectively. This establishes the specific binding isotherm. The approach also reveals latent structurally determined features of the applied physical measuring signal. A number of examples including specific binding, unspecific adsorption and insertion in two-dimensional molecular films will illustrate the methodology.

Dipole lattice membrane model for protein calculations.

A dipole lattice model for lipid membranes and their interactions with peptides is presented. It uses the Langevin dipole method to calculate electrostatic interactions in the heterogeneous membrane environment. A series of test cases are presented, including spherical charges, dipoles, side chain analogs, and helical peptides. The model consistently produces qualitatively correct results.

In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin.

The UDP-glucuronosyltransferases (UGTs) are a superfamily of membrane-bound enzymes whose active site is localized inside the endoplasmic reticulum. Glucuronidation using human liver microsomes has traditionally involved disruption of the membrane barrier, usually by detergent treatment, to attain maximal enzyme activity. The goals of the current work were to develop a universal method to glucuronidate xenobiotic substrates using microsomes, and to apply this method to sequential oxidation-glucuronidation reactions. Three assays of UGT catalytic activity estradiol-3-glucuronidation, acetaminophen-O-glucuronidation, and morphine-3-glucuronidation, which are relatively selective probes for human UGT1A1, 1A6, and 2B7 isoforms, respectively, were developed. Treatment of microsomes with the pore-forming peptide alamethicin (50 microg/mg protein) resulted in conjugation rates 2 to 3 times the rates observed with untreated microsomes. Addition of physiological concentrations of Mg(2+) to the alamethicin-treated microsomes yielded rates that were 4 to 7 times the rates with untreated microsomes. Optimized assay conditions were found not to detrimentally affect cytochrome P450 activity as determined by effects on testosterone 6beta-hydroxylation and 7-ethoxycoumarin deethylation. Formation of estradiol-3-glucuronide displayed atypical kinetics, and data best fit the Hill equation, yielding apparent kinetic parameters of K(m)(app) = 0.017 mM, V(max)(app) = 0.4 nmol/mg/min, and n = 1.8. Formation of acetaminophen-O-glucuronide also best fit the Hill equation, with K(m)(app) = 4 mM, V(max)(app) = 1.5 nmol/mg/min, and n = 1.4. Alternatively, morphine-3-glucuronide formation displayed Michaelis-Menten kinetics, with K(m)(app) = 2 mM and V(max)(app) = 2. 5 nmol/mg/min. Finally, alamethicin treatment of microsomes was found to be effective in facilitating the sequential oxidation-glucuronidation of 7-ethoxycoumarin.

Tissue distribution and interindividual variation in human UDP-glucuronosyltransferase activity: relationship between UGT1A1 promoter genotype and variability in a liver bank.

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles.

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.

Dynamic properties of Na+ ions in models of ion channels: a molecular dynamics study.

We present simulation results for the effective diffusion coefficients of a sodium ion in a series of model ion channels of different diameters and hydrophobicities, including models of alamethicin, a leucine-serine peptide, and the M2 helix bundle of the nicotinic acetylcholine receptor. The diffusion coefficient, which in the simulations has a value of 0.15(2) A2ps-1 in bulk water, is found to be reduced to as little as 0.02(1) A2ps-1 in the narrower channels, and to about 0.10(5) A2ps-1 in wider channels such as the nicotinic acetylcholine receptor. It is anticipated that this work will be useful in connection with calculations of channel conductivity using such techniques as the Poisson-Nernst-Planck equation, Eyring rate theory, or Brownian dynamics.

Peptide models for membrane channels.

Peptides may be synthesized with sequences corresponding to putative transmembrane domains and/or pore-lining regions that are deduced from the primary structures of ion channel proteins. These can then be incorporated into lipid bilayer membranes for structural and functional studies. In addition to the ability to invoke ion channel activity, critical issues are the secondary structures adopted and the mode of assembly of these short transmembrane peptides in the reconstituted systems. The present review concentrates on results obtained with peptides from ligand-gated and voltage-gated ion channels, as well as proton-conducting channels. These are considered within the context of current molecular models and the limited data available on the structure of native ion channels and natural channel-forming peptides.

Detection of peptaibols and their hydrolysis products in cultures of Trichoderma species.

Fifteen strains of Trichoderma belonging to 12 different species for potential use in biological control were examined for their ability to produce polypeptide antiobitics (peptaibols). Gas chromatography was used to detect the hydrolysis products of peptaibols after esterification with n-propanol and derivatisation with heptafluorobutyric anhydride. In particular, amino acids (alpha-aminoisobutyric acid, and isovalin) and amino alcohols (phenylalaninol, leucinol, and valinol) were used as markers for the detection of different peptaibols. alpha-Aminoisobutyric acid, an amino acid characterising all peptaibols, was detected in all cultures examined, indicating that the production of peptaibols is a common feature of the various Trichoderma species. Different amino acid/amino alcohol combinations suggested specific peptaibol production. On this basis, the peptaibol paracelsin was confirmed by HPTLC in 11 out of 15 Trichoderma strains cultures. Valinol was not found in any culture extract, indicating the lack of trichotoxins production. Culture extracts of the 6 tested Trichoderma strains were extremely toxic to Artemia salina larvae; for 5 of them this toxicity could be attributed to paracelsin (calculated LD50 = 2.2 microM).

Incorporation of synthetic peptide helices in membranes of tetraether lipids from Thermoplasma acidophilum. A calorimetric study.

Four analogues of the membrane-modifying, alpha-helical polypeptide antibiotic alamethicin were synthesized. the alpha-helical deca-, undeca-, heptadeca-, and icosapeptides were mixed with the main tetraether lipid of the Archaebacterium Thermoplasma acidophilum (MPL), dipalmitoylphosphatidylcholine (DPPC) and dihexadecylmaltosylglycerol (DHMG) in various ratios and the modification of the lipid phase transition was determined by differential thermal analysis (DTA). The polypeptides form mixed phases with MPL and DPPC, however, not with DHMG. Heptadeca- and icosapeptide exert a much stronger reduction of enthalpy (delta H) than deca- and undecapeptide and bind about 0.5 molecule of MPL (or one molecule of DPPC) per peptide molecule. delta H of the DPPC pretransition is reduced by the deca- and the undecapeptides and completely disappears with heptadeca- and icosapeptides (at 0.2 mole of peptide/mole of lipid). The modulation of the melting point Tm by the incorporation of peptides is more pronounced with MPL than with DPPC, the heptadecapeptide exhibiting the strongest reduction (with MPL) and the strongest broadening of the transition peak (with DPPC). Helix length, amphiphilicity and charge of the polypeptides can be correlated with the observed modifications of the lipid phase transitions.