Cholesterol induces surface localization of polyphenols in model membranes thus enhancing vesicle stability against lysozyme, but reduces protection of distant double bonds from reactive-oxygen species.

The main scope of the present study was to analyze the membrane interaction of members of different classes of polyphenols, i.e. resveratrol, naringenin, epigallocatechin gallate and enterodiol, in model systems of different compositions and phase states. In addition, the possible association between membrane affinity and membrane protection against both lipid oxidation and bilayer-disruptive compounds was studied. Gibbs monolayer experiments indicated that even though polyphenols showed poor surface activity, it readily interacted with lipid films. Actually, a preferential interaction with expanded monolayers was observed, while condensed and cholesterol-containing monolayers decreased the affinity of these phenolic compounds. On the other hand, fluorescence anisotropy studies showed that polyphenols were able to modulate membrane order degree, but again this effect was dependent on the cholesterol concentration and membrane phase state. In fact, cholesterol induced a surface rather than deep into the hydrophobic core localization of phenolic compounds in the membranes. In general, the polyphenolic molecules tested had a better antioxidant activity when they were allowed to get inserted into the bilayers, i.e. in cholesterol-free membranes. On the other hand, a membrane-protective effect against bilayer permeabilizing activity of lysozyme, particularly in the presence of cholesterol, could be assessed. It can be hypothesized that phenolic compounds may protect membrane integrity by loosely covering the surface of lipid vesicles, once cholesterol push them off from the membrane hydrophobic core. However, this cholesterol-driven distribution may lead to a reduced antioxidant activity of linoleic acid double bonds.

Microcin J25 membrane interaction: selectivity toward gel phase.

The interaction of the tryptophan-containing variant of microcin J25, MccJ25 I13W, with phosphatidylcholine membranes was studied by fluorescence spectroscopy techniques. The peptide was able to interact with dimiristoylphophatidylcholine and dipalmitoylphosphatidylcholine liposomes only when the membranes were in gel phase, as was demonstrated by the blue shift of the intrinsic fluorescence of MccJ25 I13W. The binding isotherm showed a cooperative partition of the peptide toward the membrane and the binding constant increased as the temperature decreased and the order parameter increased. No interaction with liquid crystalline membranes was observed. Studies of dynamic quenching of the fluorescence indicated that the peptide penetrated the lipid bilayer and was located primarily in the interfacial region. Our results suggest that MccJ25 I13W interacts with gel phase phospholipids and increases both its own affinity for the bilayer and the membrane permeability of small ions.

The PmrA/PmrB regulatory system controls the expression of the wzzfepE gene involved in the O-antigen synthesis of Salmonella enterica serovar Typhimurium.

The degree of polymerization of O-antigen from Salmonella enterica serovar Typhimurium is controlled by the products of the wzz(s)(t) and wzz(fepE) genes. In the present study we investigated the role of the PmrA/PmrB regulatory system in wzz(fepE) transcription. We report that the direct binding of the PmrA regulator to a specific promoter site induces the expression of the wzz(fepE) gene. This effect increases the amount of very long (VL) O-antigen, which is required for the resistance of Salmonella to serum human complement and polymyxin B, and for the replication of the bacteria within macrophages. The results obtained here highlight functional differences between Wzz(fepE) and Wzz(st), although the genes for both proteins are regulated in a PmrA-dependent way.

Redox-active tyrosine residue in the microcin J25 molecule.

Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal 'tail' passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H2O2 was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.

Proton motive force dissipation precludes interaction of microcin J25 with RNA polymerase, but enhances reactive oxygen species overproduction.

BACKGROUND: Microcin J25 targets the RNA polymerase as well as bacterial membranes. Because there is scarce information on the relationship between the uptake and the activity, a fluorescent microcin J25-derivative was used to further characterize its mechanism of action. METHODS: MccJ25 I13K was labeled with FITC and its uptake by sensitive cells was assessed by fluorescence measurements from supernatants of MccJ25-Escherichia coli suspensions. The interaction of the peptide with bacterial membranes was investigated by fluorescence resonance energy transfer. Oxygen consumption was measured with Clark-type electrode. RNA synthesis was evaluated in vivo by incorporation of [3H]uridine. ROS production was assayed by measuring the fluorescence emission of the ROS-sensitive probe 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. RESULTS: The protonophore 2,4-dinitrophenol decreased 80% of the MccJ25 uptake and prevented inhibition of transcriptional activity, the antibiotic intracellular target. On the other hand, peptide binding to bacterial membranes was not affected and antibacterial activity remained nearly unchanged. Proton gradient dissipation by protonophore accelerated cell oxygen consumption rates and enhanced MccJ25-related reactive oxygen species overproduction. GENERAL SIGNIFICANCE: The deleterious reactive oxygen species would be produced as a consequence of the minor fraction of MccJ25 that interacts with the bacterial plasma membrane from the periplasmic side. These results show the first evidence of the mechanism underlying ROS production in sensitive bacteria.

Transcriptional autoregulation of the RcsCDB phosphorelay system in Salmonella enterica serovar Typhimurium.

The RcsCDB (Rcs) phosphorelay system is involved in the regulation of many envelope genes, such as those responsible for capsule synthesis, flagella production and O-antigen chain length, as well as in other cellular activities of several enteric bacteria. The system is composed of three proteins: the sensor RcsC, the response regulator RcsB, and the phospho-transfer intermediary protein RcsD. Previously, we reported two important aspects of this system: (a) rcsB gene expression is under the control of P(rcsDB) and P(rcsB) promoters, and (b) rcsD gene transcription decreases when the bacteria reach high levels of the RcsB regulator. In the present work, we demonstrate that the RcsB protein represses rcsD gene expression by binding directly to the P(rcsDB) promoter, negatively autoregulating the Rcs system. Furthermore, we report the physiological role of the RcsB regulator, which is able to modify bacterial swarming behaviour when expressed under the control of the P(rcsB) promoter.

The structure and biological aspects of peptide antibiotic microcin J25.

Microcin J25 (MccJ25) is a plasmid-encoded peptide of 21 L-amino acids (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G), excreted to the medium by an Escherichia coli strain. MccJ25 is active on Gram-negative bacteria related to the producer strain, including some pathogenic strains. The four-plasmid genes mcjABCD, are involved in MccJ25 production: mcjA encodes a 58-residue precursor, mcjB and mcjC codify two processing enzymes required for the in vivo synthesis of the mature peptide and mcjD encodes the immunity protein (McjD), a member of the super family of ABC transporters. Immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level. YojI, a chromosomal protein with ATP-binding-cassette-type exporter homology, is also able to export MccJ25. The E. coli outer membrane protein, TolC, is necessary for MccJ25 secretion mediated by either McjD or YojI. The uptake of MccJ25 is dependent on the outer-membrane receptor FhuA and the four inner-membrane proteins TonB, ExbD, ExbB and SbmA. At least two mechanisms described the action of MccJ25 on the target cells: (1) inhibition of the RNA-polymerase (RNAP) activity by obstructing the secondary channel, and consequently, preventing the access of the substrates to its active sites; and (2) operating on the cell membrane, MccJ25 disrupts the electric potential inhibiting the oxygen consumption in Salmonella enterica. MccJ25 also inhibits oxygen consumption and the respiratory chain enzymes in E. coli throughout the increasing of ROS concentration. Nevertheless the exact mechanism of this phenomenon must be elucidated. The MccJ25 exhibits a prolonged antimicrobial activity in a mouse infection model, suggesting a noteworthy potential for therapeutic uses.

Microcin J25 induces the opening of the mitochondrial transition pore and cytochrome c release through superoxide generation.

Microcin J25, an antimicrobial lasso-structure peptide, induces the opening of mitochondrial permeability transition pores and the subsequent loss of cytochrome c. The microcin J25 effect is mediated by the stimulation of superoxide anion overproduction. An increased uptake of calcium is also involved in this process. Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclosporin A and Mn(2+), allowed us to establish a time sequence of events starting with the binding of microcin J25, followed by superoxide anion overproduction, opening of mitochondrial permeability transition pores, mitochondrial swelling and the concomitant leakage of cytochrome c.

Role of electrostatics on membrane binding, aggregation and destabilization induced by NAD(P)H dehydrogenases. Implication in membrane fusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a classical glycolytic protein that can promote the fusion of phospholipid vesicles and can also play a vital role on in vivo fusogenic events. However, it is not clear how this redox enzyme, which lack conserved structural or sequence motifs related to membrane fusion, catalyze this process. In order to detect if this ability is present in other NAD(P)H dehydrogenases with available structure, spectroscopic studies were performed to evaluate the capability of alcohol dehydrogenase (ADH), glutamic dehydrogenase (GDH) and sorbitol dehydrogenase (SDH) to bind, aggregate, destabilize and fuse vesicles. Based on finite difference Poisson-Boltzmann calculations (FDPB) the protein-membrane interactions were analyzed. A model for the protein-membrane complex in its minimum free energy of interaction was obtained for each protein and the amino acids involved in the binding processes were suggested. A previously undescribed relationship between membrane destabilization and crevices with high electropositive potential on the protein surface was proposed. The putative implication of the non-specific electrostatics on NAD(P)H dehydrogenases induced membrane fusion is discussed.

Membrane viscosity is a major modulating factor of the enterocin CRL35 activity.

Enterocin CRL35 activity is deeply influenced by the membrane viscosity as could be demonstrated performing determinations of the minimal inhibitory concentrations (MIC) at different temperatures and analyzing the membrane viscosity in these cells as well as in resistant bacteria. In all the cases, bacteriocin activity was linked to higher levels of viscosity. This finding was confirmed studying the interaction of enterocin CRL35 with liposomes composed of dimyristoyl phosphatidylcholine: dimyristoyl phosphatidylglycerol (9:1) in both gel and liquid-crystalline phases. It could be establish, from peptide insertion analysis following the tryptophan fluorescence and microviscosity experiments that this peptide is able to interact more efficiently with membranes having a more structured hydrophobic core.

Penetration and interactions of the antimicrobial peptide, microcin J25, into uncharged phospholipid monolayers.

Microcin J25 forms stable monolayers at the air-water interface showing a collapse at a surface pressure of 5 mN/m, 220 mV of surface potential, and 6 fV per squared centimeter of surface potential per unit of molecular surface density. The adsorption of microcin J25 from the subphase at clean interfaces leads to a rise of 10 mN/m in surface pressure and a surface potential of 220 mV. From these data microcin appears to be a poor surfactant per se. Nevertheless, the interaction with the lipid monolayer further increase the stability of the peptide at the interface depending on the mode in which the monolayer is formed. Spreading with egg PC leads to nonideal mixing up to 7 mN/m, with hyperpolarization and expansion of components at the interface, with a small excess free energy of mixing caused by favorable contributions to entropy due to molecular area expansion compensating for the unfavorable enthalpy changes arising from repulsive dipolar interactions. Above 7 mN/m microcin is squeezed out, leaving a film of pure phospholipid. Nevertheless, the presence of lipid at 10 and 20 mN/m stabilize further microcin at the interface and adsorption from the subphase proceeds up to 30 mN/m, equivalent to surface pressure in bilayers.

EFFECT OF OVIDUCAL PROTEINASE UPON BUFO ARENARUM VITELLINE ENVELOPE. A FLUORESCENCE APPROACH.

The intrinsic fluorescence of vitelline envelope from amphibian eggs was enhanced after having been treated with pars recta secretion fluid. At the same time the total number of binding sites for 8-anilino-1-naphthalene sulfonate (ANS) was increased, with the affinity constant remaining unchanged. These observations are interpreted in terms of a structural change of the vitelline envelope submitted to pars recta fluid effect.

A novel insight on signal transduction mechanism of RcsCDB system in Salmonella enterica serovar typhimurium.

The RcsCDB system of Salmonella enterica serovar Typhimurium is implicated in the control of capsule and flagella synthesis. The hybrid sensor RcsC, the phosphotransferase RcsD and the RcsB regulator, constitute the main components of the RcsCDB system. The proposed Rcs signaling cascade involves the autophosphorylation of RcsC and the transfer of the phosphate group to RcsB, mediated by RcsD. We previously reported that the overexpression of rcsB repress the transcription of rcsD by an autoregulation mechanism. Moreover, we demonstrated that during the rcsD repression, the RcsB-dependent flagellar modulation remained active. These results suggest that the Rcs phosphorelay mechanism occurs even in the absence of RcsD. In this work, we established the existence of two alternative phosphorelay pathways driving activation of this system. We demonstrated that RcsC and RcsD can act as histidine kinase proteins which, after autophosphorylated, are able to independently transfer the phosphate to RcsB. Our results suggest that these pathways could be activated by different environmental signals, leading different levels of RcsB-phosphorylated to produce a differential gene modulation. These findings contribute to a better understanding of the complexity and importance of the Rcs system activation, where more than one phosphate flow pathway increases the possibilities to exert gene regulation for a quick environmental changes response.

Resveratrol modulates ATPase activity of liposome-reconstituted ABCG1.

ABCG1 is a half-sized transporter with an unquestionable importance in cholesterol homeostasis. So far, its expression and thus its activity was suggested to be regulated at transcriptional level by LXR and PPAR agonists including polyphenols. However, it is unknown whether there are other mechanisms of up-regulation of ABCG1 activity. In the present work resveratrol was shown to induce a nearly twofold increase in ATPase activity of reconstituted ABCG1. Evidence is presented for the first time suggesting that resveratrol is able to activate ABCG1 activity by an alternative mechanism that involves an indirect interaction.

A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria.

Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35-MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35-MccV may find important applications in food or pharmaceutical industries.

Microcin J25-Ga induces apoptosis in mammalian cells by inhibiting mitochondrial RNA-polymerase.

MccJ25, an antimicrobial peptide, was unable to cause apoptosis of COS-7 cells in spite of inducing reactive-oxygen species overproduction as well as cytochrome c release from isolated mitochondria. Surprisingly, MccJ25-Ga, an amidated variant of MccJ25 that displays similar anti-mitochondrial effects, did induce apoptosis in COS-7. The only difference found between the activities of these peptides was the unpredicted inhibition of mitochondrial RNA synthesis by MccJ25-Ga. These results led us to hypothesize that both mitochondrial RNA polymerase and mitochondrial membrane might be the molecular targets of MccJ25-Ga in mitochondria and this combined effect may lead to apoptosis.

Glyceraldehyde-3-phosphate dehydrogenase tetramer dissociation and amyloid fibril formation induced by negatively charged membranes.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme related with Huntington's, Parkinson's and Alzheimer's diseases. The ability of negatively charged membranes to induce a rapid formation of GAPDH amyloid fibrils has been demonstrated, but the mechanisms by which GAPDH reaches the fibrillar state remains unclear. In this report, we describe the structural changes undergone by GAPDH at physiological pH and temperature conditions right from its interaction with acidic membranes until the amyloid fibril is formed. According to our results, the GAPDH-membrane binding induces a beta-structuring process along with a loss of quaternary structure in the enzyme. In this way, experimental evidences on the initial steps of GAPDH amyloid fibrils formation pathway are provided.

Microcin J25 triggers cytochrome c release through irreversible damage of mitochondrial proteins and lipids.

We previously showed that the antimicrobial peptide microcin J25 induced the over-production of reactive oxygen species with the concomitant release of cytochrome c from rat heart mitochondria via the opening of the mitochondrial permeability transition pore. Here, we were able to demonstrate that indeed, as a consequence of the oxidative burst, MccJ25 induces carbonylation of mitochondrial proteins, which may explain the irreversible inhibition of complex III and the partial inhibition of superoxide dismutase and catalase. Moreover, the peptide raised the levels of oxidized membrane lipids, which triggers the release of cytochrome c. From in silico analysis, we hypothesize that microcin would elicit these effects through interaction with heme c1 at mitochondrial complex III. On the other hand, under an excess of l-arginine, MccJ25 caused nitric oxide overproduction with no oxidative damage and a marked inhibition in oxygen consumption. Therefore, a beneficial anti-oxidative activity could be favored by the addition of l-arginine. Conversely, MccJ25 pro-oxidative-apoptotic effect can be unleashed in either an arginine-free medium or by suppressing the nitric oxide synthase activity.

Identification of a new promoter for the response regulator rcsB expression in Salmonella enterica serovar Typhimurium.

The RcsCDB (Rcs) phosphorelay system regulates capsule synthesis, flagella production and other cellular activities in several enteric bacteria. This system consists of three proteins: the sensor RcsC, the cognate response regulator RcsB and the histidine-containing phosphotransfer protein RcsD (YojN), which is hypothesized to act as an intermediary in the phosphotransfer from RcsC to RcsB. The rcsC gene is convergently transcribed toward rcsB, which follows rcsD in what appears to be a two-gene operon. Here, it is reported that the overproduction of the rcsB gene represses rcsD transcription, but has a weak effect on its own expression. We demonstrated that the differential rcsD and rcsB expression is due to the activity of two promoters to transcribe the rcsB gene: (1) P(rcsDB) located upstream of rcsD and (2) P(rcsB) located within the rcsD coding region. In addition, here it was demonstrated that in Salmonella typhimurium, P(rcsB) is important to activate the rcsB expression during the stationary growth phase.

Tyrosine 9 is the key amino acid in microcin J25 superoxide overproduction.

Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 L-amino acid residues (G(1)-G-A-G-H(5)-V-P-E-Y-F(10)-V-G-I-G-T(15)-P-I-S-F-Y(20)-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr(9) or Tyr(20) with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr(9), and not Tyr(20), is involved in the MccJ25 action on the respiratory chain target.