Beatriz F. de Arcuri

Microcin J25 Has Dual and Independent Mechanisms of Action in Escherichia coli: RNA Polymerase Inhibition and Increased Superoxide Production

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O(2)(*-)) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.

Phospholipase-like myotoxins induce rapid membrane leakage of non-hydrolyzable ether-lipid liposomes.

Two phospholipase-like myotoxins--ammodytin L from Vipera ammodytes and myotoxin II from Bothrops asper--are shown to be able to induce leakage of liposomes made from non-hydrolyzable ether-linked phospholipids. This demonstrates that the cytolytic activity of these toxins is completely independent of any remaining enzyme activity or contamination with active phospholipases.

Effects of the antibiotic peptide microcin J25 on liposomes: role of acyl chain length and negatively charged phospholipid

This paper reports the effects of microcin J25 (MccJ25) on the microviscosity and permeability of phospholipid vesicles of different compositions. The results obtained indicate that MccJ25 interacts with egg L-alpha-phosphatidylcholine (PC) vesicles as demonstrated by peptide intrinsic fluorescence determinations. The interaction depends on the lipid composition of the vesicles. MccJ25 interaction induces a significant fluidity increase of egg PC vesicles. This effect is time and concentration dependent. Both trimethyl ammonium 1,6-diphenyl-1,3,5-hexatriene and 1,6-diphenyl-1, 3,5-hexatriene gave the same results. The microviscosity of L-alpha-phosphatidylcholine dipalmitoyl small unilamellar vesicles (SUVs) was affected while that of L-alpha-phosphatidylcholine dimyristoyl vesicles was not, indicating that the effect was strongly dependent on the chain length of fatty acids. On the other hand, negatively charged L-alpha-phosphatidyl-DL-glycerol (PG) vesicles remarkably inhibited the peptide effect. Nevertheless vesicles composed of L-alpha-phosphatidylethanolamine:PG:cardiolipin (7:2:1), a composition resembling bacterial membrane, were sensitive to the MccJ25 effect. MccJ25 effectively dissipated the valinomycin-induced membrane potential, but induced only a modest leakage (5%) of the trapped Tb(+3)-dipicolinic acid complex. These results indicate that the peptides interact and perturb the bilayer of SUVs. The relationships between this effect and bactericidal action remain to be elucidated.

LYSOZYME INDUCED FUSION OF NEGATIVELY CHARGED PHOSPHOLIPID VESICLES

Lysozyme promotes fusion of negatively charged phospholipid vesicles prepared by ethanolic injection. Vesicle fusion was a leaky process as revealed by the release of encapsulated carboxyfluorescein or Tb-DPA complex. Extensive proteolysis of lysozyme inhibited the fusion process. The fusion process was critically dependent on the medium ionic strength; 100 mM of any salt was sufficient to inhibit totally the fusion activity of the protein. The high efficiency of lysozyme (80% RET) was almost constant in the pH range from 4.0 to 9.0, but it was sharply diminished when the pH of the medium was at the isoelectric point of the protein (pI 11.0). Fusion induced by chemically modified lysozyme, showed that the pH profile changed according to the isoelectric point of the protein derivative. These observations stress the importance of electrostatic interactions in the process of fusion induced by lysozyme.

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 Mn2+, 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.

Aggregatation, fusion and aqueous content release from liposomes induced by lysozyme derivatives: Effect on the lytic activity

Chemically modified lysozymes, namely: N-succinyl lysozyme, glycine methyl ester of N-succinyl lysozyme and oxoindole lysozyme have been prepared. Aggregation, fusion and leakage of phospholipid vesicles induced by these derivatives have been studied in comparison with the effect of the unmodified protein. The experiments were carried out with negatively charges 9PC/PA, 9:1) and uncharged (PC and PC/DOPE/Chol (10:5:5)) lipid vesicles of different packing. Fusion and aggregation of negatively charged phospholipid vesicles in induced by proteins positively charged at pH 7.0 involving electrostatic interactions, a similar pattern on fusion and aggregation of the least stably packed lipid vesicles points also to hydrophobic forces playing a role in the lipid-protein interaction. A conformational change of the protein involved increasing beta-turns, loops and unordered structure at the expenses of beta-sheet without affecting alpha helix content. The conformational effect is necessary to provoke the effects studied, since one of the derivatives (N-succinyl lysozyme) neither changes conformation nor causes aggregation and fusion of vesicles. However, there is no relationship between lysozyme activity and fusion or aggregation of lipid vesicles that catalytic and fusogenci sites of, indicating lysozyme are topographically different.

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.