Susceptibility of bacterial, eukaryotic and artificial membranes to the disruptive action of the cationic peptides Pep 5 and nisin

Abstract The cationic bactericidal peptides Pep 5 and nisin render membranes permeable to low- M _r compounds. All Gram-positive bacteria treated with these peptides showed an immediate efflux of entrapped radioactive markers. The uptake of α-[¹⁴C]methylglucoside by the phosphoenolpyruvate-dependent phosphotransferase system was stimulated by Pep 5, supporting previous results that pep 5 abolishes the membrane potential. Oxygen consumption was inhibited, presumably due to lack of ADP. Escherichia coli became sensitive to Pep 5 and nisin when the outer membrane was bypassed by osmotic shock or by formation of cytoplasmic membrane vesicles. In contrast, Mycoplasma cells and erythrocytes were unaffected by Pep 5 and nisin in concentrations up to 1 mM. Human lung fibroblasts released only small amounts of ATP when treated with Pep 5 and nisin in μM concentrations. Eukaryotic and Mycoplasma cells were disrupted more effectively by the bee venom peptide melittin, which displays overall structural similarities to Pep 5 and nisin. Various artificial membranes were not affected by Pep 5, nisin, or melittin.     

The outer membrane of gram-negative bacteria inhibits antibacterial activity of brochocin-C.

Brochocin-C is a two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754 that has a broad activity spectrum comparable to that of nisin. Brochocin-C has an inhibitory effect on EDTA-treated gram-negative bacteria, Salmonella enterica serovar Typhimurium lipopolysaccharide mutants, and spheroplasts of Typhimurium strains LT2 and SL3600. Brochocin-C treatment of cells and spheroplasts of strains of LT2 and SL3600 resulted in hydrolysis of ATP. The outer membrane of gram-negative bacteria protects the cytoplasmic membrane from the action of brochocin-C. It appears that brochocin-C is similar to nisin and possibly does not require a membrane receptor for its function; however, the difference in effect of the two bacteriocins on intracellular ATP indicates that they cause different pore sizes in the cytoplasmic membrane.     

The Outer Membrane of Gram-Negative Bacteria Inhibits Antibacterial Activity of Brochocin-C

Brochocin-C is a two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754 that has a broad activity spectrum comparable to that of nisin. Brochocin-C has an inhibitory effect on EDTA-treated gram-negative bacteria, Salmonella enterica serovar Typhimurium lipopolysaccharide mutants, and spheroplasts of Typhimurium strains LT2 and SL3600. Brochocin-C treatment of cells and spheroplasts of strains of LT2 and SL3600 resulted in hydrolysis of ATP. The outer membrane of gram-negative bacteria protects the cytoplasmic membrane from the action of brochocin-C. It appears that brochocin-C is similar to nisin and possibly does not require a membrane receptor for its function; however, the difference in effect of the two bacteriocins on intracellular ATP indicates that they cause different pore sizes in the cytoplasmic membrane.     

Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis

Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.     

Carbon dioxide and nisin act synergistically on Listeria monocytogenes

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis(r)) cells grown in broth at 4 degrees C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis(r) cells. Wild-type cells grown in 100% CO(2) were two to five times longer than cells grown in air. Nisin (2.5 microg/ml) did not decrease the viability of Nis(r) cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO(2). There was a quantifiable synergistic action between nisin and CO(2) in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 microg of nisin per ml increased from 3.1 to 12.5 microg/ml over 35 days, but this increase was markedly delayed for cultures in CO(2). This synergism between nisin and CO(2) was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO(2) and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO(2) atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4 degrees C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30 degrees C. Cells grown in the presence of 100% CO(2) and those grown at 4 degrees C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO(2) is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.     

Sensitization of Escherichia coli to nisin by maltol and ethyl maltol

F. SCHVED, M.D. PIERSON AND B.J. JUVEN. 1996. When used separately, 20 mmol 1^-1 maltol or 1600 AU ml^-1 nisin resulted in a 0–0.6 log₁₀ reduction in viable counts of Escherichia coli in a buffer system. However, when added in combination they yielded a 1.8–5. 5–log-cycle reduction in viable counts of E. coli at pH 5.0 and 6.8 respectively. It is postulated that maltol (and ethyl maltol) destabilizes the cell outer membrane by chelation of Mg²⁺ and/or Ca²⁺, thus permeabilizing the E. coli cell to nisin.     

Molecular recognition of lipopolysaccharide by the lantibiotic nisin

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli.     

Dication and trication which can increase the permeability of Escherichia coli outer membrane

Recent success in the preparation of the monomer, dimer and trimer in compound 48/80 prompted us to investigate the action of these compounds on Escherichia coli cells. It was found that compound 48/80 inhibited growth of E. coli cells, while the monomer, dimer and trimer in 48/80 did not. However, the following experiments showed that the dimer and trimer disrupted the permeability barrier of the outer membrane of E. coli. First, addition of the dimer or trimer in cell suspension stimulated the uptake of tetraphenylphosphonium cation. Second, the synergistic effect of the dimer on the action of gramicidin caused the efflux of K+. In experiments using isolated cytoplasmic membrane vesicles, addition of gramicidin alone caused the efflux of K+. Thus, it was speculated that, with whole cells, the dimer formed some defect structure in the outer membrane, through which gramicidin reached the cytoplasmic membrane and increased the K+ permeability. The temperature dependence of efflux K+ showed that the dimer in 48/80 rendered the outer membrane permeable to gramicidin at temperatures above the phase transition of the outer membrane.     

Mode of action of gramicidin S on Escherichia coli membrane

The action of a cationic antibiotic gramicidin S on the outer and cytoplasmic membranes of Escherichia coli was studied. It was found that gramicidin S disrupted the permeability barrier of the outer membrane, permitting the permeation of an antibiotic ionophore, this being similar to the action of the dimer in compound 48/80 (Katsu, T., Shibata, M. and Fujita, Y. (1985) Biochim. Biophys. Acta 818, 61-66). However, differently from the dimer, gramicidin S further stimulated the efflux of K+ through the cytoplasmic membrane of E. coli. The time course of K+ permeability change accorded well with that of change in the viability of E. coli cells. These changes occurred at temperatures above the phase transition of the cytoplasmic membrane. This temperature range differed greatly from the case of polymyxin B, a polycationic antibiotic acting at temperatures above the phase transition of the outer membrane. We discuss the mode of gramicidin S action on the cytoplasmic membrane of E. coli, in comparison with the results on red blood cells and liposomes.     

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2°C min⁻¹) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000°C min⁻¹) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU ml⁻¹ nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5 log of cell inactivation for exponentially growing cells and 1 log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.     

Molecular parasitism in the Escherichia coli-Bdellovibrio bacteriovorus system: translocation of the matrix protein from the host to the parasite outer membrane

During the intracellular maturation in Escherichia coli of the parasite Bdellovibrio bacteriovorus the outer membrane, major protein I of E. coli (i.e., the matrix protein) becomes associated with the outer membrane of the emerging parasite cells. The binding properties of this protein with the outer membrane of the host and of the parasite are identical. An analogous phenomenon also occurs during Bdellovibrio parasitism on Klebsiella pneumoniae and on Salmonella typhimurium. Possible roles for this scavenging action of Bdellovibrio, and similar phenomena in other parasitic systems, are discussed.     

Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants.

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.     

Synergistic antibacterial action of heat in combination with nisin and magainin II amide

Lactobacillus plantarum has been exposed to mild heat at temperatures between 48 and 56 °C in combination with low concentrations of the lantobiotic nisin in different sequential set-ups. Exposure to heat and nisin caused synergistic reductions of Lact. plantarum viability. Efficient antimicrobial action was dependent on the growth state of the culture as well as on levels and sequences of treatment applications. Listeria monocytogenes and Escherichia coli were treated at 55 °C in the presence of magainin II amide. Synergistic reductions in viable counts could be observed for L. monocytogenes and, after prolonged exposure, also for E. coli . The bacterial membrane could be identified by fluorometry and flow cytometry as an important target of applied treatment combinations.     

Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants.

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.     

Carbon Dioxide and Nisin Act Synergistically on Listeria monocytogenes

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis^r) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis^r cells. Wild-type cells grown in 100% CO₂ were two to five times longer than cells grown in air. Nisin (2.5 μg/ml) did not decrease the viability of Nis^r cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO₂. There was a quantifiable synergistic action between nisin and CO₂ in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 μg of nisin per ml increased from 3.1 to 12.5 μg/ml over 35 days, but this increase was markedly delayed for cultures in CO₂. This synergism between nisin and CO₂ was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO₂ and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO₂ atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO₂ and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO₂ is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment.

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Defeat of colicin tolerance in Escherichia coli ompA mutants: evidence for interaction between colicin L-JF246 and the cytoplasmic membrane.

Escherichia coli ompA mutants are tolerant to colicin L-JF246. This tolerance can be overcome by a variety of treatments that have as their target the outer membrane or the peptidoglycan layers of the cell envelope. Thus, increasing the concentration of colicin L, releasing lipopolysaccharide from the outer membrane by treatment of intact cells with ethylenediaminetetracetic acid (EDTA), converting cells to spheroplasts by treatment with lysozyme-EDTA or penicillin, or trypsin, treatment of intact cells will result in an increased colicin sensitivity. These treatments alter the outer membrane of ompA mutants and suggest that the altered outer membrane may allow the penetration of at least a portion of the colicin L molecule to a site of action located within this barrier. To substantiate this, we have demonstrated that membrane vesicles prepared from ompA mutants are sensitive to colicin L and that 14C-labeled colicin L binds rapidly to both the outer and inner membrane fractions of the cell.     

Heavy metal cation-induced increase in the antimicrobial activity of gramicidin S. Increased sensitivity of metal-resistant mutants of Escherichia coli B to the antibiotic]

Gramicidin S response of metal resistant mutants of E. coli B and the effect of concentrations of Cu2+, Ag+, Co2+ and Cd2+ on the growth and sensitivity of E. coli B to cationic antibiotics, i.e. gramicidin S2+ and streptomycin2+, were studied. It was shown that the metal-cumulating mutants of E. coli B with two different mechanisms of cross resistance to Cu2+, Cd2+ and Ag+ had higher sensitivity to gramicidin S than the initial wild type strain of E. coli B. It was found that in the threshold or higher doses the salts of Cu, Ag, Co and Cd increased the gramicidin S antimicrobial action on actively metabolizing cells of E. coli B. Analysis of the experimental data as well as the literature ones suggested that the synergic action of gramicidin S and the heavy metals stemmed from an increase in the cationic conductivity of the cytoplasma membrane modified by the metals in the threshold doses which induced an increase in the transport and accumulation of the cations in the bacterial cells by the electric field gradient (with the negative sign inside). Withdrawal of Ca2+ and Mg2+ from the E. coli outer structures into the cytoplasm impaired the barrier properties of the outer membrane and promoted binding of the gramicidin S cations to the liberated anionic groups of the E. coli outer structures and potentiation of the gramicidin S antimicrobial activity as was shown in our experiments.     

Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B.

Growing Escherichia coli release envelope material into the medium. Upon infection with T4 phage increased amounts of this material are released and at a greater rate. In order to determine whether both inner and outer membranes are present in this material, and whether the material released by growing cells differs from that released by infected cells, we have examined the protein composition of envelope released by growing and T4-infected E. coli B. Our results show: (a) the protein composition of envelope released from growing or infected cells is similar, (b) the proteins present are representative of the outer membrane, (c) the major outer membrane protein of E. coli B, protein II, is deficient in the released material. We therefore conclude that the envelope material released from growing or infected E. coli represents a special fraction of the outer membrane. This finding is discussed in relation to outer membrane structure and function. In addition, data are presented on the differing outer membrane protein composition of substrains of E. coli B obtained from different laboratories.