Interactions between Salmonella typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide

Effects of magainin 2 amide on the phase behavior of Salmonella typhimurium lipopolysaccharide were characterized by FT-IR spectroscopy. This antimicrobial cationic peptide disorders the lipopolysaccharide at molecular ratios of lipopolysaccharide to magainin greater than 4, and can induce a temperature-dependent structural reorientation. The nature of the five phosphate groups of lipopolysaccharide was determined by 31P NMR spectroscopy. At pH 7.4, the net charge on the phosphates is -7. Lipopolysaccharide undoubtedly plays an important role in modulating the interactions of magainin with the gram-negative cell envelope and may act as a molecular sponge to protect the plasma membrane.     

Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.

The reversibility and specificity of phospholipid translocation between the inner and outer membrane of Salmonella typhimurium has been investigated by incorporating exogenous lipids from phospholipid vesicles into the outer membrane of intact cells. Translocation of newly incorporated phospholipids to the inner membrane was demonstrated by decarboxylation of vesicle-derived phosphatidylserine and by recovery of vesicle constituents in both inner and outer membrane fractions. All Salmonella phospholipids tested, as well as phosphatidylcholine and cholesteryl oleate were effectively translocated to the inner membrane. However, no translocation of vesicle-derived lipopolysaccharide or an incomplete biosynthetic precursor of lipid A could be detected. Translocation of phospholipids and cholesteryl ester was rapid and extensive, and appeared to lead to equilibration of the lipids between the two membranes. The mechanism of intermembrane translocation has not been established, but the results are suggestive of diffusional flow across zones of adhesion between the inner and outer membranes.     

X-ray diffraction studies of outer membranes of Salmonella typhimurium.

X-ray diffraction studies were carried out on the outer membranes of various strains of Salmonella typhimurium. Ten distinct diffraction peaks which seem to be caused by protein assemblies were observed for most strains. Three small-angle reflections were used to determine an average structure of the protein assembly in the outer membrane of mutant HN202. An electron density distribution of the averaged assembly was obtained by means of the Fourier-Bessel transform. It has a diameter of about 100A, in agreement with the results of electron microscope observations (Smit, Kamio, and Nikaido (1975) J. Bacteriol. 124, 942--958), and exhibits a low electron density region at its center, suggesting the presence of a pore, as predicted on the basis of transmembrane transport experiments (Nakae (1976) J. Biol. Chem. 251, 2176--2178).     

Polyamines as constituents of the outer membranes of Escherichia coli and Salmonella typhimurium.

Extraction of whole cells of Salmonella typhimurium and Escherichia coli with 1 M NaCl released 8 to 13% of their total cellular polyamines (putrescine, cadaverine, and spermidine). This extraction did not cause significant cell lysis, release of outer membrane (OM) constituents, or leakage of periplasmic beta-lactamase. The extraction released nearly equal amounts of polyamines from mdo (membrane-derived oligosaccharide) mutants and wild type. These findings suggest that the released polyamines are apparently bound to the cell envelope. NaCl (1 M) was as effective as trichloroacetic acid in releasing polyamines from isolated OM and lipopolysaccharide (LPS). Isolated OM contained four times more polyamines than the cytoplasmic membrane. The increased binding to the OM is apparently due to the association of polyamines with the polyanionic LPS. Nearly identical amounts of polyamines were found in the OM and LPS preparations (as quantified per milligram of LPS). These amounts are equal to those released from the intact cells by 1 M NaCl (quantitation as above). However, redistribution of polyamines took place after cell disruption, because the relative proportions of different polyamines varied in the OM and LPS preparations. These results indicate that polyamines released from intact cells during 1 M NaCl extraction are preferentially derived from the OM.     

Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments.

Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents.     

Bactericidal activity of magainin 2: use of lipopolysaccharide mutants

Salmonella typhimurium and a series of rough lipopolysaccharide mutants derived from it were used as target bacteria to examine the antimicrobial capacity of magainin 2. Magainin 2 demonstrated a dose-related bactericidal activity against the smooth parent strain and the series of lipopolysaccharide mutants. The lipopolysaccharide mutant series showed an ordered increase in sensitivity to the magainin 2 as the depth of the rough lesion in the lipopolysaccharide increased.     

Detoxification of Salmonella typhimurium lipopolysaccharide by ionizing radiation

The efficiency of ionizing radiation in detoxifying the lethal determinant(s) of the lipopolysaccharide (LPS) of Salmonella typhimurium, S. enteritidis, and Escherichia coli in aqueous solution and associated with heat-killed S. typhimurium cells in suspension decreased with doses above 1 Mrad. The 50% end point of inactivation was more than 7.0 Mrad for heat-killed salmonellae and 4.8, 4.5, and 1.0 Mrad for the LPS of S. typhimurium, S. enteritidis, and E. coli, respectively. After exposure to 20 Mrad, S. typhimurium LPS retained a small portion of its lethal properties although the ld(50) was much greater than 9.5 mg per 20-g mouse. However, at -184 C, no inactivation of the lethal determinant(s) occurred after exposure to as much as 20 Mrad. This demonstrated the significance of the indirect effect and the mobility and formation of free radicals. At 22 C, the optical density at 400 mmu increased and the pH decreased with increasing radiation dose, but no qualitative changes were observed in the infrared spectrum. No change was observed in the pyrogenicity of S. typhimurium LPS; a slight decrease in antigenicity was revealed when 6 days, but not when 1 day, elapsed between vaccination and challenge in the mouse protection test. The results were interpreted as evidence of the existence of two or more lethal and antigenic determinants. The differential effect of radiation on these properties and on the pyrogenic component(s) probably are indicative of separate functional sites for lethal, antigenic, and pyrogenic activities.     

Energy dependence of lipopolysaccharide translocation in Salmonella typhimurium

Energy inhibitors block translocation of pulse-labeled core lipopolysaccharide to outer membrane under conditions which allow maintenance of constant specific radioactivity of intracellular precursor pools throughout the chase period. Under the conditions used, approximately 75% of the total cellular label was membrane-bound at initiation of chase. Translocation of core lipopolysaccharide from inner to outer membrane showed apparent first order kinetics (t1/2 = 1.2 min, 32 degrees C). Translocation was blocked by arsenate (5-10 mM) under conditions where proton motive force was unchanged, while the uncouplers 2,4-dinitrophenol (0.1 mM to 0.8 mM) and carbonyl cyanide-m-chlorophenyl hydrazone (12-30 microM) inhibited translocation with no apparent effect on the ATP pool. Therefore, core lipopolysaccharide translocation appears to require maintenance of both proton motive force and high energy phosphate pools. Electron microscopic experiments show no gross disruption of zones of adhesion, the putative sites of lipopolysaccharide translocation, in the presence of arsenate or 2,4-dinitrophenol suggesting that energy is not required simply for maintenance of these structures.     

Molecular organization of the outer membrane of Salmonella typhimurium different release of lipopolysaccharide from wild type and lipopolysaccharide mutant cells by EDTA treatment

Cells of Salmonella typhimurium wild type and of several well defined lipopolysaccharide mutants were treated with EDTA. The percentage release of lipopolysaccharide and phospholipid was determined. The results obtained show that the release of lipopolysaccharide by EDTA declines along with the gradually diminishing chain length of the lipopolysaccharide, althought the total amount of lipopolysaccharide was found to increase at the same time in the respective mutants. Implications of these findings for the organization of the outer membrane are discussed.     

Immunomodulation of macrophages by radio-detoxified lipopolysaccharide of Salmonella typhimurium

Lipopolysaccharide (LPS) and ratio-detoxified LPS (Rd-LPS) from Salmonella typhimurium were analysed for their ability to stimulate murine peritoneal exudate cells (PEC) and macrophages. Rd-LPS induced much more inflammatory response as compared to LPS. PEC numbers/mouse obtained were significantly higher (3-fold) in response to Rd-LPS than LPS. The haemorrhage was induced in mice by LPS but not by Rd-LPS. Activation of macrophages in vivo by Rd-LPS was significantly higher as compared to LPS. This was evident from the increase levels of their lysosomal enzymes and cytokines. Rd-LPS induced 10-fold increase in acid phosphatase contents of macrophages as compared to controls while only 7-fold increase was obtained with LPS. Arylsulfatase and beta-glucuronidase increased by about 2-fold by Rd-LPS and LPS. Macrophages incubated with Rd-LPS in vitro showed 16-fold and 20-fold increase in the cell associated levels of arylsulfatase and beta-glucuronidase respectively as compared to unstimulated cells. On the other hand, only 6-fold increase was observed in response to LPS in the levels of both the enzymes. TNF-[symbol: see text] and IL-1 secreted by macrophages increased considerably in response to Rd-LPS as compared to those released by LPS. Rd-LPS, thus seems to be a better immunomodulator than untreated LPS.     

Bacteriophage P22 is not a likely probe for zones of adhesion between the inner and outer membranes of Salmonella typhimurium.

Thin-section electron micrographs of plasmolyzed Salmonella typhimurium infected with bacteriophage P22 demonstrated that phage adsorbed to cells over sites of inner- and outer-membrane contact. Efforts were made to isolate such adsorption sites by infection of cells with 35S- and 32P-labeled phage and by separation of the membranes on sucrose gradients. At 37 degrees C, about 75% of the 35S radioactivity could be recovered in a region of intermediate density between the inner and outer membranes. This region (phi band) did not contain 32P. The gradient profile was independent of the multiplicity of infection (between 0.2 and 50) and of the presence or absence of chloramphenicol, dinitrophenol, or cyanide. However, ethylenediaminetetraacetate, when present during the infection step, prevented the formation of phi band. The density of phi band was at least 1.30 g/cm3, as demonstrated by prolonged centrifugation on a D2O-sucrose gradient. phi Band was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy to contain empty phage heads and contaminating cellular debris. In purified preparations, phage heads were the only structures, visible by negative staining, and very little cellular phospholipid or protein was associated with the phage proteins (less than 2% and 30% by weight, respectively, as determined by using [3H]glycerol or [3H]leucine). The residual cellular protein included all of the major outer-membrane proteins rather than any one specific protein. These results are interpreted as indicating that phi band probably does not contain adhesion site material stably associated with phage heads.     

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.     

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.     

TonB protein of Salmonella typhimurium. A model for signal transduction between membranes

The tonB gene product is required for several outer membrane transport processes in bacteria. The tonB gene from Salmonella typhimurium was sequenced and found to be similar to that of Escherichia coli. The TonB protein is highly proline-rich and includes an unusual segment consisting of multiple X-Pro dipeptide repeats. A synthetic peptide corresponding to this segment has been used to raise anti-TonB antibodies. TonB was shown to be associated with the cytoplasmic membrane, apparently anchored via a single hydrophobic N-terminal segment. Protease accessibility studies, and the use of a series of TonB-beta-lactamase fusions, showed that the rest of the TonB protein is periplasmic. Unusually, export of TonB is not accompanied by cleavage of the N-terminal signal peptide. In the accompanying paper, we show that TonB interacts directly with the outer membrane FhuA (TonA) receptor. Thus, TonB must span the periplasm, providing a link between the cytoplasmic membrane and receptors in the outer membrane. On the basis of these data, and those published by other laboratories, we propose a model whereby TonB serves as a "mechanical" linkage that, by transmitting protein conformational changes from the cytoplasmic membrane across the periplasm, acts as a means of coupling energy to outer membrane transport processes. Such a mechanism has general implications for signal transduction within and between proteins.     

Binding of antibacterial magainin peptides to electrically neutral membranes: thermodynamics and structure.

Magainins are positively charged amphiphatic peptides which permeabilize cell membranes and display antimicrobial activity. They are usually thought to bind specifically to anionic lipids, and binding studies have been performed almost exclusively with negatively charged membranes. Here we demonstrate that binding of magainins to neutral membranes, a reaction which is difficult to assess with spectroscopic means, can be followed with high accuracy using isothermal titration calorimetry. The binding mechanism can be described by a surface partition equilibrium after correcting for electrostatic repulsion by means of the Gouy-Chapman theory. Unusual thermodynamic parameters are observed for the binding process. (i) The three magainin analogues that were investigated bind to neutral membranes with large exothermic reaction enthalpies DeltaH of -15 to -18 kcal/mol (at 30 degrees C). (ii) The reaction enthalpies increase with increasing temperature, leading to a large positive heat capacity DeltaC(p) of approximately 130 cal mol(-)(1) K(-)(1) (at 25 degrees C). (iii) The Gibbs free energies of binding DeltaG are between -6.4 and -8.6 kcal/mol, resulting in a large negative binding entropy DeltaS. The binding of magainin to small unilamellar vesicles is hence an enthalpy-driven reaction. The negative DeltaH and DeltaS and the large positive DeltaC(p) contradict the conventional understanding of the hydrophobic effect. CD experiments reveal that the membrane-bound fraction of magainin is approximately 80% helical at 8 degrees C, decreasing to approximately 60% at 45 degrees C. Since the random coil --> alpha-helix transition in aqueous solution is known to be an exothermic process, the same process occurring at the membrane surface is shown to account for up to 65% of the measured reaction enthalpy. In addition to membrane-facilitated helix formation, the second main driving force for membrane binding is the insertion of the nonpolar amino acid side chains into the lipid bilayer. It also contributes a negative DeltaH and follows the pattern for the nonclassical hydrophobic effect. Addition of cholesterol drastically reduces the extent of peptide binding and reveals an enthalpy-entropy compensation mechanism. Membrane permeability was measured with a dye assay and correlated with the extent of peptide binding. The level of dye efflux is linearly related to the amount of surface-bound peptide and can be traced back to a membrane perturbation effect.