John H. Freer

Hydrophobic adsorptive and hemagglutinating properties ofEscherichia coli possessing colonization factor antigens (CFA/I or CFA/II), type 1 pili, or other pili

Hydrophobic and hemagglutinating activities of piliated enterotoxigenicEscherichia coli possessing colonization factor antigens (CFA)/I and putative CFA/II, strains with type 1 pili, and piliated strains of nonenterotoxigenicE. coli from urinary tract infections were compared. When passed through columns of hydrophobic Phenyl Sepharose in the presence of buffered ammonium sulfate, the strains with CFA adsorbed most strongly. Similarly, the CFA strains showed a tendency to autoagglutinate at a lower (NH4)2SO4 concentration than the other strains studied. The degree of hydrophobicity of the strains tested is in the order CFA/I>CFA/II>type 1 pili>urinary tract strains. Rough variants ofE. coli strains were more hydrophobic than their smooth parents. Electron microscopy showed large numbers of pili on CFA strains, whereas type 1 piliated strains possessed fewer pili. CFA-negative clones possessed few or no, pili and did not adsorb to the gel. A highly piliated mutant strain (PAK/2PfS) ofPseudomonas aeruginosa bound to the Phenyl Sepharose while the poorly piliated wild-type strains did not. Strains, lost their adsorptive capacity after blending, sonication, heating, or trypsin treatment. It is concluded that the hydrophobicity of enteric organisms, as measured by hydrophobic interaction chromatography, is a function of the type and number of pili on the cell surface.

Interaction of Clostridium perfringens θ-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study

Commercially available preparations of phospholipase C from Clostridium perfringens are commonly contaminated with theta haemolysin, one of a group of bacterial haemolysins called oxygen labile (O-labile) haemolysins. Treatment of erythrocyte ghosts and a mixed lipid dispersion containing cholesterol with commercially available phospholipase C in the absence of Ca-2+ and the presence of phosphate buffer and/or EDTA resulted in the formation and release of ring or arc-shaped structures. Highly purified phospholipase C, free of theta-haemolysin, produced no changes in the morphology of erythrocyte ghosts or lipid dispersions in the presence of phosphate or EDTA, but caused the formation of typical diglyceride droplets in the presence of Ca-2+ in the absence of these inhibitors. Ring structures, identical to those caused by commercial phospholipase C, were formed on addition of highly purified theta-haemolysin to erythrocyte ghost membranes, lipid dispersions containing cholesterol and cholesterol dispersions, but not on treatment of membranes from Micrococcus lysodeikticus. Heat-inactivated O-haemolysin (60 degrees C for 10 min) produced no such effects. The dimensions of rings and arcs displayed heterogeneity. The outside diameters in various preparations varied from approx. 27-58 nm with border thickness of 4.1-7.8 nm.

Delta-haemolysin from Staphylococcus aureus and model membranes. A solid-state 2H-NMR and 31P-NMR study.

Solid-state 2H NMR and 31P NMR of 2H-enriched chains and polar head groups, respectively, of dipalmitoylglycerophosphatidylcholine/water dispersions were undertaken to investigate the action of delta-haemolysin from Staphylococcus aureus on biomembranes. When the lipid/toxin molar ratio, Ri, is greater than or equal to 10, the gel-phase 2H powder patterns and the temperature of the gel-fluid phase transition, tc, are unchanged by the presence of the toxin whereas the 31P powder spectra of polar head groups are perturbed. At t greater than tc, a detailed analysis of methylene ordering indicates that delta-haemolysin orders the lipid chains near tc and disorders them for t much greater than tc. These findings are interpreted in terms of peptide location with regard to the membrane and suggest that the position of the toxin depends on the temperature relative to tc. Experiments carried out at Ri = 4 exhibit sharp, isotropic 2H-NMR lines, at t greater than tc, indicating that delta-haemolysin promotes the appearance of very small objects undergoing fast isotropic reorientation which average to zero the quadrupolar interaction. Below tc, one observes gel-phase powder patterns which indicate that the bacterial toxin is unable to form such small structures with ordered dipalmitoylglycerophosphocholine phospholipids. From comparison of the action of delta-haemolysin with that of melittin on same lipids [Dufourc et al. (1986) Biochemistry 25, 6448-6455] it results that both toxins perturb similarly fluid-phase lipids at elevated temperature, but they behave differently with gel-phase lipids, the former toxin being less efficient in membrane restructuring than the latter.

Consequences of sphingomyelin degradation in erythrocyte ghost membranes by staphylococcal β-toxin (sphingomyelinase C)

Abstract The purified β-toxin (sphingomyelinase C) of Staphylococcus aureus released water-soluble organic phosphorus from aqueous dispersions of sphingomyelin, and bovine and human erythrocyte ghosts in the presence of Mg 2+ , in amounts indicating extensive sphingomyelin degradation. Extensive ultrastructural changes were evident in both human and bovine freeze-etched ghosts after sphingomyelinase C treatment. Pools of apparently solid particle-free material, possibly the ceramide product of hydrolysis, accumulated in the hydrophobic region of the bilayer. These observations can be explained in terms of a membrane in which sphingomyelin is preferentially located in the outer half of the bilayer. This staphylococcal toxin should prove useful in further studies on the distribution of phospholipids in various membranes and their relationship to membrane function.

Cytolytic toxins and surface activity

Surface-active properties of alpha-toxin and delta-lysin, two cytolytic protein exotoxins of the bacterium Staphylococcus aureus are summarised. The relevance of differences in surface charge density on membranes is discussed in relation to possible mechanisms of binding and membrane penetration by alpha-toxin. An hypothesis for the mechanism of membrane disruption by delta-lysin involving the formation of hydrophilic transmembrane pores is proposed.

[3] Purification and assay of staphylococcal δ-lysin

Publisher Summary This chapter describes the purification and assay of staphylococcal δ-lysin. The δ-1ysin (also known as δ-hemolysin, delta toxin). Two immunologically distinct forms are described, one produced by S. aureus strains isolated from humans and a variant form produced by strains of S. aureus of canine origin. Comparison of the amino acid sequences of the lysins from strain 186X (human origin) with that of strain CN7450 (canine origin) shows changes in the 26 amino acids of the peptide but conservation of charge distribution and both peptides have similar biological properties.. Unlike the other lysins of S. aureus , the δ-1ysin is of relatively low specific lytic activity yet is active against a wide range of cells, subcellular organelles, and artificial membrancs. Its membrane-damaging activity is conveniently assayed against erythrocytes by measuring the release of hemoglobin (hemolysis) as a result of membrane damage. Although erythrocytes from a wide range of species are sensitive to the action of δ-1ysin, those most sensitive are from marine fish, particularly the mackerel ( Scomber scombrus ).

Some properties of IgG1 and IgG1 globulins from normal and adjuvant stimulated guinea-pigs

Abstract After electrofocussing of guinea-pig anti-ovalbumin serum stimulated with Freund complete (FCA) and incomplete (FIA) adjuvant IgG 1 and IgG 2 were completely separated. Disc gel electrophoresis and densitometry showed the presence of three peaks two of which possessed IgG 1 and one which prossessed IgG 2 immunoelectrophoretic mobility. Although variation i the E 280nm profiles was oberved after electrofocussing of normal sera it was possible to detect the three peaks containing IgG. The ratio of IgG 2 : IgG 1 in normal guinea-pig serum determined by polyacrylamide disc-gel electrophoresis, densitometry and combined with E 280nm spectroscopy was also viable. More IgG 1 than IgG 2 was present in the serum of animals stimulated by FCA. The serum of animals stimulated with FIA did not show greatly increased levels of IgG 1 . Electron micrographs of IgG 1 -containing fractions from sera stimulated with either Freund complete or incomplete adjuvant showed the presence of dispersed material. The IgG 2 -containing fractions from the same sera always showed the presence of highly aggregated material. The IgG 1 fractions from normal sera contained either dispersed or a mixture of dispersed and loosely-aggregated material. The IgG 2 component of normal guinea-pig serum always contained highly aggregated material.