Approximate dimensions of membrane lesions produced by streptolysin S and streptolysin O

Membrane lesions produced by the streptococcal membranolysins streptolysin S and streptolysin O were investigated. Escape of labeled marker molecules of various sizes from resealed sheep erythrocyte ghosts treated with the toxins for 30 min allowed estimation of the sizes of the primary channels formed. Streptolysin S formed lesions ranging in size up to 45 A in diameter, and even high toxin concentrations did not result in larger channels. The lesions produced by streptolysin O exceeded 128 A in diameter. Kinetics experiments demonstrated that the primary streptolysin O lesions were formed rapidly (1-2 min), but release of marker molecules from streptolysin S-treated vesicles began only after a 5-15-min lag period. Label release from large unilamellar liposomes treated with streptolysin S suggested that membrane fluidity does not affect the size of the streptolysin S lesions.     

Sterol structural requirements for inhibition of streptolysin O activity

Reduced streptolysin O, a toxin produced by certain beta-haemolytic streptococci, lyses human erythrocytes. The reaction is inhibited by cholesterol at concentrations of about 1.0mug/ml. Other sterols inhibit the lysin and there is a specific requirement for a 3beta-hydroxyl group. Inhibition was obtained with 3beta-hydroxychol-5-en-24-oic acid, containing a hydrophilic group at C-24. The mode of inhibition is likely to involve attachment to the fixation site of the lysin which attaches the molecule to cell membranes, probably to membrane cholesterol. A second streptolysin site, concerned in the final haemolytic event, may also be involved. Inhibitors of the latter site have not been characterized, other than antibody with specificity for the site.     

Klebsiella pneumoniae haemolysin adsorption to red blood cells

It was necessary to incubate the Klebsiella pneumoniae haemolysin with erythrocytes at 37 degrees C to produce the whole lytic action. The amount of attached klebolysin at 4 degrees C increased as its concentration in the medium increased, until the erythrocyte surface was saturated. Treatment with 2-mercaptoethanol was necessary to permit the adsorption; it was inhibited by low concentrations of cholesterol. Klebsolysin was immunogenic and its antiserum neutralized its own haemolytic effect and streptolysin O. Anti-streptolysin serum also neutralized klebsolysin. Streptolysin O attachment to erythrocytes impeded the posterior klebolysin adsorption in the same way that klebsolysin adsorption interfered with streptolysin O attachment.     

Klebsiella pneumoniae haemolysin adsorption to red blood cells

It was necessary to incubate the Klebsiella pneumoniae haemolysin with erythrocytes at 37°C to produce the whole lytic action. The amount of attached klebolysin at 4°C increased as its concentration in the medium increased, until the erythrocyte surface was saturated. Treatment with 2-mercaptoethanol was necessary to permit the adsorption; it was inhibited by low concentrations of cholesterol. Klebsolysin was immunogenic and its antiserum neutralized its own haemolytic effect and streptolysin O. Anti-streptolysin serum also neutralized klebsolysin. Streptolysin O attachment to erythrocytes impeded the posterior klebolysin adsorption in the same way that klebsolysin adsorption interfered with streptolysin O attachment.     

Biochemical and ultrastructural study of the disruption of blood platelets by streptolysin O

The membrane-damaging protein toxin, streptolysin O, proved highly lytic on human, guinea-pig and rabbit platelets. About 15 molecules of toxin were sufficient to lyse one cell. Platelet disruption was assessed by electron microscopy, clearing of cell suspensions and assay of lactate dehydrogenase, serotonin, monoamine oxidase and glutathione peroxidase released in the extracellular fluid. This egress reflected the damage of both plasmic and organelle membranes. A quantitative study of lactate dehydrogenase and serotonin liberation taken as respective markers of the cytosol and dense bodies was undertaken as a function of toxin concentration. No platelet aggregation or shape change was elicited by streptolysin O. The ghosts resulting from platelet lysis retained properties of the native membrane such as aggregability and serotonin uptake. Dense bodies were easily separated after gentle disruption of the plasmic membrane by small amounts of toxin. Platelet lysis by streptolysin O proved a useful procedure for the determination of protein content, enzyme activities and serotonin assay on the same lysate in contrast to usual methods.     

Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin.

Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by microM concentrations of free Ca2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.     

Rat pancreatic acini permeabilised with streptolysin O secrete amylase at Ca2+ concentrations in the micromolar range, when provided with ATP and GTP gamma S.

The aim of this study was to define the conditions required for exocytosis in pancreatic acini permeabilised with the bacterial toxin streptolysin O. Treatment of a suspension of acini with streptolysin O caused the release of both the cytoplasmic enzyme lactate dehydrogenase and the zymogen granule enzyme amylase. The release of amylase occurred more quickly than that of lactate dehydrogenase and was smaller in magnitude. In addition, a component of amylase release occurred only in the presence of Ca2+ (at concentrations in the micromolar range), ATP and GTP gamma S. We conclude that this component represents an exocytotic event, but that the release of lactate dehydrogenase occurs through toxin-generated lesions. The concentrations of Ca2+, ATP and GTP gamma S causing half-maximal exocytosis were 0.7 microM, 0.2 mM and 10 microM, respectively. This system should permit a study of the mechanisms underlying regulated exocytosis in this cell type.     

The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells

The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography. Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O. Purified light chain inhibited the Ca2+-stimulated [3H]noradrenaline release with a half-maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.     

Interaction of steptolysin O with sterols.

A quantitative study of the specific inhibitory power of cholesterol and other sterols on the hemolytic properties of streptolysin O is reported. This streptococcal exocellular protein is a cytolytic toxin which disrupts cytoplasmic membranes of eukaryote cells. The structural characteristics, particularly the stereochemical ones required for a steroid molecule to inhibit the cytolytic activity of streptolysin O, have been investigated in detail. By immunodiffusion techniques, in agar gel plates or tubes containing sterols, the formation of hydrophobic complexes between streptolysin O and inhibitory steroids, but not non-inhibitory steroids except lanosterol, is shown. Upon interaction with inhibitory steroids streptolysin O loses its immunoreactive properties towards neutralizing and precipitating homologous antibodies. An interpretation of the mechanism of biomembrane disorganization by streptolysin O is discussed in the light of its steroid binding properties.     

Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin

RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration.     

Enterotoxin B Synthesis by Replicating and Nonreplicating Cells of Staphylococcus aureus

Although 95% of the enterotoxin B produced by Staphylococcus aureus appears during the latter part of the exponential phase of growth, growth per se is not necessary for toxin synthesis. A procedure is described whereby a concentrated suspension (at least 6 x 10(10) cells per ml) of a 16-hr culture of S. aureus was found to be capable of producing toxin, without replication, when air and glucose were present. This technique allows the growth requirement to be separated from toxin formation. Although higher (100 mug/ml) concentrations of toxin appeared in the medium when nitrogen was present, lower levels (30 mug/ml) were produced in the absence of N-Z-amine A. Toxin production proceeded without any net increase in deoxyribonucleic acid, ribonucleic acid, or protein. Chloramphenicol did not inhibit toxin formation in a nitrogen-free medium. The optimal pH for toxin production in a nitrogen-free medium was 8.0 to 8.5; for synthesis in a medium where nitrogen was available, the optimal pH was 7.0 to 7.5. Increasing the rate of aeration increased toxin release during growth, but decreased the amount of toxin subsequently produced when the bacteria were resuspended. These results suggest the presence of a precursor pool in the cells collected after 16 hr of growth.     

Molecular cloning and characterization of Bacillus alvei thiol-dependent cytolytic toxin expressed in Escherichia coli

A chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (Mr 60,000, pI 5.0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64 kDa polypeptide of pI 8.2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.     

温度、盐度、光照对海洋卡盾藻生长和产毒的影响

在正交试验条件下,分析了盐度、温度、光照强度对海洋卡盾藻生长和产毒的影响.结果表明：在盐度22、33、45和温度20 ℃、25 ℃、30 ℃以及光照强度2000、3000、4500 lx条件下,三因素对海洋卡盾藻生长的影响均不显著,盐度是影响海洋卡盾藻产毒的主要因子;盐度45、温度30 ℃、光照2000 lx下海洋卡盾藻的比生长速率最大,盐度22、温度20 ℃、光照4500 lx下海洋卡盾藻的产毒能力最强;低盐条件不利于海洋卡盾藻的生长,但有利于溶血毒素的合成;当海洋卡盾藻生长受到限制时,其溶血毒素合成增多.     

The tetanus toxin light chain inhibits exocytosis

The intracellular action on exocytosis of various forms of tetanus toxin was studied using adrenal medullary chromaffin cells, the membrane barrier of which has been removed by permeabilization with streptolysin O. Such cells still release catecholamines on stimulation with calcium. The two-chain form of tetanus toxin (67 nmol/l) strongly inhibited exocytosis, but only if dithiothreitol was present as a reducing agent. Purified light chain completely prevented [3H]noradrenaline release with a half-maximal effect at about 5 nmol/l. Heavy chain (up to 11 nmol/l) and unprocessed single-chain toxin (up to 133 nmol/l) were without effect. It is concluded that the original single-chain form of tetanus toxin has to be processed by proteolysis and reduction to yield a light chain which inhibits transmitter release.     

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.     

Regulation of toxinogenesis in Corynebacterium diphtheriae. I. Mutations in bacteriophage beta that alter the effects of iron on toxin production

Diphtherial toxin is produced in maximal yields by Corynebacterium diphtheriae (C7(beta tox+) only when iron is present in growth-limiting amounts. Toxin production is markedly decreased under high-iron conditions. We studied the role of the bacteriophage beta genome in this apparent regulation of toxin production by iron. Using a passive immune hemolysis assay to detect toxin antigen production in individual plaques, we identified rare phage mutants that were toxinogenic in high-iron medium. Lysogenic derivatives of C. diphtheriae C7 harboring such phage mutants were constructed. The lysogens were compared with wild-type strain C7(beta) for their ability to produce toxin in deferrated liquid medium containing varying amounts of added iron. Quantitative tests for extracellular toxin were performed by competitive-binding radioimmunoassays. We identified phenotypically distinct mutant strains that produced slightly, moderately, or greatly increased yields of toxin antigen under high-iron conditions. The toxin produced by the mutant lysogens was biologically active and immunochemically indistinguishable from wild-type toxin. Complementation experiments demonstrated that the phage mutation designated tox-201 had a cis-dominant effect on the expression of the toxin structural gene of phage beta. The characteristics of the tox-201 mutation suggest that it defines a regulatory locus of phage beta that is involved in control of toxinogenesis by iron in C. diphtheriae.     

Influence of the interaction of temperature and water activity on the production of ochratoxin A and the growth of Aspergillus niger, Aspergillus carbonarius and Aspergillus ochraceus on coffee-based culture medium

In the present study, the effect of temperature and water activity on fungal growth and ochratoxin production on coffee-based medium was assessed. Optimal growth of three Aspergillus strains was observed in the same ecological conditions, namely 30 degrees C and 0.99 water activity. Maximal daily growth is 11.2, 6.92, and 7.22 mm/day for Aspergillus niger, Aspergillus carbonarius, and Aspergillus ochraceus, respectively. However, ecological conditions for optimal ochratoxin production vary according to the toxinogenic strain, with water activity as a limiting factor. Such an ochratoxin A production is inhibited at 42 degrees C and 0.75 water activity. Correspondence between laboratory tested water activity and that measured on a sun-dried ripe cherry batch shows that the first 5 days of drying are critical for fungal growth and ochratoxin A production. Accordingly, artificial drying of cherries at temperatures above 42 degrees C will impede not only fungal growth but also contamination with ochratoxin A.     

In vitro inhibition of diphtheria toxin action by ammonium salts and amines

Kim, K. (University of Washington, Seattle), and N. B. Groman. In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J. Bacteriol. 90:1552-1556. 1965.-An inhibitor for diphtheria toxin action on HeLa cells was demonstrated in the growth supernatant fractions of both toxinogenic and nontoxinogenic strains of Corynebacterium diphtheriae and in the Mueller and Miller medium in which these organisms were grown. The inhibitor in the growth supernatant fractions of the nontoxinogenic strain was dialyzable, stable to autoclaving, and stable on storage in the refrigerator for a period of many months, but was destroyed by ashing. When the components of Mueller and Miller medium were analyzed, only the Casamino Acids proved inhibitory. Further study with artificial mixtures of amino acids revealed that glutamine alone inhibited toxin. It was subsequently shown that ammonium salts and the aliphatic amines, glycamine and prolamine, could also function as inhibitors. Histamine and 16 amino acids tested individually were ineffective. The effectiveness of the amines and the ineffectiveness of sodium or potassium ions indicates that there is a specific requirement for inhibition.