Interaction of Staphylococcal α-Toxin with Artificial and Natural Membranes

Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal alpha-toxin indicated the presence of a lytic factor other than alpha-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified alpha-toxin (HP alpha-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure alpha 12S-toxin. The interaction of HP alpha-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The alpha 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An alpha 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the alpha 12S molecule. Lipid monolayer experiments showed that HP alpha-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP alpha-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to alpha-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to alpha 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.     

Purification and Properties of Staphylococcal δ-Hemolysin I. Production of δ-Hemolysin

Concentrated preparations of staphylococcal delta-hemolysin were obtained by growing selected hemolytic colonies from the 146P strain of Staphylococcus aureus on dialysis membranes laid over Brain Liver Heart agar plates at 37 C for 20 hr under 10% CO(2) and harvesting the growth from five such membranes in 1.0 ml of deionized distilled water. Incubation in a humid environment facilitated this harvesting procedure. Incubation longer than 40 hr or incubation under CO(2) higher than 10 to 20% gave lower yields of delta-lysin. Addition of a sugar, fermented by the organisms, resulted in lower yields of delta-hemolysin. Agar, although separated from the growing cells by the dialysis membrane, did potentiate delta-hemolysin production. Addition of 0.1% agar to the inoculum further enhanced this potentiation. delta-Hemolysin produced in broth or semisolid cultures was excessively diluted with the media. Dialysis membranes prevented this dilution and thus yielded concentrated preparations of delta-hemolysin.     

Platelet Damaging Factor, a Fifth Activity of Staphylococcal α-Toxin

Crude and purified staphylococcal alpha-toxin were used to demonstrate that the platelet-damaging effect of crude alpha-toxin represents a fifth activity of the alpha-toxin molecule. The homogeneity of the purified toxin employed was demonstrated by ultracentrifugation, Ouchterlony, and immunoelectrophoretic methods. Continuous-flow electrophoretic migration studies demonstrated under a variety of conditions that the platelet-damaging and the alpha-hemolytic activities migrated as a unit. Fractionation studies with the use of Sephadex G-100, carboxymethyl cellulose, and diethylaminoethyl cellulose failed to separate these two activities. Further, when alpha-toxin of demonstrated purity and crude toxin were adjusted to the same hemolytic activity, they possessed the same platelet-damaging activity. In addition, heat-reactivation studies with crude alpha-toxin revealed that the platelet-damaging effect was inactivated and reactivated in parallel with alpha-hemolytic activity. Comparable studies with purified alpha-toxin showed parallel inactivation of both activities at 60 C. Additional heating at 100 C failed to reactivate either activity. Electron micrographs revealed that purified alpha-toxin produced distinct degenerative changes in rabbit platelets. These studies also provided definite evidence that purified alpha-toxin has a damaging effect on human platelets. Monovalent alpha-antisera prevented platelet damage.     

Biochemical and genetic studies of plasma and leukocyte α-L-fucosidase

Deficiency of alpha-L-fucosidase in plasma and leukocytes has been demonstrated in three patients affected with fucosidosis. The measurement of plasma fucosidase activity alone is of little diagnostic value. Several normal individuals were found to have extremely low plasma alpha-L-fucosidase activity, but normal activity in leukocyte preparations. The low plasma enzyme activity exhibited by clinically normal individuals appears to be an inherited characteristic. The plasma enzyme was found to be different from that of leukocytes in terms of electrophoretic mobility.     

Relation of In Vitro Inhibition by Chelates of Clostridium perfringens α-Toxin to Their Ability to Protect Against Experimental Toxemia

The inhibition of Clostridium perfringens alpha-toxin by ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentacetate (DTPA) was studied utilizing three different in vitro assay procedures: diffusion on egg yolk-agar, disintegration of muscle sections, and manometric assay with partially purified lecithin as substrate. DTPA was 10 to 20 times more efficient as an inhibitor than EDTA in systems containing relatively large amounts of calcium; these observations were similar to those observed in previous in vivo protection studies. A number of other chelating agents were tested for their ability to inhibit alpha-toxin in vitro and protect mice against it; the chelating agents which were the most efficient in vitro inhibitors had the greatest in vivo protective ability.     

Inhibition studies of bacterial α-carbonic anhydrases with phenols

The α-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) were investigated for their inhibition by a panel of phenols and phenolic acids. Mono-, di- and tri-substituted phenols incorporating additional hydroxyl/hydroxymethyl, amino, acetamido, carboxyl, halogeno and carboxyethenyl moieties were included in the study. The best NgCAα inhibitrs were phenol, 3-aminophenol, 4-hydroxy-benzylalcohol, 3-amino-4-chlorophenol and paracetamol, with K_I values of 0.6–1.7 µM. The most effective VchCAα inhibitrs were phenol, 3-amino-4-chlorophenol and 4-hydroxy-benzyl-alcohol, with K_I values of 0.7–1.2 µM. Small changes in the phenol scaffold led to drastic effects on the bacterial CA inhibitory activity. This class of underinvestigated bacterial CA inhibitors may thus lead to effective compounds for fighting drug resistant bacteria.     

Lytic Action of β(1-3)-Glucanase on Yeast Cells

Candida utilis, Saccharomyces cerevisiae, S. fragilis, Pichia polymorpha, and Hansenula anomala yeast cells, harvested in the early logarithmic phase, were attacked with purified beta(1-3)-glucanase from Micromonospora chalcea, which resulted in the liberation of protoplasts. The treated cells were observed under the electron microscope before the protoplasts were liberated. Differences in the cell walls of the enzyme-treated and untreated cells were observed. The action of the glucanase was also tested against isolated walls of C. utilis. The enzyme attacked the S. cerevisiae cell wall in a uniform manner. The attack on S. fragilis was located in certain zones of the cell wall, where breakage occurred and through which the protoplast emerged. On the other three yeasts, an intermediate attack was observed, not as definitely located as in S. fragilis, yet less uniformly than in S. cerevisiae.     

Purification and Characterization of Yeast β-Glucosidases

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.     

β-Galactosidase of Propionibacterium shermanii

Ten strains of Propionibacterium shermanii were tested for beta-galactosidase (beta-gal) activity. Of these ten strains, five yielded enhanced enzyme activity when cell suspensions were treated with toluene-acetone; on solvent treatment, the remaining five lost a considerable portion of the activity found in whole-cell suspensions. By using a strain yielding decreased activity upon solvent treatment, explanations for the loss in activity were sought through assays for possible alternative beta-galactoside utilization mechanisms. When this strain was assayed for beta-D-phosphogalactoside galactohydrolase by using orthonitrophenyl-beta-D-galactopyranoside-6-P04 as a substrate, the activity was wither lower or indiffernt as compared with beta-gal activity determined simultaneously. Cell suspensions of P. shermanii 7 and 22 (strains chosen for further work) grown separately on the individual substrates (lactose, glucose, galactose, and sodium lactate) did not show significant differences in beta-gal activity. Optimal temperature for beta-gal activity in untreated and toluene-acetone-treated cell suspensions of strain 7 was 52 C. With strain 22, of the temperatures tested, maximal activity in untreated cell suspensions was noted at 58 C and with solvent-treated cells at 32 C. In the cell-free extract (CFE) system, both strains exhibited maximal activity at 52 C. Optimal pH for untreated and solvent-treated cell suspensions of both strains was around 7.5. In the P. shermanii 22 CFE system, maximal activity occurred at pH 7.0; pH had very little effect on enzyme activity in P. shermanii 7 CFE. Sodium or potassium phosphate buffers in the assay system yielded the best activity. In the CFE system of these two strains, Mn2+ was definitely stimulatory, but in untreated and solvent-treated cell systems of these strains presence or absence of Mn2+ in the assay system had variable effects on enzyme activity. Maximal beta-gal activity was noted in P. shermanii 7 cells harvested after 28 h of growth at 32 C in sodium lactate broth. Sulfhydryl-group blocking agents inhibited enzyme activity in P. shermanii 22 CFE; the inhibition was partly reversed by dithiothreitol.     

Coumarins effectively inhibit bacterial α-carbonic anhydrases

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with K_Is in the range of 28.6–469.5 µM, whereas VchCAα with K_Is in the range of 39.8–438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with K_Is of 137–948.9 µM for hCA I and of 296.5–961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.     

NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival

The α_vβ₃ integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β₃ integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β₁-integrin ligand) did not induce NF-κB activity. The α_vβ₃ integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β₃ integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in α_vβ₃ integrin-mediated endothelial cell survival.     

Bile Salt 3α- and 12α-Hydroxysteroid Dehydrogenases from Eubacterium lentum and Related Organisms

Thirty-two strains of Eubacterium lentum and phenotypically similar anaerobic gram-positive bacilli were screened for intracellular bile salt 3α- and 12α-hydroxysteroid dehydrogenase (HSDHase) activities. These organisms were categorized into four groups: (A) those containing 12α-HSDHase only (10 strains), (B) those containing 3α- and 12α-HSDHase (13 strains), (C) those containing 3α-HSDHase only (2 strains), and (D) those devoid of any measurable HSDHase activity (7 strains). Of the respective four groups, 9/10, 13/13, 0/2, and 0/7 were like the neotype strain of E. lentum (ATCC 25559) in that they produced H₂S in a triple sugar iron agar butt, reduced nitrate to nitrite, and weakly decomposed hydrogen peroxide. The other strains were variable for nitrate reduction and activity on hydrogen peroxide, but all the organisms in the first three categories (with one exception) were H₂S producers (triple sugar iron agar butt) and all (with one exception) were designated E. lentum, whereas the organisms of category B were non-H₂S producers (triple sugar iron agar butt). Five of these seven were not stimulated by arginine and are designated “phenotypically similar organisms.” Thin-layer chromatography of extracted spent bacterial medium of four representative strains from each group grown in the presence of cholate revealed the presence of (A) 12-oxo product, (B) 12-oxo and 3-oxo products, (C) 3-oxo product, and (D) the absence of any of these products. The 12α-HSDHase of category B organisms was unstable unless 10⁻³ M dithioerythritol was added to the buffer. With the exception of 3 out of 32 strains, there was a positive correlation between the production of measurable amounts of 12α-HSDHase and H₂S production. Growth curves and the effect of arginine on growth and the production of 3α- and 12α-HSDHase were examined in representative strains of categories A, B, and C. Both enzymes were shown to bind onto a nicotinamide adenine dinucleotide-Sepharose column and could be eluted by high-ionic-strength buffer, resulting in approximately 25-fold and 18-fold purification, respectively. Molecular weight estimations by Sephadex G-200 gave values of 205,000 and 125,000 for the 3α- and 12α-HSDHase, respectively. Purified 12α-HSDHase was investigated with respect to pH requirement, substrate specificity, and enzyme kinetics.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine αs1-, β-, and κ-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine α_s1-, β-, and κ-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM₁, was concluded. The action of the HP-type proteinase P₁ (also detectable in strains Wg₂, C₁₃, and TR) was established by electrophoretic methods to be directed preferentially towards β-casein. The AM₁-type proteinase P_III (also detectable in strain SK₁₁) was also able to degrade β-casein, but at the same time split α_s1- and κ-casein more extensively than did P_I. Strain FD₂₇ exhibited mainly P_I activity but also detectable P_III degradation characteristics. The cell wall proteinase preparation of strain E₈ showed low P_I as well as low P_III activity. All proteinase preparations produced from κ-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P_I and P_III in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-¹⁴C-labeled β-casein and by the effect of α_s1- plus κ-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C₁₈) to the C₁₀ precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents.     

Accumulation of Poly (β-Hydroxybutyrate) by Halobacteria

Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas fragi, Escherichia coli, and Salmonella "anatum" were challenged with butylated hydroxyanisole (BHA). Susceptibility was measured as the concentration of BHA required to cause a 90% reduction in bacterial survivors. Staphylococcus aureus LP and P. fragi were two of the most resistant species examined; C. perfringens and P. fluorescens were the most susceptible. Gram stain reaction was found not to be a strict indicator of bacterial susceptibility to BHA. There was no obvious relationship between individual fatty acids and susceptibility. The ratio of saturated to unsaturated fatty acids in the total lipid fraction of only the gram-positive species was related to susceptibility. The ratios of saturated to unsaturated fatty acids of other fractions were not related to susceptibility.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled beta-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.     

Production of Epoxides from α,β-Halohydrins by Flavobacterium sp

The relative activity of Flavobacterium whole cells on the enzymatic synthesis of epoxides from α,β-chlorohydrins, -bromohydrins, and -iodohydrins is described.     

Activation of β-Glucan Synthases by Wall-Bound Purple Acid Phosphatase in Tobacco Cells

Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of β-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.