Staphylococcal Beta-hemolysin Research Articles

Inhibition of Endothelial Interleukin-8 Production and Neutrophil Transmigration byStaphylococcus aureusBeta-Hemolysin

Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-alpha). Migration of neutrophils across TNF-alpha-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response.

Identification of nonlipophilic corynebacteria isolated from dairy cows with mastitis.

Nonlipophilic corynebacteria associated with clinical and subclinical mastitis in dairy cows were found to belong to four species: Corynebacterium amycolatum, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, and Corynebacterium minutissimum. These species may easily be confused. However, clear-cut differences between C. ulcerans and C. pseudotuberculosis were found in their acid production from maltotriose and ethylene glycol, susceptibility to vibriostatic agent O129, and alkaline phosphatase. Absence of growth at 20 degrees C and lack of alpha-glucosidase and 4MU-alpha-D-glycoside hydrolysis activity differentiated C. amycolatum from C. pseudotuberculosis and C. ulcerans. The mastitis C. pseudotuberculosis strains differed from the biovar equi and ovis reference strains and from caprine field strains in their colony morphologies and in their reduced inhibitory activity on staphylococcal beta-hemolysin. C. amycolatum was the most frequently isolated nonlipophilic corynebacterium.

Cooperative Haemolysis between Weakly Beta-haemolytic Human Intestinal Spirochaetes and Staphylococcus aureus

Weakly beta-haemolytic spirochaetes related to human intestinal spirochaetosis produced a cooperative haemolysis together with S. aureus consisting of an enhanced haemolysis in the zone of the spirochaetal growth which was overlapped by the zone of activity of the staphylococcal beta-haemolysin. The cooperative haemolysis was observed in sheep blood agar media when the concentration of spirochaetes ranged from 1.5 x 10(3) to 1.5 x 10(8) CFU/ml and the concentration of S. aureus from 4 x 10(3) to 4 x 10(8) CFU/ml. With the increase of the distance between the streaks of the spirochaetes and S. aureus from 3 to 10 mm, the period of incubation needed to observe the cooperative haemolysis also increased from 18 to 72 hours. When the spirochaetes and S. aureus were streaked at the same time and when S. aureus was streaked earlier than the spirochaetes, the phenomenon was observed after anaerobic incubation of the plates for 24-72 hours but not after incubation in 10% CO2 under atmospheric conditions. A cooperative haemolysis was also observed between S. aureus and spirochaetes related to the porcine and avian intestinal spirochaetosis and the spirochaete causing swine dysentery when the same experimental conditions were used which allowed an observation of the phenomenon involving human spirochaetes and S. aureus.

Antigenicity of Dermatophilus congolensis hemolysin.

The separated cell-free form of hemolytic exosubstance was obtained from five strains of Dermatophilus congolensis. Three strains produced exosubstance with high activity, two strains produced exosubstance with lower intensity of activity. The separated forms exhibited the same hemolytic interactions as the native forms produced by growing strains, namely the antagonism with staphylococcal beta hemolysin and the synergism with staphylococcal delta hemolysin, streptococcal CAMP factor and rhodococcal equi factor. Rabbit sera obtained after intravenous or intraperitoneal application of the separated forms contained precipitation and neutralization antibodies. Cross tests of precipitation and neutralization proved antigen identity of hemolysins of different D. congolensis, strains which makes the serodiagnostics of this species possible.

Exotoxic activities ofCorynebacterium pseudotuberculosis

Several methods were used to investigate the relationships between staphylococcal beta-hemolysin inhibitory (BHI) activity, phospholipase D, dermonecrosis, and lethality, which have all been used as indicators of exotoxin in culture filtrate ofCorynebacterium pseudotuberculosis. Culture filtrate was subjected to treatment with formalin, heat, and fractionation by (NH4)2SO4 precipitation, ultrafiltration, and Sephadex G-100 chromatography. Formalin treatment of culture filtrate resulted in loss of phospholipase D and dermonecrotic activities, but no decrease in BHI activity. Culture filtrate and formalin-treated culture filtrate blocked complement-mediated immune hemolysis. Heat treatment of culture filtrate destroyed phospholipase-D activity and reduced BHI activity. Phospholipase-D and dermonecrotic activities eluted from Sephadex G-100 as two distinct adjacent peaks preceding the main protein peak. Maximum lethal activity did not correspond to phospholipase D, but was closely associated with dermonecrotic and maximum BHI activity. Dermonecrotic and BHI, but not phospholipase-D activities were detected in a 1000-dalton filtrate of culture filtrate.

Biochemical markers of the penicillin-induced L phase of a group B, type III Streptococcus sp

The penicillin-induced L phase of growth of the group B, type III streptococcal strain 76-043 was examined for biochemical properties used for the identification of group B streptococci. After numerous serial subcultures in the cell wall-defective state, this stable L phase continued to produce hemolysin, hippuricase, and CAMP factor in addition to the group- and type-specific antigens. Hemolysin production by the L-phase cells was observed on solid and in liquid media containing sheep erythrocytes. Washed whole L-phase cells hydrolyzed hippuric acid. CAMP factor was detected by the characteristic hemolysis produced on blood agar by L-phase cells or filtered culture supernatants. CAMP factor activity was quantitated by an agar well diffusion system and a macrotube assay with partially purified preparations of CAMP factor and staphylococcal beta-hemolysin. Hyperimmune cow serum neutralized the CAMP activity of the L phase and parent bacterial phase to the same degree, suggesting identity of the CAMP factors. Production of hemolysin, hippuricase, and CAMP factor confirmed the bacterial origin of this L phase. Assay for these biological markers could be used to identify L-phase organisms derived from group B streptococci.

The exotoxins of Corynebacterium ulcerans.

The exotoxins produced by ten strains of C. ulcerans (two human, six bovine and two equine) have been studied. On the criteria of toxin-antitoxin neutralisation and immunoprecipitation tests using highly specific diphtheria and C. ovis antitoxins with crude toxic filtrates, (NH4)2SO4 concentrates, and partially purified chromatographic preparations of these, together with the presence or absence of inhibition of the action of staphylococcal beta-haemolysin, and the reaction produced when injected intradermally into rabbits, two toxins could be identified, namely diphtheria toxin and C. ovis toxin. There was no evidence for the production of a third toxin specific for C. ulcerans. Five strains produced both diphtheria and C. ovis toxins. In four diphtheria toxin predominated, but in the fifth C. ovis toxin predominated. Two strains produced only diphtheria toxin and two only C. ovis toxin, though there was good but not complete evidence that a third strain (Revell) also fell into this latter group. Considerable variation occurred in the concentration of each toxin and, where both were present, in the proportion of each.

Modified Christie-Atkins-Munch-Petersen (CAMP) test for direct identification of hemolytic and nonhemolytic group B streptococci on primary plating.

A primary plating medium, using sheep blood agar with preformed zones of cell-free staphylococcal beta-hemolysin, was used for direct identification of both hemolytic and nonhemolytic group B streptococci by the direct elicitation of the Christie-Atkins-Munch-Petersen (CAMP) reaction. A positive CAMP reaction consisted of a semilunar-shaped area or circle of complete hemolysis of sheep erythrocytes and occurred only when colonies of group B streptococci developed along the periphery of the preformed zone of staphylococcal beta-hemolysin. Of 104 simulated specimens containing group B streptococci in combination with a variety of microbial species and of 85 vaginal specimens examined, direct recognition of group B streptococci was possible in 64 (61.5%) simulated specimens and in 12 (100%) vaginal cultures. Group B streptococcal colonies not developing along the active edge of staphylococcal beta-hemolysin were still identified by subculture. Of particular value was the facilitated recognition of colonies of nonhemolytic group B streptococcal strains and of hemolytic group B streptococci few in number or overgrown by other microbial species.

Purification and partial characterization of the exotoxin of Corynebacterium ovis

1. The toxin from Corynebacterium ovis, a phospholipase D (sphingomyelin phosphodiesterase D) that acts on 2-lysophosphatidylcholine and sphingomyelins, was purified by about 400-fold to homogeneity as judged by several criteria. [The EC number of the toxin (EC 3.1.4.41) has been allotted by the Nomenclature Committee of IUB, but has not yet been published.] 2. A new assay method performed in vitro, based on inhibition by the toxin of erythrocyte lysis by staphylococcal beta-haemolysin, was developed to facilitate the purification. 3. The toxin was found to be a basic (pI9.1) glycoprotein of mol.wt. 14,500 +/- 1,000. 4. The amino acid composition of the toxin was highly reminiscent of that of collagen, since it contained hydroxyproline, hydroxylysine and a high proportion of glycine, but preliminary tests showed no other similarities to collagen or proteins with similar compositions.

Effect of microwave radiation on cells treated with membrane-injuring substances

WISH cells grown in vitro were pretreated with subcytotoxic concentrations of digitonin, cortisol and purified bacterial toxins -- staphylococcal beta-haemolysin or Clostridium perfringens alpha-toxin and irradiated with 3 GHz electromagnetic wave (microwaves) at the field power densities 5 or 40 mW/cm2. At 40 mW/cm2 increase in temperature of the culture medium of about 2-3 degrees C was noted, while at 5 mW/cm2 no detectable increase in temperature was found. Control and pretreated WISH cells after irradiation in the microwave field were used for evaluation of their viability, incorporation of tritiated thymidine, glycine and uridine and level of intracellular cyclic AMP. Irradiation with microwaves resulted in lowering of thymidine and glycine incorporation along with changes in the intracellular amount of cAMP (decrease in cells exposed to 5 mW/cm2 and increase in those exposed to 40 mW/cm2). Under both conditions viability of the cultures was normal. Pretreatment of cells with digitonin or purified bacterial toxins followed by irradiation with microwaves resulted in enhancement of the cytotoxic effect with lowering of cell viability, especially after exposition to power density of 40 mW/cm2. Cortisol led to decrease in 3H-glycine and 3H-uridine incorporation into WISH cells, but did not influence the reaction of the cells to microwave radiation. In view of the results presented it may be concluded that substances injuring cell membranes sensitize cell cultures to electromagnetic radiation of the microwave range and may enhance the specific (non-thermal) effect of microwaves.

CAMP factor of group B streptococci: production, assay, and neutralization by sera from immunized rabbits and experimentally infected cows.

Quantitative assay methods for CAMP factor activity of group B streptococci and for neutralization of this activity by animal sera are described. The course of the CAMP reaction is influenced by preincubation of red cells with staphylococcal beta-hemolysin and the rate of the reaction by the concentration of CAMP factor. The production of CAMP factor is enhanced by substitution of maltose for glucose in culture broth. Rising inhibitory titers to CAMP activity in sera of rabbits injected with sterile culture supernates of group B streptococci suggest that CAMP factor is an antigen in the rabbit. Cows with experimentally induced group B streptococcal mastitis also demonstrated significant rises in neutralizing titers for CAMP activity.

A chemically defined medium for the production of staphylococcal beta hemolysin.

A synthetic medium, containing L-proline, glycine, L-arginine, L-cystine, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, nicotinamide, thiamine, glucose, sodium citrate, Na2HPO4, KH2PO4, (NH4)2SO4, MgSO4∙7H2O, FeSO4∙7H2O, and CaCl2∙2H2O, was developed. It supported growth and beta hemolysin production by the 681C strain of Staphylococcus aureus when the culture was incubated under carbon dioxide. L-Threonine, L-tyrosine, i-inositol, folic acid, riboflavin, pyridoxal HCl, choline Cl, and D-Ca pantothenate were added for obtaining good growth and toxin formation under air. L-Proline, L-glutamine, and L-cystine were the absolute requirements for the production of beta hemolysin under carbon dioxide. Carbon dioxide stimulated the production of beta hemolysin, but invariably resulted in lower cell yield.

Purification and characterization of Corynebacteruum ovis exotoxin

A scheme for the purification of Corynebacterium ovis toxin by fractional precipitation with ammonium sulphate has been described. Based on four experiments a mean yield of 219·1 per cent. of the original activity was obtained. The mean concentration effected was 96-fold. Expressing the activity of the toxin per mg. of non-dialysable nitrogen, the mean purification value achieved was 361·3 per cent. The dermo-necrotic and staphylococcal beta-haemolysin inhibition activities of C. ovis toxin behaved identically to precipitation by ammonium sulphate, inactivation with formaldehyde and heat, neutralization by antitoxin and fractionation by Sephadex G-100. These two activities are considered to be the manifestation of the same protein moiety. The toxin appears to be composed of two components, one a stable high molecular weight and the other an unstable low molecular weight protein.

Purification and Properties of Staphylococcal Delta Hemolysin

Large amounts (200 mg per liter of culture supernatant fluid) of highly purified staphylococcal soluble delta hemolysin were obtained by adsorption to and selective elution from hydroxyapatite followed by exhaustive dialysis against water, concentration by polyvinylpyrrolidone or polyethylene glycol 20,000 dialysis, and a final water dialysis. No carbohydrate, phosphorus, or inactive 280-nm absorbing material was detected in the preparation; however, analysis by density gradient centrifugation, gel filtration, analytical ultracentrifugation, carboxymethyl cellulose chromatography, polyacrylamide disc gel electrophoresis, isoelectric focusing, and electron microscopy revealed that the lysin was molecularly heterogeneous. The preparation contained an acidic fibrous lysin (S(20,w) of 11.9) and a basic lysin component composed of a population of granular aggregates of various sizes, with a maximum S(20,w) of approximately 4.9. No other staphylococcal products were detected in the preparation. The lysin was active against erythrocytes from many animal species and acted synergistically with staphylococcal beta hemolysin against sheep erythrocytes. It was soluble in chloroform-methanol (2:1), was inactivated by various phospholipids, normal sera, and proteolytic enzymes, but was partially resistant to heat inactivation. Activity was not affected by Ca(2+), Mg(2+), citrate, ethylenediaminetetraacetic acid, or cysteine. The lysin preparation also disrupted bacterial protoplasts and spheroplasts, erythrocyte membranes, lysosomes, and lipid spherules, was growth-inhibitory for certain bacteria, and clarified egg yolk-agar. Large amounts produced dermonecrosis in rabbits and guinea pigs. The minimum lethal intravenous dose for mice and guinea pigs was approximately 110 and 30 mg/kg, respectively.

Purification and properties of staphylococcal beta hemolysin. II. Purification of beta hemolysin.

Staphylococcal beta hemolysin from the 681 strain of Staphylococcus aureus grown in a Heart Infusion dialysate semisolid medium under 10% carbon dioxide was obtained in an immunoelectrophoretically pure form by a combination of procedures of precipitation with 2 volumes of acetone followed by chromatography on diethylaminoethyl cellulose at pH 6.0. The acetone precipitation procedure did not show any deleterious effect on the hemolytic activity of the beta hemolysin unless the precipitate was left in contact with the acetone for at least 4 hr. The crude preparations contained two types of beta hemolysin. One of these represented the major portion of the total activity of beta hemolysin and behaved as a cation. The other represented a minor (1/5,000) portion of the total beta hemolysin activity and behaved as an anion. These active principles were designated as cationic and anionic beta hemolysins, respectively. An unexpected increase in the total beta hemolysin activity of the crude preparations was noted when these were concentrated by dialysis against polyethylene glycol (20 m). This effect was probably due to polyethylene glycol. A further unexpected increase in the titer of the acetone-precipitated preparations occurred when these were lyophilized. The reason for this incremental increase is not known. It may be due to fragmentation of the beta hemolysin.

The nature of staphylococcal beta hemolysin. II. Effect on mammalian cells.

Purified beta hemolysin A was toxic to human amnion and monkey kidney and KB cells on the basis of trypan blue uptake, absence of pH changes in the medium, and inability of the cells to adhere to glass surfaces. Cells treated with the hemolysin were smaller than controls with granular cytoplasm and prominent nuclei. Rabbit and sheep leucocytes were unable to reduce the dye dichlorophenol–indophenol in the presence of the hemolysin but their ability to take up bacteria seemed unimpaired. Morphological changes in other mammalian cells were not found in leucocytes.

Saphylococcal beta-hemolysin. I. Purification of beta-hemolysin.

The purification of staphylococcal beta-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic beta-hemolysin yielded one line of precipitate when tested against the original crude beta-hemolysin. The purified cationic beta-hemolysin was stable in the lyophilized state. Crude beta-hemolysin was dermonecrotic, whereas purified cationic beta-hemolysin was not dermonecrotic even after Mg(++) activation.

The nature of staphylococcal beta hemolysin. I. Mode of action.

The mode of action of highly punned beta hemolysin derived from the R-1 strain of Staphylococcus aureus has been investigated. Sphingomyelin was absent from lipid extracts of sheep erythrocytes treated with beta hemolysin when compared to normal cells. A correlation was also established between the sphingomyelin content of other erythrocyte species and their sensitivity to beta hemolysin. Further investigations revealed that sphingomyelin is hydrolyzed to yield phosphorylcholine and N-acyl sphingosine. Thus, the mode of action of the beta hemolysin is like that of phospholipase C. Various phosphate compounds other than sphingomyelin, including RNA, glycerophosphate, phenyl-phosphate, phosphatidylethanolamine, and phosphatidylcholine were tested as substrates, but virtually no hydrolysis was observed. In contrast with the results of other workers, R-1 beta hemolysin did not release detectable amounts of carbohydrate from staphylococcal cell walls.

PURIFICATION AND PROPERTIES OF STAPHYLOCOCCAL BETA-HEMOLYSIN. I. PRODUCTION OF BETA-HEMOLYSIN.

Haque, Riaz-ul (Ohio State University, Columbus), and Jack N. Baldwin. Purification and properties of staphylococcal beta-hemolysin. I. Production of beta-hemolysin. J. Bacteriol. 88:1304-1309. 1964.-Highest activity of beta-hemolysin was observed when buffered saline (pH 7.0) containing 0.001 m magnesium sulfate was used as a diluent, and the tubes were incubated at 37 C for 80 min and then refrigerated for 30 min. Either Heart Infusion semisolid agar or a dialysate of Heart Infusion containing 0.3% agar was suitable for the production of large quantities of beta-hemolysin. The concentration of beta-hemolysin in semisolid and broth cultures was greatest after incubation for 24 hr. Continued incubation resulted in a loss of active hemolysin in broth cultures but not in semisolid agar cultures. Incubation in atmospheres containing 20% carbon dioxide greatly enhanced the production of beta-hemolysin. The presence of fermentable sugars inhibited the production of beta-hemolysin. Highest yields of beta-hemolysin were obtained when the initial pH of the medium was 5.5 to 5.8.