Regulation of Staphylococcal Enterotoxin B

Several factors influenced the formation of enterotoxin B by Staphylococcus aureus strain S-6. In the standard casein hydrolysate medium, toxin was not produced in detectable quantities during exponential growth; it was produced during the post-exponential phase when total protein synthesis was arithmetic. The rate of toxin synthesis was much greater than the rate of total protein synthesis. The appearance of enterotoxin was inhibited by chloramphenicol; thus, the presence of toxin was dependent on de novo protein synthesis. When low concentrations of glucose (<0.30%) were added to the casein hydrolysate medium, growth was diauxic; glucose was completely metabolized during the first growth period. During the second growth period, enterotoxin was synthesized. In unbuffered casein hydrolysate medium containing excess glucose, toxin synthesis was completely repressed. The absence of toxin production under such conditions might be explained by the low (4.6) pH resulting from the acid end products of glucose metabolism. At pH <5.0, little or no toxin was produced. Toxin synthesis was initiated in the presence of glucose when the medium were buffered at any pH above 5.6. In such media, the differential rates of toxin synthesis, with respect to the rates of total protein synthesis, were lower than the differential rates in casein hydrolysate medium alone. Addition of glucose to a culture synthesizing toxin resulted in an immediate decrease in the differential rate without any change in pH. Thus, toxin synthesis appeared to be regulated by catabolite repression.     

Staphylococcal Enterotoxin B: Solid-Phase Radioimmunoassay

An immunoassay employing (125)I-labeled enterotoxin B and polystyrene tubes coated with specific antibody was used for assaying purified and crude enterotoxin. Antibody was adsorbed to untreated polystyrene tubes. Unlabeled enterotoxin competed with (125)I-labeled enterotoxin for antibody-combining sites. The uptake of (125)I-labeled toxin reflected the concentration of unlabeled toxin present. The test is sensitive to 1 to 5 ng of purified and crude enterotoxin B per ml, and cross-reactions with heterologous enterotoxins did not interfere with the specificity. This test possesses the combination of sensitivity and objectivity absent in current methods for assaying enterotoxin and provides a model for investigating other enterotoxin serotypes.     

Microtiter Hemagglutination-Inhibition Assay for Staphylococcal Enterotoxin B

The rapid microtiter technique was investigated as a means of facilitating the detection of staphylococcal enterotoxin B. With this technique, many samples were assayed simultaneously, and readable results were obtained in 3 hr. Other advantages of this method, in addition to speed, were the small quantity of reactants used, ease of reading, and reproducibility.     

Detection of Staphylococcal Enterotoxin B via Biomolecular Interaction Analysis Mass Spectrometry

Detection of Staphylococcus enterotoxin B (SEB) by biomolecular interaction analysis mass spectrometry (BIA/MS) is presented in this work. The BIA/MS experiments were based on a surface plasmon resonance (SPR) MS immunoassay that detects affinity-captured SEB both via SPR and by means of exact and direct mass measurement by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Experiments were performed with standard samples and food samples to assess the BIA/MS limit of detection for SEB and to set the experimental parameters for proper quantitation. Single and double SPR referencing was performed to accurately estimate the amount of the bound toxin. Reproducible detection of 1 ng of SEB per ml, corresponding to affinity capture and MS analysis of ~500 amol of SEB, was readily achieved from both the standard and mushroom samples. A certain amount of SEB degradation was indicated by the signals in the mass spectra. The combination of MS with SPR-based methods of detection creates a unique approach capable of quantifying and qualitatively analyzing protein toxins from pathogenic organisms.     

New Technique for Rapid Purification, Entrapment, and Recovery of Enterotoxin A from a Liquid Chamber by Polyacrylamide Gel Electrophoresis

Electrophoreis of enterotoxin into a middle liquid section of a polyacrylamide gel column enhances recovery for serological assay.     

Effect of Staphylococcal Enterotoxin B on the Electroencephalogram of Monkeys

A highly purified preparation of staphylococcal enterotoxin B was administered intravenously, 1 mg/kg, to rhesus monkeys. Electroencephalograms (EEG) were recorded from electrodes attached to the skin or implanted on the dura. The dose of toxin employed consistently produced a sequence of vascular collapse followed by death; in control studies, animals were bled periodically to produce a similar pattern of shock. Regardless of the time to death following administration of the enterotoxin, there were essentially no changes from base line EEG patterns until shortly before death. With the development of preterminal severe shock, there was a marked decrease in EEG wave frequency and an initial increase in amplitude. The latter diminished progressively to produce an isoelectric tracing immediately prior to death. This could be reversed for a brief period by epinephrine. An identical sequence of EEG changes was observed during the terminal period of hemorrhagic shock. It is postulated that cerebral anoxia, caused by inadequate blood flow, is the primary cause of the altered EEG patterns that accompany enterotoxin toxicity. In this respect, staphylococcal enterotoxin B produces changes apparently similar to bacterial endotoxin but distinctly different from the EEG effects reported after botulinum toxin, anthrax toxin, or rattlesnake and cobra venom.     

Thermal Inactivation of Staphylococcal Enterotoxin B in Veronal Buffer

The times and temperatures required to inactivate staphylococcal enterotoxin B were studied by use of the double-gel-diffusion technique to assay enterotoxin. Enterotoxin B (99 +% pure) was suspended in 0.04 M Veronal buffer, dispensed into borosilicate vials, and the vials were sealed and heated in an oil bath. An amount of 30 mug/ml of this toxin was reduced to less than 0.7 mug/ml in 103.0, 87.1, 70.5, 57.2, 39.1, 27.6, 16.4, and 12.0 min, respectively, at temperatures of 96, 99, 101.7, 104.4, 110, 115.6, 121, and 126.7 C. The end point for enterotoxin inactivation by gel diffusion was identical to that by intravenous injection of cats. Limited studies with crude enterotoxin B showed that the crude preparation was slightly more thermostable. The respective D values of crude and purified enterotoxin B were 64.5 and 52.3, 40.5 and 34.4, 29.7 and 23.5, 18.8 and 16.6, and 11.4 and 9.9 min at temperatures of 99, 104.4, 110, 115.6, and 121 C. The z value for purified enterotoxin B was 32.4 C. The experimental activation energy was 20,700 cal/g mole, standard enthalpy of activation at 120 C was 19,900 cal/g mole, standard entropy of activation at 120 C was -21.4 cal/g mole K, and the standard free energy of activation at 120 C was 28,200 cal/g mole.     

Modified Radioimmunoassay Determination for Staphylococcal Enterotoxin B in Foods

The sensitivity of solid-phase radioimmunoassay for the measurement of staphylococcal enterotoxin B (SEB) in foods was decreased by food constituents that react with rabbit anti-SEB with an equivalency of over 2 ng/ml. This activity was minimized by a conditioning step for anti-SEB and by removal of interfering compounds in the sample by extraction. The assay was a sequential solid-phase radioimmunoassay technique in which polystyrene test tubes were initially incubated with antisera and then with bovine serum albumin. The tubes were then conditioned with either a centrifuged aqueous cheese extract or, equally effective, reconstituted nonfat dry milk for 16 h at 4°C. Samples of milk or heat-treated and CHCl₃-extracted cheese or chicken salad slurries were incubated in the assay tubes for 6 h at 37°C. The samples were replaced by ¹²⁵I-labeled SEB and incubated for a further 2 to 4 h before the contents were removed and the tubes were washed and counted. A buffer solution containing known concentrations of toxin served as standards for assaying SEB in the food extracts. The entire assay can be accomplished within 24 h with a sensitivity of 1 ng/ml in milk and in the cheese extract or 1.3 ng/ml in the chicken salad extract.     

Regulation of Staphylococcal Enterotoxin B: Effect of Anaerobic Shock

The metabolism of glucose during enterotoxin B synthesis in Staphylococcus aureus S-6 was examined under anaerobic conditions in the presence and absence of nitrate. The repression of enterotoxin synthesis which occurs during the oxidative metabolism of glucose was relieved after a shift to anaerobic conditions; glucose was then converted primarily to lactic acid and was metabolized more rapidly, presumably to obtain the equivalent amount of energy available aerobically. A greater proportion of oxidized end products and evidently more energy per glucose molecule was produced in the presence of oxygen. Thus, available energy as judged by a change in the type and proportion of end products appears to be related to the degree of toxin repression. As expected, the addition of nitrate during anaerobic glucose metabolism prevented derepression of toxin synthesis.     

Regulation of Staphylococcal Enterotoxin B: Effect of Thiamine Starvation

During the transition between the exponential and stationary phases of growth, there was a rapid accumulation of both cell-associated and extracellular enterotoxin B. Extracellular enterotoxin was synthesized until the cells entered the stationary phase during which cell-bound toxin was not detected. The differential rate of toxin synthesis relative to that of total protein synthesis was greater at pH 7.7 than at 6.0. Addition of glucose decreased the differential rate of toxin synthesis. This decrease was greater at pH 7.7 than at 6.0. Addition of pyruvate decreased the differential rate at pH 7.7 but not at 6.0. Analysis of the nongaseous end products of glucose and pyruvate metabolism showed that conditions which favor the oxidative decarboxylation of pyruvate also favor the repression of toxin synthesis. Elimination of thiamine from the medium prevented the oxidative decarboxylation of pyruvate by Staphylococcus aureus S-6 and partially or completely reversed the repression of toxin synthesis by glucose and pyruvate. In the absence of an added energy source, thiamine starvation caused a decrease in protein synthesis but an increased differential rate of toxin synthesis which was greater at pH 7.7 than at 6.0. In the absence of thiamine, pyruvate was not metabolized but caused a decrease in the rate of protein synthesis. This resulted in a twofold increase in the differential rate of toxin synthesis. Thus, conditions which altered the oxidative decarboxylation of pyruvate or decreased the rate of protein synthesis increased the rate of enterotoxin B synthesis.     

从噬菌体表面展示肽库中筛选葡萄球菌B型肠毒素抑制剂

通过生物淘选,从噬菌体表面展示12肽肽库中筛选能与葡萄球菌B型肠毒素(staphylococcalenterotoxinB ,SEB)结合且能抑制其肠毒活性的特异性短肽.采用Phage ELISA和MTT鉴定所得目的肽的亲和性;根据优势噬菌体阳性克隆序列合成相应多肽.利用竞争ELISA研究合成肽与SEB单克隆抗体竞争结合SEB的情况;通过动物实验考察其抑制SEB的超抗原特性和肠毒活性情况.筛选所得短肽在一定浓度范围内可以抑制SEB对鼠脾淋巴细胞的激活;合成肽与SEB质量比为16 0∶1时,合成肽可较好地抑制SEB对乳猫的肠毒活性,并对SEB引起的小鼠致死具有明显保护作用.结果表明,初步得到了能与SEB特异结合并能抑制SEB超抗原特性和肠毒活性的短肽,为进一步研制SEB高效抑制剂奠定了基础.     

Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.     

Identification of a Fourth Staphylococcal Enterotoxin, Enterotoxin D

A fourth staphylococcal enterotoxin was identified serologically with antiserum to the very crude enterotoxic products of growth of a strain which also produces enterotoxin C, and then with antiserum to the considerably purified enterotoxic antigen of a strain which produces only the new enterotoxin. The identification of this antigen as enterotoxin D was based on the following observations. It was produced by strains which do not produce enterotoxins A, B, or C; it was absent in the growth products of nonenterotoxigenic strains; when appreciably purified, it was associated with emetic activity in the cat, and its biological activity was neutralized only by antisera containing its specific antibody and not by antibodies to enterotoxins A, B, and C. Staphylococcal strain 494 (ATCC 23235) was selected as the prototype strain. The production of this enterotoxin alone and together with enterotoxin A by strains of food-poisoning origin indicates that its role in food poisoning is second in frequency only to that of enterotoxin A. The incidence of production of enterotoxins A, B, C, and D, and of unidentified cat emetic substances by strains from several source categories, is presented.     

Pathogenesis of Lethal Shock After Intravenous Staphylococcal Enterotoxin B in Monkeys

The pathogenesis of shock in the rhesus monkey given intravenous staphylococcal enterotoxin B (SEB) is not understood. Several cardiovascular changes produced by a highly purified preparation of SEB were studied after administration of doses ranging from 50 to 1,000 mug/kg. Irreversible arterial hypotension was found consistently at the higher doses. Arterial blood pressure and cardiac output declined substantially as shock developed. Total peripheral vascular resistance did not rise at any time, but showed a significant fall during the late stages of shock. Portal and central venous pressures remained essentially unchanged. Venous O(2) content and pO(2) declined gradually throughout the period of toxemia, but arterial O(2) content remained constant until just prior to death, when a slight fall was noted in some monkeys. These changes were consistent with a pooling of blood in the peripheral vascular beds and seemed to resemble cardiovascular responses reported to occur in monkeys during shock due to bacterial endotoxin. Epinephrine, administered in the late stages of shock, caused arterial pressure to increase almost immediately and cardiac output to return to normal about 1 min later. Although life could occasionally be prolonged for several hours by continuous or intermittent epinephrine infusions, this therapy never succeeded in reversing the lethal effects of high doses of SEB.     

Analysis of Arbovirus Ribonucleic Acid Forms by Polyacrylamide Gel Electrophoresis

Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained by sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., the 42S, 26S, replicative form, and replicative intermediate) were very clearly separated. The high resolution of the electrophoretic method permitted the identification of two new single-stranded RNA species. In addition, the replicative form was shown to be heterogeneous and to consist of at least two forms. The results suggested that the replicative forms occur in vivo although in relatively small amounts.     

Effect of Formaldehyde on the Immunogenicity of Staphylococcal Enterotoxin B for Macaca mulatta

Monkeys were immunized with enterotoxin and enterotoxoid by intracutaneous injection or by feeding. Identical schedules were used to compare the effectiveness of the two antigens and the two routes. Enterotoxin administered intracutaneously was the most effective antigen, whereas oral administration of enterotoxoid was least effective. Intracutaneous injection of toxoid and oral feeding of toxin were intermediate and not too dissimilar in effectiveness. Antibody titers and protection persisted for at least 1 year at a relatively high level. Monkeys that had preimmunization hemagglutinins showed an anamnestic response after immunization. The development of protection and the appearance of antibodies subsequent to feeding toxin or toxoid suggest that ingestion of food contaminated by staphylococci or their metabolites may be one cause for the appearance of antitoxin in the serum of supposedly unexposed animals and man.     

Staphylococcal Enterotoxin B and Nuclease Production Under Controlled Dissolved Oxygen Conditions

Enterotoxin B, nuclease, and total exoprotein production by Staphylococcus aureus strain S-6 was studied in a 0.5-liter fermentor system. While these extracellular products were elaborated over a wide range of aeration rates, maximal production occurred within the very narrow range of 125 to 150 cm(3) of air per min. The levels attained at the optimal aeration rate were not increased by maintaining a constant pH, although yield of enterotoxin:cell mass was highest at a constant pH of 7.0. During the growth cycle of the cultures, when aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen (DO) levels, initially set at 100% of saturation, decreased to 5 to 10% 4 to 5 h after inoculation. The oxygen demand of the culture then maintained this level for an additional 4 to 6 h. This interval of low DO was characterized by maximal growth and exoprotein production. When the DO was controlled at a constant value throughout growth (by increasing or decreasing the airflow rate as appropriate), the culture demonstrated different optima for maximal growth and exoprotein production. A constant DO of 100% stimulated growth to extremely high densities, but the accumulation of toxin and nuclease was not observed. On the other hand, maintaining constant DO levels at 50 or 10% raised exoprotein levels higher than those achieved in a culture grown at the optimal aeration rate. Compared to the optimal aeration rate culture, the 10% DO culture yielded 20% more nuclease, 25% more toxin, and 40 to 50% more total exoprotein. These results indicate that it is the DO and not the aeration rate, per se, that is influential in controlling growth, toxin, nuclease, and total exoprotein production.     

Production of Antiserum for Staphylococcal Enterotoxin

Immunization of rabbits with approximately 95% pure enterotoxin B and approximately 20 and 50% pure enterotoxin A yielded specific antisera which required no and little absorption, respectively, when used in the slide double-diffusion test. Methods for purifying enterotoxin A and for the production and absorption of antisera are presented.     

Detection of Staphylococcal Enterotoxin in Foods

A short procedure for the extraction of staphylococcal enterotoxins from food materials has been developed. The procedure involves extraction of the food at pH 4.5, centrifugation, extraction of the supernatant with CHCl(3) (pH 7.5), extraction of the enterotoxin from the water layer with CG-50 ion exchange resin (pH 5.4 to 5.9), and treatment of the eluate with agar and concentration with Carbowax 20-M. The concentrate was extracted with CHCl(3), and the water layer was lyophilized. The dried material was dissolved in a 1% trypsin solution and placed on microslides, which were incubated 24 h at 37 C. The time required for enterotoxin analysis was 3 days with microslides and 1 day with the reversed passive hemagglutination technique.