Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.     

Detection of staphylococcal enterotoxin in foods.

A method has been developed for the detection of staphylococcal enterotoxin A in the boiled rice extract. The procedure utilized was the batch adsorption of enterotoxin from the cell-free culture supernatant by CG-50 ion exchange resin at pH 5.6. The enterotoxin was eluted by various concentrations of elution solution with different pH values. The lyophilized eluate was dissolved in Phosphate Buffer Saline (PBS) solution and analyzed with a quantitative double diffusion method. The desorption of enterotoxin from ion exchange resin appeared to be less effective by increasing the concentration of elution solution than by elevating the pH value of elution solution. The pH below 6.2 seemed to lose the ability to elute the enterotoxin from ion exchanger but enough to elimate non-specific extra proteins. The quantitative double diffusion method was able to detect enterotoxin in food with approximation in quantitation.     

Interaction of staphylococcal enterotoxin B with cell cultures of human embryonic intestine

Schaeffer, W. I. (Massachusetts Institute of Technology, Cambridge), J. Gabliks, and R. Calitis. Interaction of staphylococcal enterotoxin B with cell cultures of human embryonic intestine. J. Bacteriol. 91:21-26. 1966.-The cytotoxic effect of staphylococcal enterotoxin B upon human embryonic intestine cell cultures is characterized by retraction of cells from the monolayer. This is followed by clumping of the retracted cells to form clear areas in the monolayer and finally by sloughing of the clumps from the glass surface. The 50% effective dose of the toxin, determined by protein analysis of the cultures used in titration studies, was found to be between 40 and 60 mug/ml. The cytotoxic property of the enterotoxin was completely neutralized by 3.9 x 10(-5) ml of specific antitoxin per mug of toxin. The cytotoxicity was found to be slightly enhanced by 2.2 g of bicarbonate per liter of Eagle's basal medium (Earle's salt solution level), the absence of serum, the absence of penicillin and streptomycin, and the presence of 2.8 mmoles of calcium in the medium. The cytotoxicity was profoundly influenced by the age of the culture. No cytotoxicity was evident until after 2 days of growth had taken place, when the cell number was approximately 4.0 x 10(5) cells per culture.     

Studies on staphylococcal enterotoxin B. II. Production and regulation (author's transl)]

Enterotoxigenic strain of Staphylococcus aureus (ATCC 14458) was grown under various conditions with constant shaking to determine the requirements for maximum toxin production. It was evident that 3% tryptic soy broth, 3% NZ-Amine NAK + 3% casein hydrolysate, 3% NZ-Amine NAK + 1% yeast extract, and 3% NZ-Amine NAK + 1% yeast extract + 0.2% glucose are most available toxin production media. But concentration of glucose could strictly triggered the enterotoxin producing efficiency. When glucose concentration was less than 0.5%, although with higher yield, the toxin production was delayed for certain period of time. However, if glucose concentration was up to more than 0.5%, the enterotoxin production was almost inhibited. Some metabolites of glucose to elucidate the inhibitory effect have also investigated. Our results indicated that glycerol and citric acid inhibited the toxin production directly, while the inhibitory effect of lactic acid and acetic acid were due to those acidic metabolites, decreased the pH value of media, and adversely suppressed the bacterial growth.     

Transport and processing of staphylococcal enterotoxin A.

A larger-molecular-weight precursor of enterotoxin A was found in membranes of Staphylococcus aureus and was shown to be the kinetic precursor to the extracellular form of the toxin. Subcellular fractionation revealed that mature enterotoxin A was transiently associated with the cell wall before being released to the extracellular environment.     

Immunosuppressive activity of staphylococcal enterotoxin B. II. Activation of suppressor-effector cells by a staphylococcal enterotoxin B-induced suppressor factor

The capacity of staphylococcal enterotoxins to stimulate all T cells bearing certain T cell receptors has recently generated a great deal of interest. These toxins are believed to bind directly both to the TCR:CD4 complex via its V beta domains and to class II MHC molecules on accessory cells prior to T cell activation. Previous studies from this laboratory have demonstrated that staphylococcal enterotoxin B (SEB) is capable of inducing multiple T suppressor cell populations which can inhibit in vitro antibody responses. Additional studies have demonstrated that the suppressive activity of these cells is mediated, at least in part, by an I-J-restricted suppressor factor. Efforts to characterize the inhibitory activity of this factor have demonstrated that the suppressive element is capable of activating both early and late acting suppressor cell populations in vitro. Analysis by both positive and negative selection shows that cells bearing the Lyt1-2+ surface marker phenotype are active early, whereas Lyt1+2+ cells are active both early and late in the antibody response. Additional experiments using various strains of mice as sources of suppressor factor and of naive splenocyte populations have demonstrated that activation of suppressor-effector cells by this suppressor factor is restricted at the I-J, but not Igh, gene locus. These studies suggest that this SEB-induced suppressor factor alone provides the signals necessary for the induction and activation of suppressor-effector cell activity.     

Transport and processing of staphylococcal enterotoxin B.

A larger, membrane-bound form of staphylococcal enterotoxin B was shown by in vivo pulse-chase analysis to be the kinetic precursor to extracellular enterotoxin B. Processing of the enterotoxin B precursor molecules can apparently occur either cotranslationally or posttranslationally. Subcellular fractionation of cells revealed that all of the precursor toxin was associated with the membrane fraction. Once processed and released from the membrane, it was transiently associated with the cell wall before being released into the extracellular environment. The cell-wall-associated enterotoxin B was completely resistant to protease treatment and to extraction by high- or low-salt solutions at 0 to 2 degrees C, although it could be easily released from the cell by removal of the cell wall with lysostaphin. These data imply that newly formed enterotoxin B may be temporarily sequestered in specialized regions that require cell wall integrity before being released into the extracellular environment.     

Immunosuppressive activity of staphylococcal enterotoxin B. I. Characterization of staphylococcal enterotoxin-B-induced suppressor cells.

Staphylococcal enterotoxin B (SEB) binds specifically to major histocompatibility complex class II molecules on the surface of accessory cells and stimulates virtually all T cells bearing certain, but not all, T cell-receptor V beta alleles. We have previously shown that this superantigen is a potent inducer of multiple regulatory T cell populations. In the present report we show that SEB induces a population of suppressor T cells which inhibits the generation of alloantigen-induced cytotoxic T cell activity. Using both negative- and positive-selection analysis, we found that this suppressor population is a CD4- CD8- CD5+ IL-2R+ T cell. This cell population inhibited both syngeneic and allogeneic cytotoxic T lymphocyte activity, but the cell population which inhibited allogeneic CTL activity was radiation sensitive. In addition, allogeneic SEB-primed cells appeared to develop cytolytic activity as a result of the additional stimulation in the mixed-lymphocyte reaction culture. The relationship of the SEB-primed CD4- CD8- CD5+ T cells to related regulatory T cell populations is discussed.     

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

金黄色葡萄球菌超抗原肠毒素临床应用研究进展

金黄色葡萄球菌肠毒素(staphylococcal enterotoxins,SEs)是由金黄色萄萄球菌产生的一类典型超抗原(Superantigen,SAg)。与传统抗原不同,超抗原无须经抗原提呈细胞(Antigen presenting cell,APC)加工处理,可直接与主要组织相容性复合体II类分子(Major histocompatibility complex class II molecules,MHC II)及T细胞受体Vβ区(Variable pacts of the T cells receptor,TCR Vβ)特异性结合,极低浓度即可刺激大量T细胞增殖,产生大量有生物学活性的细胞因子(如TNF-α、TNF-β、IFN-γ、IL-2等)和抗肿瘤效应。本文综述了近年来超抗原金葡球菌肠毒素在恶性肿瘤的临床应用、存在问题和解决策略等的进展,针对Trousseau综合征利用超抗原开发新药进行了展望,并介绍了SEC2在临床用于骨病治疗的情况。