Staphylococcal Toxic Shock Syndrome Toxin-1 Research Articles

An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.

Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody

Toxic shock syndrome toxin 1 (TSST-1), a 22-kilodalton protein made by strains of Staphylococcus aureus harboring the chromosomal toxin gene, may elicit toxic shock syndrome in humans. In vitro, TSST-1 induces T cells to proliferate and macrophages to secrete interleukin-1. To conduct a structure-function analysis, point mutations on the TSST-1 gene were generated by site-directed mutagenesis to identify amino acids critical for activity of the toxin. Specific tyrosine and histidine residues were replaced by alanines. Wild-type and mutant TSST-1 gene constructs were expressed in Escherichia coli, and the products were tested for their mitogenic potential and reactivity with a TSST-1 neutralizing monoclonal antibody (MAb 8-5-7). Four of the mutants were similar to the wild type; i.e., the mutant toxins stimulated murine T cells and reacted with MAb 8-5-7 equally as well as the wild type. Two mutants exhibited a decrease in mitogenic activity, but one of these retained the capacity to bind with MAb 8-5-7 while the other was no longer recognized by the same antibody. One double mutant demonstrated minimal mitogenic activity and did not react in enzyme-linked immunosorbent and immunoblot assays with MAb 8-5-7. The data show that specific residues near the carboxy terminus of TSST-1 are essential for mitogenic activity and in forming the epitope recognized by neutralizing MAb 8-5-7.

Binding of toxic shock syndrome toxin‐1 to murine major histocompatibility complex class II molecules

The staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) has potent stimulatory effects on murine and human lymphocytes. This is the consequence of TSST-1 binding to major histocompatibility complex (MHC) class II molecules and the engagement in a V beta-restricted fashion of the T cell receptor by the TSST-1-MHC class II complex. Using radioligand and functional assays we have recently shown that TSST-1 binds to all HLA-DR (n = 14), HLA-DQ (n = 2) and HLA-DP (n = 2) phenotypes tested. In this study, we have examined the ability of murine MHC class II molecules to bind TSST-1. Specific high-affinity binding of TSST-1 was detectable to unfractionated BALB-c (H-2d) and C57BL/6 (H-2b), but not to C3H (H-2k) spleen cells. The Kd of this binding estimated from Scatchard analysis was in the same nanomolar range as the Kd of binding of TSST-1 to HLA-DR. Binding of 125I-labeled TSST-1 to BALB/c-derived B cell lymphoma lines and to L cell transfectants correlated with the expression of I-A molecules, but not with the expression of I-E molecules. Furthermore, I-A+, I-E- cells but not I-A-, I-E+ cells were able to support TSST-1-induced T cell proliferation. The binding affinity of TSST-1 for I-Ak appears to be much lower than for I-Ad. L cell transfectants expressing hybrid DR alpha: I-E beta k molecules, but not those expressing I-E alpha k: DR1 beta molecules, could bind TSST-1 and efficiently support TSST-1-induced T cell proliferation. This suggests that minor differences in the highly homologous I-E alpha and DR alpha chains are critical in determining the affinity of the MHC class II molecule for TSST-1. These results demonstrate that the binding of TSST-1 to MHC class II molecules in the mouse, in contrast to humans, is strongly influenced by phenotype. Analysis of the molecular basis of these differences may help to localize staphylococcal exotoxin binding sites on MHC class II molecules.

Molecular requirements for T cell activation by the staphylococcal toxic shock syndrome toxin-1.

The activation of Ag-specific, Ia molecule-restricted, TCR V beta 3+ T cell clones by staphylococcal toxic shock syndrome toxin-1 (TSST-1), was investigated. The results show that although Ag- and TSST-1-induced activation of T cell clones both require TCR expression and similar biologic activation signals, the Ia molecule requirement for TSST-1 recognition was much less stringent than that observed for antigenic peptide recognition. In addition, T cell clones recognized TSST-1 without processing by APC. These results suggest that the ability of TSST-1 to polyclonally activate T cells is dependent on TCR recognition of the intact toxin molecule bound to a nonpolymorphic region(s) of the Ia molecule resulting in the same activation events induced by Ag recognition.

Streptococcal Toxic Shock Syndrome

Staphylococcal toxic shock syndrome toxin-1 (TSST-1) is an important factor of pathogenicity, but further unknown toxins may exist because some strains isolated from such cases dit not produce TSST-1. The mechanism of action of TSST-1 is not fully understood but induction of mediators like TNF and IL-1 seems to play an important role. Infections with TSST-1-producing Staphylococcus aureus strains do not implicate the development of a toxic shock syndrome

The staphylococcal toxic shock syndrome toxin 1 triggers B cell proliferation and differentiation via major histocompatibility complex-unrestricted cognate T/B cell interaction.

The Staphylococcus aureus exotoxin toxic shock syndrome toxin 1 (TSST-1) is a potent activator of T cells and monocytes. We have recently demonstrated that TSST-1 is a superantigen that binds monomorphic determinants on MHC class II molecules. In the present study, we have examined the effect of TSST-1 on the activation and differentiation of high density human tonsillar B cells. TSST-1 bound to tonsilar B cells with high affinity and saturation kinetics. This binding was effectively inhibited by a combination of anti-HLA-DR and anti-HLA-DQ mAbs. Treatment of purified B cells with TSST-1 failed to induce B cell proliferation or Ig production. However, in the presence of irradiated T cells, TSST-1 induced resting B cells to proliferate and differentiate into Ig secretory cells. TSST-1 mimicked nominal antigen in that its induction of B cell responses was strictly dependent on physical contact between T and B cells, and was profoundly inhibited by anti-MHC class II mAbs, anti-CD3 mAbs, and, to a lesser extent, by anti-CD18 mAbs. However, unlike nominal antigen, TSST-1-mediated T/B cell interactions were MHC unrestricted. These results suggest that TSST-1 induces T cell-dependent B cell proliferation and differentiation by virtue of its ability to mediate MHC-unrestricted cognate T/B cell interaction via the TCR/CD3 complex and MHC class II antigens.

Immunohistological localisation of staphylococcal toxic shock syndrome toxin (TSST-1) antigen in sudden infant death syndrome.

A polyclonal antiserum to toxic shock syndrome toxin (TSST-1) and a standard immunoperoxidase technique were used on formalin fixed tissues from 50 cases of sudden infant death syndrome (SIDS) to determine if the syndrome was associated with bacterial infection. There was strong specific staining in the renal tubular cells in nine (18%) cases. A similar pattern of staining was seen in three of a series of 50 kidneys selected for comparison from a wide range of necropsy cases. The staining was finely granular within the cytoplasm of proximal convoluted tubular cells and diffuse in tubular cell nuclei. In an attempt to validate the staining pattern the immunoperoxidase technique was also performed on formalin fixed kidneys from rats which had been given intravenous injections of crude bacterial products containing TSST-1. These showed coarse granular cytoplasmic staining in proximal convoluted tubules with some diffuse nuclear staining. This pattern was not seen in controls injected with saline. These results indicate that TSST-1 might have a pathogenic role in some cases of SIDS.

Mitogenic properties of two distinct forms of toxic shock syndrome toxin‐1 separated on hydroxyapatite by high‐performance liquid chromatography

The homogeneity of a purified staphylococcal toxic shock syndrome toxin-1 (TSST-1) was tested by high-performance methods. This preparation was homogenous in ion-exchange chromatography and isoelectric focusing (pI = 7.4), but was resolved into two distinct peaks by high-performance hydroxyapatite chromatography. Both components, TSST-1hA and TSST-1hB had similar molecular weights (22 kD) and amino acid compositions. TSST-1 did not dimerize or polymerize upon heating at 60 degrees C for 30 min or in solutions with pH varying from 4.0 to 8.5. TSST-1hA and TSST-1hB showed similar immunological reactivity to native TSST-1 goat polyclonal antibodies. TSST-1hA and TSST-1hB as well as staphylococcal enterotoxin A and staphylococcal exfoliative toxin were potent mitogens in lymphocyte proliferation assays. The lymphocyte proliferative response to 10 pg of TSST-1hB was comparable to a response elicited by 10 ng of TSST-1hA, suggesting that the former component is a more potent mitogen. Rabbit or goat polyclonal antibodies to native TSST-1 efficiently neutralized both TSST-1 components. Heat treatment at 80 degrees C for 15 min had minimal or no effect on the mitogenic properties of TSST-1hA and TSST-1hB.

Toxicity of staphylococcal toxic shock syndrome toxin 1 for germ-free and conventional piglets.

The susceptibilities of conventional and germ-free miniature pigs to staphylococcal toxic shock syndrome toxin 1 (TSST-1) were studied and compared. TSST-1 at doses of 100 micrograms/kg evoked reactions similar to those observed in rabbits, i.e., hyperthermia, diarrhea, apathy, conjunctival hyperemia, and various changes indicating toxic damage of different organs, but it was never lethal at this dose. Germ-free piglets were less susceptible than were corresponding conventional animals, a finding that may support the concept that endogenous endotoxin is involved in the pathogenesis of toxic shock syndrome.

Damaging effect of toxic shock syndrome toxin 1 on chick embryo cells in vitro.

The lethal effect of staphylococcal toxic shock syndrome toxin 1 (TSST-1) on rabbits and chick embryos is enhanced in the presence of lipopolysaccharide (LPS). In an investigation of the mode of action of TSST-1, its effect-both singly and in combination with LPS-on tissue culture cell lines was examined. Of a variety of cell lines examined for sensitivity to TSST-1 treatment, only primary chick embryo cells were susceptible. At a critical concentration (0.2 microgram/mL), TSST-1 alone caused detachment of the cell monolayer. In contrast, LPS per se had no visible effect on the cells at any concentration tested. TSST-1 in combination with LPS caused monolayer detachment at all concentrations of TSST-1 employed; thus detachment was independent of TSST-1 concentration in the presence of LPS. The ability of TSST-1 to disrupt the monolayer was neutralized in the presence of polyclonal rabbit antiserum to TSST-1. In a time course study over 24 hours, the effect of the toxin on the cells was initially visible by light microscopy after 4-7 hours. Clear differences in cellular morphology between TSST-1 treated monolayers and untreated controls were observed by scanning electron microscopy. Treated cells lost their normal spindle-shaped appearance before detachment.

Direct effects of purified staphylococcal toxic shock syndrome toxin 1 on myocardial function of isolated rabbit atria.

Toxic shock syndrome is associated with reversible cardiomyopathy. The cardiac dysfunction may be mediated by toxic shock syndrome toxin 1 (TSST-1), an exotoxin generated by Staphylococcus aureus. However, the effects of purified TSST-1 on cardiac function are unknown. In a study of the toxin's effect on myocardial function, TSST-1 (200 ng/mL) was added to muscle baths containing isolated rabbit atria. TSST-1 caused time-dependent inhibition of developed force (systolic function) but did not alter resting force (an index of muscle stiffness or cardiac compliance). These data show that TSST-1 can directly inhibit myocardial function, but the significance of this effect in patients with toxic shock syndrome remains to be determined.

Induction of tumour necrosis factor by staphylococcal toxic shock toxin 1.

Staphylococcal toxic shock syndrome toxin 1 (TSST1) induced the release of tumour necrosis factor (TNF) from human and rabbit monocytes in vitro. Nanogram amounts of TSST1 were sufficient to induce TNF release. There was considerable variation in response between cells from different rabbits and different donors. Rabbit monocytes were slightly more sensitive to TSST1 than were human monocytes. Release of TNF in vivo could explain many of the symptoms of toxic shock syndrome.

48] Chick embryo assay for staphylococcal toxic shock syndrome toxin-1

Publisher Summary This chapter describes the chick embryo assay for staphylococcal toxic shock syndrome toxin-1 (TSST-1). TSST-1 is produced by the strains of Staphylococcus aureus associated with toxic shock syndrome, an acute, febrile disease in man leading to cardiovascular shock. TSST-1 is pyrogenic, causes shocklike symptoms, and is lethal in rabbits. Other biological effects include lymphocyte mitogenicity, release of interleukin-1 from monocytes, and potentiation of chick embryo lethality because of gram-negative lipopolysaccharide (LPS). The enhancement of chick embryo lethality can be used as a bioassay for TSST-1 in the presence of a fixed sublethal amount of LPS. Assays based on the mitogenic effect and the ability to induce release of interleukin-1 from monocytes, although sensitive, are fairly complex to perform and require the use of tissue culture facilities. The chick embryo assay is easily executed, sensitive, and reproducible. However, it should be noted that the assay may not be suitable for crude culture supernatants from TSST-1 + strains because staphylococcal products other than TSST-I may be lethal in chick embryos.

Comparison of two newly developed forms of an enzyme-linked immunosorbent assay for the detection of staphylococcal toxic shock syndrome toxin-1 (TSST-1)

Two modifications of an enzyme-linked immunosorbent assay (ELISA) were applied to the quantitative determination of TSST-1 in culture supernatants of S. aureus strains. In both techniques, biotinylated antibodies (anti-TSST-1), obtained by means of either glutaraldehyde or N,N-dimethyl formamide were used. IgG and biotin were conjugated in various ratios and the different conjugates thus obtained were examined for their reactivity in the ELISA tests. The application of the glutaraldehyde-method resulted in more active, sensitive and stable conjugates. Furthermore, acceptable TSST-1 standard curves under fixed conditions were only obtained when the appropriate ratio of biotin to IgG had been determined before the biotinylated antibodies were employed.

Cloning and expression of streptococcal pyrogenic exotoxin A and staphylococcal toxic shock syndrome toxin-1 in Bacillus subtilis.

The genes encoding streptococcal pyrogenic exotoxin type A (SPE A) and staphylococcal toxic shock syndrome toxin-1 (TSST-1) were stably cloned and expressed in Bacillus subtilis. In the non-pathogenic Bacillus background, the recombinant speA clone expressed 32-fold more SPE A than the native streptococcus, and similarly, the recombinant plasmid harboring tst expressed 4-fold more TSST-1 in Bacillus than in the native Staphylococcus aureus. The Bacillus-derived products were secreted into the culture fluid, were resistant to proteolytic degradation and their biological activities mimicked native preparations.

Antibodies to staphylococcal enterotoxins and toxic shock syndrome toxin 1 in sera of patients and healthy people in Rio de Janeiro, Brazil.

Sera from 33 persons with staphylococcal infections and from 37 healthy persons were surveyed for the presence of antibody to staphylococcal enterotoxins A, B, C, D, and E and toxic shock syndrome toxin 1. Thirty-one (93.9%) of the patients and 35 (94.6%) of the control group had antibodies to one or more of the enterotoxins. The numbers of patients with antibody to the enterotoxins were as follows: A, 8; B, 9; C, 7; D, 17; E, 21; and toxic shock syndrome toxin 1, 11. The numbers of healthy individuals with antibody to the enterotoxins were as follows: A, 6; B, 12; C, 8; D, 27; E, 21; and toxic shock syndrome toxin 1, 9.

Regulation of staphylococcal toxic shock syndrome toxin-1 and total exoprotein production by magnesium ion.

The effect of Mg2+ on in vitro production of extracellular proteins and, specifically, of toxic shock syndrome toxin-1 (TSST-1), by Staphylococcus aureus in a chemically defined medium was examined. As previously observed, the organisms did not proliferate in the absence of divalent cations. Low levels of Mg2+ (0.02 to 0.04 mM) permitted submaximal proliferation and elevated production of exoproteins. When the Mg2+ concentration was raised to 0.4 mM, multiplication was optimal and exoprotein levels were depressed. Ca2+ and Mn2+ diminished the effect of limiting Mg2+. The increased levels of exoproteins were not due to cell lysis or leakage since intracellular TSST-1 levels were not high enough to account for the increase in extracellular TSST-1 and since the intracellular enzyme, lactate dehydrogenase, was not found in culture supernatants. Cells cultured in low levels of Mg2+ remained in logarithmic growth longer than did those cultured in high concentrations of Mg2+ and, unlike the latter, produced exoproteins throughout the logarithmic growth phase. Low Mg2+ had no effect on cultures in the stationary phase, and organisms cultured in low Mg2+ recovered fully when transferred to high Mg2+. We conclude that, when cultured in medium deficient in Mg2+, S. aureus responds early in the growth cycle by increasing production of many extracellular proteins, including TSST-1.

Clearance of endotoxin from blood of rabbits injected with staphylococcal toxic shock syndrome toxin-1.

This study was undertaken to examine some of the properties of staphylococcal toxic shock syndrome toxin 1 (TSST-1) with regard to the clearance of endotoxin from blood. The concentration of endotoxin in blood was measured by using a chromogenic limulus test and modified perchloric acid method. When TSST-1, which produces fever in rabbits, was injected (100 ng/ml or 100 micrograms/ml per kg) intravenously (i.v.) into the animals, no measurable level of endotoxin was detected in the blood. In control animals, which were given 5 micrograms of endotoxin per ml per kg i.v., endotoxin could be detected in the blood at rapidly declining levels. These results suggested that TSST-1 might not lead bacterial endotoxin from other body sites into the blood. When the animals were given TSST-1 (1 to 100 ng/ml per kg) i.v. and then endotoxin (5 micrograms/ml per kg) i.v. 4 h later, endotoxin was detected in the blood at a high level, depending on the dose of TSST-1 injected. These results showed that TSST-1 inhibited the clearance of endotoxin in the blood; this clearance is thought to be mainly done by the reticuloendothelial system. In the animals given TSST-1 (100 ng/ml per kg) and endotoxin (5 micrograms/ml per kg) simultaneously, the endotoxin level in the blood was found to be higher than that in control animals given endotoxin only but lower than that in the animals given TSST-1 and then endotoxin at the same doses.

Latex agglutination test for staphylococcal toxic shock syndrome toxin 1.

A reversed passive latex agglutination method, in which latex particles were sensitized with specific anti-toxic shock syndrome toxin-1 (TSST-1) immunoglobulin, was found to be a simple and sensitive method for the detection of TSST-1 production by Staphylococcus aureus strains. The minimum amount of TSST-1 detectable was approximately 1.0 ng/ml. Of 41 S. aureus isolates from toxic shock syndrome patients and controls, 23 were positive for TSST-1 production, whereas only 20 strains were positive for TSST-1 production by an Ouchterlony immunodiffusion method. The reversed passive latex agglutination method was used to examine S. aureus strains isolated in Japan from staphylococcal infections, feces from healthy individuals, food from poisoning outbreaks, and market food.

Affinity purification of staphylococcal toxic shock syndrome toxin 1 and its pathologic effects in rabbits.

Toxic shock syndrome toxin 1 (TSST-1) was purified to apparent homogeneity by chromatofocusing and affinity chromatography. The amino acid composition of the toxin was very similar to that reported for TSST-1 by other investigators. The amino-terminal amino acid was serine. A partial specific volume of 0.73 ml/g was calculated for the toxin from the amino acid data, and a molecular weight of 19,200 +/- 1,300 was determined by hydrodynamic methods. New Zealand white rabbits of both sexes were equally susceptible to the lethal effects of the toxin; however, older rabbits (greater than 12 months) were far more susceptible than young adults or weanlings. The 50% lethal dose of TSST-1 in older rabbits was 50 to 60 micrograms/kg when injected subcutaneously and 20 to 30 micrograms/kg when injected intravenously. Enhancement of lethal endotoxin shock by TSST-1 could not be demonstrated when both toxins were injected subcutaneously; however, lethal shock did occur when endotoxin (10 micrograms/kg) was injected intravenously after TSST-1 had been injected by either the subcutaneous (50 to 60 micrograms/kg) or the intravenous (20 to 30 micrograms/kg) route. Endotoxin alone was not lethal at a dose of 500 micrograms/kg of body weight when injected subcutaneously. When injected intravenously, endotoxin at a dose of 500 micrograms/kg was not lethal in weanling males or in females in any age group; however, young (6 to 7 months) and adult (greater than 12 months) males were killed by endotoxin doses as low as 45 to 50 micrograms/kg. Histopathologic studies of rabbits by both sexes which died as a result of TSST-1 alone or in combination with endotoxin showed extensive damage to organs rich in lymphoid and mononuclear phagocytic cells such as the thymus, mesenteric lymph nodes, liver, and spleen. Severe congestion of these organs as well as erythrophagocytosis and lymphoid depletion in the spleen and mesenteric lymph nodes were noted. Congestion and hemorrhage were also found in the heart, lungs, trachea, and thymus. The systemic pathology produced by TSST-1 was strikingly similar to that seen in humans who had died of toxic shock syndrome and in rabbits with subcutaneous chamber inoculated with toxic shock case strains of Staphylococcus aureus. Rabbits that were not killed by the toxin suffered a very rapid and severe leukopenia followed by leukocytosis with a left shift. Lymphopenia was also noted as was a mild but persistent anemia. With the exception of the early leukopenia, very similar hematologic findings have been noted in humans with toxic shock syndrome.