A mutation at histidine residue 135 of toxic shock syndrome toxin yields an immunogenic protein with minimal toxicity.

Structure-function studies have revealed that the region between amino acids 115 and 141 of toxic shock syndrome toxin 1 (TSST-1) constitutes a biologically active domain. A critical residue appears to be histidine 135, since a site-directed mutation that alters the histidine to alanine (H135A) results in a loss of mitogenic activity and an absence of toxicity as measured in a rabbit infection model of toxic shock syndrome. We have characterized the mutant toxin further and report here on its immunogenic activity in rabbits and on the protective ability of mutant-specific antibodies in two animal models of toxin-mediated shock. Antibodies raised in rabbits by immunization with the purified H135A are fully cross-reactive with staphylococcal TSST-1 and wild-type recombinant TSST-1 (rTSST-1) expressed in Escherichia coli. The H135A antibodies neutralized the mitogenic activity for murine splenic T cells equally well as did TSST-1-specific polyclonal and monoclonal antibodies. In addition, the H135A antibodies blocked the production of tumor necrosis factor by spleen cells stimulated with rTSST-1. The toxicities of rTSST-1 and H135A were compared in D-galactosamine (D-GalNH2)-sensitized MRL-lpr/lpr mice. The nontoxicity of H135A was confirmed in this murine model of superantigen-induced septic shock. No toxicity of H135A was demonstrable at doses of 60 micrograms, while doses of rTSST-1 as low as 2 micrograms caused significant mortality within 24 to 72 h after challenge. Furthermore, subsequent to challenge of mice with H135A, no elevation in the serum levels of interleukin-2 or tumor necrosis factor was measurable. Passive immunization with H135A antibodies also protected MRL-lpr/lpr mice against lethal challenge with rTSST-1. Finally, rabbits actively immunized with purified H135A did not succumb to infection with a transformed strain of Staphylococcus aureus expressing rTSST-1. Additional animal studies will be required to confirm the immunizing potential of H135A and the efficacy of H135A antibodies as a neutralizing antitoxin.

A fas antigen receptor mutation allows development of toxic shock syndrome toxin-1-induced lethal shock in V beta 8.2 T-cell receptor transgenic mice.

Recombinant toxic shock syndrome toxin-1 (rTSST-1) administered to MRL-lpr/lpr TCR V beta 8.2 transgenic mice at doses of 0.1 microgram/mouse resulted in 100% mortality. This was an unexpected finding since TSST-1 does not activate V beta 8.2 T cells. In contrast, control mice heterozygous at the lpr locus and also for the transgene (MRL-lpr/+; V beta 8.2/0) survived doses of superantigen 100 times higher. The transgenic mice which succumbed to rTSST-1 challenge exhibited histopathology of the liver consistent with toxic shock (generalized inflammation and hepatocellular necrosis) as well as substantially elevated serum TNF-alpha, IL-2, and IL-6 cytokine levels. Splenic T cells derived from transgenic mice stimulated with rTSST-1 in vitro did not undergo detectable proliferation as measured in a standard mitogen assay. However, PCR amplification of cDNA prepared from the V beta 8.2 splenocytes revealed the presence of minor populations of TSST-1-reactive V beta elements (i.e. V beta 3 and V beta 15). Furthermore, an expansion of the V beta 3 and V beta 15 T-cell families was detected by PCR assay of spleen cell cultures stimulated with rTSST-1. These results suggested that the exquisite sensitivity of the MRL-lpr/lpr V beta 8.2 transgenic animals to rTSST-1 was not dependent exclusively on T-cell proliferation but was augmented by the influence of a defective fas antigen receptor expressed in homozygous lpr mice. To test this hypothesis more directly, we compared the sensitivity of MRL-lpr/lpr mice (not carrying the V beta 8.2 transgene) to MRL-+/+ mice. The MRL-lpr/lpr fas antigen-defective mice were substantially more susceptible to rTSST-1 challenge. Mice carrying the lpr mutation on another genetic background (C57BL/6.C3H-lpr/lpr) were also more sensitive to rTSST-1 challenge than were C57BL/6.C3H-+/+ mice. Although induction of toxic shock is clearly associated with T-cell proliferation, defects in fas antigen receptor or ligand may also contribute substantively to superantigen-mediated lethal shock by still undefined mechanisms.

Toxicity of recombinant toxic shock syndrome toxin 1 and mutant toxins produced by Staphylococcus aureus in a rabbit infection model of toxic shock syndrome.

Menstrually associated toxic shock syndrome (TSS) is attributed primarily to the effects of staphylococcal exotoxin toxic shock syndrome toxin 1 (TSST-1). A region of the 194-amino-acid toxin spanning residues 115 through 144 constitutes a biologically active site. Several point mutations in the TSST-1 gene in that region result in gene products with reduced mitogenic activity for murine T cells. In this study we evaluated the toxicity of recombinant TSST-1 and several mutants of TSST-1 made by transformed Staphylococcus aureus during in vivo growth in a rabbit infection model of TSS. The toxicities of the transformed strains of S. aureus for rabbits correlated with the mitogenic activities of the recombinant toxins. An isolate originally obtained from a patient with a confirmed case of TSS (S. aureus 587) implanted in a subcutaneous chamber served as a positive control. TSST-1 produced in vivo led to lethal shock within 48 h, and a TSST-1-neutralizing antibody (monoclonal antibody 8-5-7) administered to rabbits challenged with S. aureus 587 prevented fatal illness. Rabbits infected with transformed S. aureus RN4220 expressing wild-type toxin (p17) or mutant toxins retaining mitogenic activity for T cells succumbed within a similar time frame. Blood chemistries of samples obtained from infected animals before death indicated abnormalities in renal and hepatic functions similar to those induced by parenteral injection of purified staphylococcal TSST-1. Mutant toxin 135 (histidine modified to alanine at residue 135) possessed only 5 to 10% of the mitogenic activity of wild-type toxin. Rabbits challenged with transformed S. aureus RN4220 expressing mutant toxin 135 exhibited only mild transient illness. Mutant toxin 135 retained reactivity with monoclonal antibody 8-5-7 and by several criteria was conformationally intact. Toxin from a double mutant, 141.144, with alanine substitutions at residues 141 (histidine) and 144 (tyrosine), also was devoid of mitogenic activity. In this case, antibody recognition was lost. Mutant toxins 115 and 141 were found to possess approximately half-maximal mitogenic activity. Rabbits challenged with S. aureus RN4220 expressing either 115 or 141 toxin succumbed to lethal shock. We conclude that the ability of TSST-1 to activate murine T cells in vitro and its expression of toxicity leading to lethal shock in rabbits are related phenomena.