B-160. Toxic Shock Syndrome Toxin-1 (TSST-1) Amino Acid Residues Required for Interaction with Human Vaginal Epithelial Cells (HVECs)

A. J. Brosnahan, P. M. Schlievert, M. M. Schaefers, M. L. Peterson;
Univ. of Minnesota, Minneapolis, MN.

Background: The superantigen (SAg) TSST-1 causes menstrual TSS. The ability of SAgs to cause TSS systemically is dependent on their capacities to cross mucosal surfaces, as causative Staphylococcus aureus typically remain localized. A dodecapeptide region of TSST-1, relatively conserved among SAgs, is implicated in penetration of SAgs across epithelial barriers. This region is separate from SAg regions involved in T cell receptor and major histocompatibility complex II binding. The purpose of this study was to identify amino acids in this dodecapeptide that are critical for TSST-1 interaction with human vaginal mucosa. Methods: Single site mutations were made in amino acids of the dodecapeptide (D120-D130) in S. aureus RN4220 (pCE107). The TSST-1 mutants were isolated by ethanol precipitation and isoelectric focusing. Mutants were tested for superantigenicity using a 4-day assay based on 3H-thymidine incorporation into DNA. Mutants were tested by ELISA for ability to induce IL-8 production by HVECs. Mutants with interesting phenotypes were tested for lethality in intravenous (IV) and intravaginal rabbit models. Results: All mutants retained wild-type (WT) superantigenicity, indicating the dodecapeptide region is not required for this activity. IL-8 production from HVECs was inhibited primarily by mutants in the C-terminal end of the region; S127A, T128A, and D130A stimulated only low level production of IL-8, compared to WT toxin. Interestingly, L129A retained WT activity. These same 4 mutants showed WT lethality when administered IV to rabbits. Intravaginally, S127A, T128A, and L129A induced TSS to varying degrees; however D130A was inactive. Conclusion: Specific residues within the TSST-1 dodecapeptide region are required to elicit cytokines from HVECs, suggesting their interaction with a novel epithelial cell receptor, and to induce TSS systemically through their importance in SAg penetration of mucosa.