Analysis of the interaction between the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) and the human T‐cell receptor

Abstract

SummaryStreptococcus pyogenes that produces the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) is associated with outbreaks of streptococcal toxic shock syndrome (STSS) in the United States and Europe. SpeA stimulates V␤2.1, 12.2, 14.1, and 15.1-positive T cells, and the lymphokine production from the activated T cells is believed to result in the symptoms associated with STSS. The T-cell receptor (TCR)-SpeA interaction is crucial for superantigenic activity, and studies were undertaken to determine regions of both SpeA and the TCR involved in the formation of MHC/SpeA/TCR complexes. Previously, recombinant toxins encoded by speA alleles 1, 2, and 3 as well as toxins resulting from 19 distinct point mutations in speA1 were generated. Here, these 22 toxin forms were incubated with human peripheral blood mononuclear cells (PBMCs), and the percentages of T-cell blasts bearing V␤ chains 2.1, 12.2, and 14.1 were quantified by flow cytometry. The analysis indicates that the residues of SpeA needed for a productive TCR interaction differ for each V␤ chain examined. An amino acid substitution at only one site significantly affected the toxin's ability to stimulate V␤2.1-expressing T cells, three individual amino acid substitutions resulted in significant loss of ability to stimulate V␤12.2-expressing T cells, and substitution at 13 individual sites significantly affected the ability to stimulate V␤14.1-expressing T cells. To elucidate the regions of the V␤ chains that interacted with SpeA, synthetic peptides representative of the human V␤12.2 complementary-determining regions (CDRs) 1, 2, and 4 were used to block the SpeA-mediated proliferation of human PBMCs. The CDR1, CDR2 and CDR4 peptides were each able to block proliferation, with the activity of CDR1 > CDR2 > CDR4. Combinations of CDR1 peptide with CDR2 or CDR4 peptides allosterically enhanced the ability of each to block proliferation, suggesting SpeA has distinct binding sites for the CDR loops.

Toxoids of Streptococcal Pyrogenic Exotoxin A Are Protective in Rabbit Models of Streptococcal Toxic Shock Syndrome

“…In selection of sites for mutagenesis, residues that were known to be important for superantigenicity, enhancement of endotoxin shock, and lethality (when administered subcutaneously to rabbits in miniosmotic pumps) were identified (16,17,29). For example, in this study, residue Asn 20 was changed to Asp.…”

“…The residue is important in TCR binding in both SEB and SEC as well as SPE A (9,19). Similarly, SPE A residues 41, 42, and 45 are in the O/B fold region of the molecule and are important for MHC class II binding (16,17,29). Importantly, by selecting such key residues, we obtained three multiple-site mutants, N20D/C98S, N20D/D45N/C98S, and Q19H/N20D/L41A/L42A/D45N/C98S, that had all of the desired properties of toxoids.…”

Mutational effects on protein folding stability and antigenicity: the case of streptococcal pyrogenic exotoxin A

“…The naturally occurring substitution V76I, known as the SPEA3 allele [13], has been shown to possess an eightfold higher affinity for HLA-DQ than SPEA1 [7,8]-even though it is not located at the predicted MHC-binding surface. Residues N20 [5,14] and L24 have been predicted to be at or near the TCR interaction surface, while residue L42 is believed to be located in the SPEA contact site for MHC class II molecules [7,8]. Residue N20 is observed to contact the TCR ␤ chain in the structure of Sundberg et al (2002) [9].…”

“…1). Modeling and mutational analyses [7,8] have predicted that the disulfide loop is important for interactions with both TCR and MHC in the ternary SPEA-TCR-MHC complex. A recent cocrystal structure of the C90S mutant of SPEA with a mouse TCR ␤ chain [9] confirmed the involvement of loop residues in binding to TCR.…”

“…Mutation of SPEA L42 substantially decreases toxicity [7,8], which is a necessary prerequisite for vaccination without lethality. Therefore, for testing immunogenicity we concentrated our efforts on using this mutation alone and in combination with other substitutions.…”