Kline J B, Collins C M
Department of Microbiology and Immunology, University of Miami School of Medicine, Florida 33101, USA.
Mol Microbiol. 1997 Apr;24(1):191-202. doi: 10.1046/j.1365-2958.1997.3381696.x.
Streptococcus 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 beta 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 beta 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 beta chain examined. An amino acid substitution at only one site significantly affected the toxin's ability to stimulate V beta 2.1-expressing T cells, three individual amino acid substitutions resulted in significant loss of ability to stimulate V beta 12.2-expressing T cells, and substitution at 13 individual sites significantly affected the ability to stimulate V beta 14.1-expressing T cells. To elucidate the regions of the V beta chains that interacted with SpeA, synthetic peptides representative of the human V beta 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.
产生细菌超抗原链球菌致热外毒素A（SpeA）的化脓性链球菌与美国和欧洲的链球菌中毒性休克综合征（STSS）暴发有关。SpeA刺激Vβ2.1、12.2、14.1和15.1阳性T细胞，并且认为活化T细胞产生的淋巴因子会导致与STSS相关的症状。T细胞受体（TCR）-SpeA相互作用对于超抗原活性至关重要，因此开展了研究以确定参与MHC/SpeA/TCR复合物形成的SpeA和TCR的区域。此前，已产生了由speA等位基因1、2和3编码的重组毒素以及speA1中19个不同点突变产生的毒素。在此，将这22种毒素形式与人外周血单核细胞（PBMC）一起孵育，并通过流式细胞术对携带Vβ链2.1、12.2和14.1的T细胞母细胞百分比进行定量。分析表明，对于所检测的每条Vβ链，产生有效TCR相互作用所需的SpeA残基各不相同。仅一个位点的氨基酸取代就显著影响毒素刺激表达Vβ2.1的T细胞的能力，三个单独的氨基酸取代导致刺激表达Vβ12.2的T细胞的能力显著丧失，而在13个单独位点的取代显著影响刺激表达Vβ14.1的T细胞的能力。为了阐明与SpeA相互作用的Vβ链区域，使用代表人类Vβ12.2互补决定区（CDR）1、2和4的合成肽来阻断SpeA介导的人PBMC增殖。CDR1、CDR2和CDR4肽均能够阻断增殖，其活性为CDR1>CDR2>CDR4。CDR1肽与CDR2或CDR4肽的组合变构增强了各自阻断增殖的能力，表明SpeA对CDR环具有不同的结合位点。
