Chung Soo-Young, Schöttelndreier Dennis, Tatge Helma, Fühner Viola, Hust Michael, Beer Lara-Antonia, Gerhard Ralf
Institute of Toxicology, Hannover Medical School, Hanover, Germany.
Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany.
Front Microbiol. 2018 Oct 26;9:2314. doi: 10.3389/fmicb.2018.02314. eCollection 2018.
toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in -associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdB reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdB showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdB to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101-1836) of TcdB and TcdB, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin-receptor interaction.
毒素TcdA和TcdB是大型梭菌属葡糖基转移酶，它们是相关疾病中的主要致病因素。所有大型梭菌属葡糖基转移酶中都存在四个高度保守的半胱氨酸。在本研究中，我们聚焦于参考菌株VPI10463（进化枝I）的TcdB的组合重复寡肽结构域内保守的半胱氨酸2232。高毒力菌株R20291（进化枝II）的TcdB中不存在半胱氨酸2232，取而代之的是一个酪氨酸。TcdB中用酪氨酸取代半胱氨酸2232会降低与两种已知TcdB受体卷曲蛋白-2（FZD2）和脊髓灰质炎病毒受体样蛋白-3/连接蛋白-3（PVRL3）的可溶性片段的结合。与此一致的是，在下拉试验中TcdB与PVRL3的结合较弱，当将酪氨酸2232换成半胱氨酸时结合增加。令人惊讶的是，我们未观察到TcdB与FZD2的结合，这表明该受体对这种毒素型不太重要。用TcdB和TcdB的受体结合片段（氨基酸1101 - 1836）以及通过抗体噬菌体展示新开发的抗体进行的竞争试验，揭示了TcdB中携带第二个受体结合区域的尚未充分描述的传递结构域的不同特征。总之，我们发现TcdB中保守的半胱氨酸-2232间接促进毒素 - 受体相互作用。
