Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 565-0871 Osaka, Japan.
Graduate School of Engineering, Osaka University, Suita, 565-0871 Osaka, Japan.
Proc Natl Acad Sci U S A. 2018 Jul 10;115(28):7422-7427. doi: 10.1073/pnas.1805671115. Epub 2018 Jun 25.
Initial attachment and subsequent colonization of the intestinal epithelium comprise critical events allowing enteric pathogens to survive and express their pathogenesis. In enterotoxigenic (ETEC), these are mediated by a long proteinaceous fiber termed type IVb pilus (T4bP). We have reported that the colonization factor antigen/III (CFA/III), an operon-encoded T4bP of ETEC, possesses a minor pilin, CofB, that carries an H-type lectin domain at its tip. Although CofB is critical for pilus assembly by forming a trimeric initiator complex, its importance for bacterial attachment remains undefined. Here, we show that T4bP is not sufficient for bacterial attachment, which also requires a secreted protein CofJ, encoded within the same CFA/III operon. The crystal structure of CofB complexed with a peptide encompassing the binding region of CofJ showed that CofJ interacts with CofB by anchoring its flexible N-terminal extension to be embedded deeply into the expected carbohydrate recognition site of the CofB H-type lectin domain. By combining this structure and physicochemical data in solution, we built a plausible model of the CofJ-CFA/III pilus complex, which suggested that CofJ acts as a molecular bridge by binding both T4bP and the host cell membrane. The Fab fragments of a polyclonal antibody against CofJ significantly inhibited bacterial attachment by preventing the adherence of secreted CofJ proteins. These findings signify the interplay between T4bP and a secreted protein for attaching to and colonizing the host cell surface, potentially constituting a therapeutic target against ETEC infection.
初始附着和随后的肠上皮定植是允许肠道病原体存活并表达其发病机制的关键事件。在肠产毒性（ETEC）中，这些是由一种长的蛋白质纤维称为 IVb 型菌毛（T4bP）介导的。我们已经报道过，定植因子抗原/III（CFA/III），ETEC 中一个操纵子编码的 T4bP，具有一个次要菌毛，CofB，其在其尖端带有 H 型凝集素结构域。尽管 CofB 对于形成三聚体起始复合物的菌毛组装至关重要，但它对细菌附着的重要性仍未确定。在这里，我们表明 T4bP 不足以进行细菌附着，这还需要一个在同一 CFA/III 操纵子中编码的分泌蛋白 CofJ。CofB 与包含 CofJ 结合区的肽复合物的晶体结构表明，CofJ 通过将其柔性 N 端延伸锚定到 CofB H 型凝集素结构域的预期碳水化合物识别位点中，与 CofB 相互作用。通过将该结构和溶液中的物理化学数据相结合，我们构建了一个 CofJ-CFA/III 菌毛复合物的合理模型，该模型表明 CofJ 通过结合 T4bP 和宿主细胞膜作为分子桥起作用。针对 CofJ 的多克隆抗体的 Fab 片段通过防止分泌的 CofJ 蛋白的附着显著抑制了细菌附着。这些发现标志着 T4bP 和分泌蛋白之间相互作用以附着和定植宿主细胞表面，这可能成为针对 ETEC 感染的治疗靶标。
