Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459.
Department of Microbiology, New York University School of Medicine, New York, New York 10016.
J Biol Chem. 2015 Nov 20;290(47):28402-28415. doi: 10.1074/jbc.M115.675967. Epub 2015 Sep 28.
Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell surface receptors that increase the local toxin concentration on cell membranes, thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. Although it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell lines, we show that VCC is unlikely to utilize high affinity protein receptors as do structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding interactions.
细菌孔形成毒素（PFT）是结构多样的病原体分泌蛋白，它们在宿主细胞的膜中形成细胞损伤通道。大多数 PFT 以水溶性单体的形式释放，首先在膜上寡聚化，然后插入跨膜通道。为了调节特异性并提高效力，许多 PFT 识别特定的细胞表面受体，这些受体增加细胞膜上毒素的局部浓度，从而促进通道形成。霍乱弧菌细胞溶素（VCC）是一种由人类病原体分泌的毒素，该病原体导致流行的霍乱病，并作为宿主免疫系统的防御剂。尽管已经表明 VCC 在细胞表面利用特定的聚糖受体，但与膜的直接接触也必须在毒素结合中发挥作用。为了更好地了解这些相互作用的性质，我们对 VCC 的膜结合表面进行了系统研究，以确定对细胞靶向很重要的其他膜相互作用。通过在几种源自人类的细胞系上进行细胞测定，我们表明 VCC 不太可能像来自金黄色葡萄球菌的结构相似的毒素那样利用高亲和力的蛋白质受体。接下来，我们确定了许多特定的氨基酸残基，这些残基极大地降低了 VCC 对细胞的效力，并研究了聚糖结合与这些直接脂质接触之间的相互作用。最后，我们使用模型膜来解析这些关键残基在脂质和胆固醇结合中的重要性。我们的研究提供了 VCC 膜结合表面的完整功能图谱，并深入了解糖、脂质和胆固醇结合相互作用的整合。
