Ruhe Frederike, Olling Alexandra, Abromeit Rasmus, Rataj Dennis, Grieschat Matthias, Zeug Andre, Gerhard Ralf, Alekov Alexi
Institute for Neurophysiology, Hannover Medical School Hannover, Germany.
Institute for Toxicology, Hannover Medical School Hannover, Germany.
Front Cell Infect Microbiol. 2017 Mar 13;7:67. doi: 10.3389/fcimb.2017.00067. eCollection 2017.
Virulent toxins TcdA and TcdB invade host intestinal epithelia by endocytosis and use the acidic environment of intracellular vesicles for further processing and activation. We investigated the role of ClC-5, a chloride/proton exchanger expressed in the endosomes of gastrointestinal epithelial cells, in the activation and processing of toxins. Enhanced intoxication by TcdA and TcdB was observed in cells expressing ClC-5 but not ClC-4, another chloride/proton exchanger with similar function but different localization. In accordance with the established physiological function of ClC-5, its expression lowered the endosomal pH in HEK293T cells by approximately 0.6 units and enhanced approximately 5-fold the internalization of TcdA. In colon HT29 cells, 34% of internalized TcdA localized to ClC-5-containing vesicles defined by colocalization with Rab5, Rab4a, and Rab7 as early and early-to-late of endosomes but not as Rab11-containing recycling endosomes. Impairing the cellular uptake of TcdA by deleting the toxin CROPs domain did not abolish the effects of ClC-5. In addition, the transport-incompetent mutant ClC-5 E268Q similarly enhanced both endosomal acidification and intoxication by TcdA but facilitated the internalization of the toxin to a lower extent. These data suggest that ClC-5 enhances the cytotoxic action of toxins by accelerating the acidification and maturation of vesicles of the early and early-to-late endosomal system. The dispensable role of electrogenic ion transport suggests that the voltage-dependent nonlinear capacitances of mammalian CLC transporters serve important physiological functions. Our data shed light on the intersection between the endocytotic cascade of host epithelial cells and the internalization pathway of the large virulence toxins. Identifying ClC-5 as a potential specific host ion transporter hijacked by toxins produced by pathogenic bacteria widens the horizon of possibilities for novel therapies of life-threatening gastrointestinal infections.
强毒毒素TcdA和TcdB通过内吞作用侵入宿主肠道上皮细胞，并利用细胞内囊泡的酸性环境进行进一步加工和激活。我们研究了ClC-5（一种在胃肠道上皮细胞内体中表达的氯/质子交换体）在毒素激活和加工中的作用。在表达ClC-5的细胞中观察到TcdA和TcdB的中毒增强，但在表达ClC-4（另一种功能相似但定位不同的氯/质子交换体）的细胞中未观察到。根据ClC-5已确定的生理功能，其表达使HEK293T细胞内体pH降低约0.6个单位，并使TcdA的内化增强约5倍。在结肠HT29细胞中，34%内化的TcdA定位于含ClC-5的囊泡，这些囊泡通过与Rab5、Rab4a和Rab7共定位确定为早期和早期到晚期内体，但不是含Rab11的再循环内体。通过删除毒素CROPs结构域来损害TcdA的细胞摄取并没有消除ClC-5的作用。此外，运输无活性的突变体ClC-5 E268Q同样增强了内体酸化和TcdA中毒，但在较低程度上促进了毒素的内化。这些数据表明，ClC-5通过加速早期和早期到晚期内体系统囊泡的酸化和成熟来增强毒素的细胞毒性作用。电生离子转运的非必需作用表明，哺乳动物CLC转运体的电压依赖性非线性电容具有重要的生理功能。我们的数据揭示了宿主上皮细胞内吞级联与大型毒力毒素内化途径之间的交叉点。将ClC-5鉴定为一种被病原菌产生的毒素劫持的潜在特异性宿主离子转运体，拓宽了危及生命的胃肠道感染新疗法的可能性范围。
