Aullo P, Giry M, Olsnes S, Popoff M R, Kocks C, Boquet P
Unité des Toxines Microbiennes URA CNRS 557, Institut Pasteur, Paris, France.
EMBO J. 1993 Mar;12(3):921-31. doi: 10.1002/j.1460-2075.1993.tb05733.x.
We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.
我们开发了一种用于研究rho在肌动蛋白应激纤维形成中作用的新工具。通过对p21 rho进行ADP核糖基化来影响肌动蛋白微丝组装的肉毒杆菌外毒素C3，以多种方式与白喉毒素（DT）进行基因融合。将所得的嵌合毒素在Vero细胞上进行测试。C3与白喉毒素的A和B片段的嵌合体具有降低的细胞结合活性，但显然能够通过与DT相同的机制进入Vero细胞。暴露于低pH值时，C3与DT B片段的融合蛋白DC3B对DT受体具有高亲和力，但在酸化后显然无法转运至细胞质。尽管如此，向生长培养基中添加皮摩尔浓度的DC3B会导致与纽蛋白相关的细胞微丝系统破坏，并有效阻断细胞生长，这表明DC3B的C3部分到达了细胞质，尽管其机制与完整的白喉毒素不同。嵌合的DC3B毒素也应用于被单核细胞增生李斯特菌感染的Vero细胞，这是一种致病细菌，它利用未知的肌动蛋白聚合机制在细胞质中快速移动。DC3B抑制了细菌诱导的微丝组装，表明单核细胞增生李斯特菌在此过程中利用了细胞内rho依赖性机制。
