A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages

BackgroundThe C3bot1 protein (∼23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes.Methodology/Principal FindingsHere, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages.Conclusions/SignificanceIn summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.背景 肉毒梭菌（Clostridium botulinum）来源的C3bot1蛋白（约23 kDa）可通过ADP核糖基化修饰并灭活Rho蛋白。该蛋白可选择性被单核细胞/巨噬细胞的胞浆摄取，而无法进入上皮细胞、成纤维细胞等其他细胞类型的胞浆。其内化过程极有可能经由酸化内体的特异性内吞通路完成。 方法与主要结果 本研究旨在探究酶学失活的C3bot1E174Q能否作为巨噬细胞选择性转运系统，将具有酶学活性的蛋白递送至此类细胞的胞浆中。在确认C3bot1E174Q不会诱导巨噬细胞活化后，我们选用肉毒梭菌来源的、可对肌动蛋白进行ADP核糖基化修饰的C2I蛋白（约50 kDa）作为报告酶，用于检测C3bot1E174Q介导的向巨噬细胞的蛋白递送效果。我们将重组C3bot1E174Q-C2I融合毒素克隆为谷胱甘肽S-转移酶（GST）融合蛋白，并在大肠杆菌（Escherichia coli）中进行表达。纯化后的C3bot1E174Q-C2I融合蛋白可被针对C2I与C3bot的抗体识别，且在体外实验中展现出C2I特异性的酶活性。当将该融合蛋白作用于培养细胞时，C3bot1E174Q-C2I可对J774A.1、RAW264.7细胞系以及原代培养人巨噬细胞等巨噬细胞的胞浆内肌动蛋白进行ADP核糖基化修饰，但无法作用于上皮细胞。结合共聚焦荧光显微镜实验结果，上述生化数据证实重组C3融合毒素可选择性被巨噬细胞胞浆摄取。 结论与意义 综上，本研究证实C3bot1E174Q可作为递送系统，快速、选择性且特异性地将酶类转运至活巨噬细胞的胞浆中。因此，基于C3的融合毒素可成为实验性巨噬细胞药理学与细胞生物学研究中的宝贵分子工具，同时也有望成为开发针对巨噬细胞相关疾病的新型治疗策略的理想候选载体。