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

Background
The 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 Findings
Here, 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/Significance
In 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.

研究背景
来自肉毒梭菌（Clostridium botulinum）的C3bot1蛋白（分子量约23 kDa）可对Rho蛋白进行ADP核糖基化修饰，进而使其失活。C3bot1可选择性地被单核细胞/巨噬细胞的细胞质摄取，而无法进入上皮细胞、成纤维细胞等其他细胞类型的细胞质。其内化过程极有可能通过依赖酸化内体的特异性内吞途径完成。
研究方法与主要结果
本研究旨在验证酶失活的C3bot1E174Q是否可作为巨噬细胞选择性转运系统，将具有酶活性的蛋白递送至此类细胞的细胞质中。在确认C3bot1E174Q不会诱导巨噬细胞活化后，我们选用来自肉毒梭菌（Clostridium botulinum）、分子量约50 kDa的肌动蛋白ADP核糖基化酶C2I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融合毒素克隆至表达载体，并在大肠杆菌（Escherichia coli）中以谷胱甘肽S-转移酶（GST）融合蛋白的形式进行表达。纯化后的C3bot1E174Q-C2I可被针对C2I与C3bot的抗体识别，且在体外实验中表现出C2I特异性的酶活性。当将该融合毒素施加于培养细胞时，C3bot1E174Q-C2I可在包括J774A.1、RAW264.7细胞系以及原代培养人巨噬细胞在内的各类巨噬细胞的细胞质中对肌动蛋白进行ADP核糖基化修饰，但无法在上皮细胞中完成此过程。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融合毒素可选择性地被巨噬细胞的细胞质摄取。
研究结论与意义
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物质转运至活巨噬细胞的细胞质中。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重要分子工具，同时也是开发针对巨噬细胞相关疾病的新型治疗策略的极具潜力的候选方案。