Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.

基于蛋白质的亲和试剂（例如抗体或替代性结合支架）为基础研究和治疗方法提供了广泛的应用。然而，尽管小化学分子有效地到达细胞内靶标，但是将大分子递送到细胞的胞质溶胶中仍然是主要挑战。因此，基于蛋白质的试剂在细胞质中的应用受到很大限制。一些致病细菌已进化出保守的III型分泌系统（T3SS），该系统可将效应蛋白递送到真核细胞中。在这里，我们增强了鼠伤寒沙门氏菌无毒菌株的T3SS以可复制的方式将多种重组蛋白递送到真核细胞中。用DARPins和单克隆抗体检测该系统的功效，以在功能上抑制该范式和很大程度上不可消耗的RAS信号通路。因此，我们开发了一种细菌分泌系统，用于治疗性大分子的有效胞质递送。