SpaP and SpaR interaction partner identification

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called needle complex. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central “cup” substructure of a Salmonella Typhimurium secretion system, however, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we show that a SpaP pentamer forms a 15 Å wide pore and present a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further demonstrate the formation of a continuous conduit for substrate translocation and injection by intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, refine the current view of export apparatus assembly and consolidate transmembrane topology models for SpaP and SpaR.

细菌III型蛋白质分泌系统可将效应蛋白注入真核宿主细胞，以促进革兰氏阴性致病菌与共生体的存活及定殖。细菌细胞包膜的跨膜分泌以及向宿主细胞的注射过程，由所谓的针状复合物（needle complex）介导完成。已有研究表明，其小型疏水分泌装置组分SpaP与SpaR可成核针状复合物的组装，并构成鼠伤寒沙门氏菌分泌系统的中央“杯状”亚结构，但这类组分在针状复合物中的体内定位以及其在分泌过程中的功能，此前仍未得到清晰阐释。
本研究证实SpaP五聚体可形成15埃宽的孔道，并绘制了SpaP与分泌装置组分SpaQ、SpaR及SpaS相互作用的详细图谱。我们还通过SpaP与SpaR的周质结构域与内杆蛋白PrgJ的紧密相互作用，证明了底物转运与注射所需连续通道的形成；同时修正了当前关于分泌装置组装的认知，并完善了SpaP与SpaR的跨膜拓扑结构模型。