Elucidating the architecture of the type III secretion system export apparatus

Abstract

Type III secretion systems (T3SS) are a widespread virulence factor in Gram negative bacteria. They contain an inner membrane spanning sub-complex termed the export apparatus, made up of five proteins. The export apparatus translocates effector proteins designated for the host cytoplasm across the inner membrane, is involved in substrate recognition and in substrate specificity switching. Knowing the structure of their components is critical for investigating makeup, assembly, and function of macromolecular machines. This has remained a technical challenge in particular for large, hydrophobic membrane-spanning protein complexes like the T3SS. I determined the stoichiometry of the complete SPI-1 T3SS of Salmonella enterica serovar Typhimurium and the topology of the export apparatus proteins. For the stoichiometric analysis, I used a mass spectrometry approach based on two complementary protocols for gentle complex purification combined with stable isotope-labelled standards. Previous structural analyses have revealed the stoichiometry of base components, but the stoichiometry of the essential hydrophobic export apparatus components and of the ’inner rod’ protein PrgJ remained unknown. Here, I provide evidence that the export apparatus of T3SS contains five SpaP, one SpaQ, one SpaR, and one SpaS. Additionally I can confirm the suggested stoichiometries of InvA and the base components in situ. Furthermore, I present evidence that no more than six PrgJ are involved in the formation of the ’inner rod’. I assessed the topology of the five export apparatus transmembrane proteins using computer predictions and a substituted cysteine accessibility method. The position of the trans- membrane helices and orientation of the loops of InvA, SpaS and one of the minor export apparatus proteins, SpaP, were mapped experimentally. The prediction could be largely confirmed for SpaS and partly for InvA, while one large periplasmic loop could be confirmed for SpaP. Providing this structural information will facilitate efforts to obtain an atomic view of T3SS. The topology and stoichiometry identification of these proteins alongside with recent interaction studies are important steps in determining the exact placement of the export apparatus in T3SS and ultimately facilitates elucidation of the function of each component.