Visualization and characterization of individual type III protein secretion machines in live bacteria

Significance Type III protein secretion systems are essential virulence factors for many bacterial pathogens. Cryo-electron microscopy has provided important details about the architecture and molecular organization of the type III secretion machine in isolation or in fixed samples. However, little information is available about the organization of the type III secretion machine and its individual substructures in live bacteria. Using 2D and 3D single-molecule switching superresolution microscopy, we have visualized individual type III secretion machines in live bacteria and obtained unique insight into the assembly and function of these machines. These studies bridge a major resolution gap in the visualization of type III secretion machines and may serve as a paradigm for the examination of other bacterially encoded molecular machines. Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine’s assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.

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