Dynamic Assembly of the Type-3 Secretion System in Yersinia enterocolitica Probed by Super-Resolution Fluorescence Imaging.

The bacterial Type III Secretion System (T3SS) is a complex membrane spanning molecular machine comprised of over 20 different proteins. The T3SS is employed by pathogenic bacterial species to deliver effector proteins though a hollow needle-like structure, providing the mechanism of eukaryotic host cell infection. While effector proteins differ between species, the structural components of the T3SS remain largely conserved, making the machinery an attractive drug target. However, how secretion substrates are selected and transported by type 3 secretion remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sub-complex of the system located within the bacterial cytosol, called the sorting platform. Recent work has suggested that a dynamic interaction network of cytosolic sorting platform proteins play a role in effector protein secretion. To examine the diffusive behavior of sorting platform proteins within the bacterial cytosol, I employed 3D single-molecule localization microscopy on fluorescently labeled proteins in live Yersinia enterocolitica cells. To extract prevalent diffusive states of sorting platform proteins from a large population of single-molecule trajectories, I developed and thoroughly tested a diffusion analysis framework. By observing the prevalent diffusive states of sorting platform proteins in a variety of genetic backgrounds, we were able to construct a model on cytosolic sorting platform complex formation, further supporting the hypothesis that secretion is regulated through a dynamic interaction network of sorting platform proteins.