Resolving Cytosolic Interactions and Injectisome Binding Dynamics of the Yersinia enterocolitica Sorting Platform and ATPase via Single Molecule Tracking

The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins, also known as the sorting platform and ATPase, are required for export of effectors and display both stationary, injectisome-bound populations and freely diffusing cytosolic populations. In Yersinia enterocolitica, evidence supports an effector protein shuttling model, in which effector proteins are shuttled from the cytosol to the injectisome prior to secretion through the hollow needle complex. However, how the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Additionally, it is difficult to reconcile the observed exchange rate of YeSctQ, the primary component of the sorting platform, with reported rates of substrate secretion. To determine the subcomplexes formed by the sorting platform and ATPase in the cytosol of live Y. enterocolitica, I utilized 3D single-molecule localization and tracking microscopy. Specifically, I developed a novel data analysis pipeline that relies on the diffusion coefficient spectrum, which provided a straightforward approach towards interpreting complex intracellular diffusion data. I also developed a data processing pipeline for 2D single-molecule bound-time data, which allowed for quantitative analysis of an individual protein’s binding kinetics with stationary injectisomes. Results support a model in which distinct complexes readily form among the sorting platform and ATPase in the cytosol of live Y. enterocolitica, where the propensity for complex formation changes in the presence or absence of injectisomes. Additionally, bound-time analysis of YeSctQ supports the effector protein shuttling mechanism. Our data indicates that YeSctQ can deliver effector proteins to the injectisome at a maximum secretion rate of approximately one effector protein every 0.6 seconds. Further research is needed to support these findings, which has important implications for how the sorting platform and ATPase functionally regulate secretion.