Towards a molecular-level understanding of bacterial type 3 secretion: Resolving cytosolic complex formation in living cells by tracking single-molecules

About a third of bacterial proteins are either transported across or integrated into the cell membranes, so that they can perform functions that are vital for bacterial survival in specific environmental niches. Directional transport of selected proteins often relies on large membrane-embedded biomolecular assemblies. For example, the dual membrane-spanning Type 3 Secretion System (T3SS) enables Gram-negative bacterial pathogens to inject so-called effector proteins directly into the cytosol of eukaryotic host cells – a virulence mechanism that currently results in more than 1 million human deaths per year. While the cocktail of injected effector proteins differs for each pathogen, the structural proteins of T3SSs are highly conserved, making Type 3 secretion systems a key target for anti-virulence treatments. Our research focuses on providing a detailed understanding of how T3SSs are functionally regulated at the molecular level and how the T3SS and similar assemblies ultimately enable bacterial survival and virulence. 
In this talk, I will describe how single-molecule localization and tracking microscopy in different genetic backgrounds provides a path towards describing the molecular mechanism(s) of type 3 secretion in living cells. Through computational aberration correction and quantitative numerical analysis, we determine the subcellular locations and 3D motion trajectories of single fluorescently labeled proteins. Our results indicate that the structural T3SS proteins also exist in freely diffusing cytosolic complexes with different molecular compositions. Some, but not all, of these complexes only form in the presence of other T3SS proteins and their abundances change upon activation of secretion. Resolving the cytosolic diffusion states of T3SS proteins provides important insights into the dynamic regulatory network that controls type 3 secretion.