Single-Molecule Super-Resolution Imaging Of Tcpp Dynamics In Vibrio Cholerae In Response To Virulence Pathway Deactivation By Increased Cell Density

The agent of the epidemic human disease cholera is Vibrio cholerae, a bacterium which produces cholera toxin via a virulence pathway that has previously been described biochemically. Various environmental stimuli affect this pathway, which is halted when the cell density is low. One virulence pathway component, TcpP, is a transcription activator that acts in concert with other membrane proteins, ToxS, TcpH and ToxR to regulate ToxT expression. TcpP regulation is itself mediated by various cellular signaling pathways. One popular model for the life of each TcpP molecule suggests that TcpP is coupled to a complex made up of ToxR, TcpH and DNA during transcription, and that TcpP is decoupled from this group upon the deactivation of the virulence pathway before degradation by regulated intramembrane proteolysis. This volume change of the diffusing TcpP particle should cause a measurable change in the rate of diffusion as predicted by the Einstein-Stokes equation. Here, we use single-molecule super-resolution fluorescence microscopy to measure the motion of individual TcpP molecules labeled with the photo-activatable fluorescent protein PAmCherry in live V. cholerae cells. Furthermore, we prepare our samples for imaging within an agarose microfluidic device, in which we set up a linear gradient of spent media inside to simulate increasing cell density. By imaging single cells at different signal concentrations on a single slide, variations in preparation or initial cell state will be decoupled from the effect of cell density on the mobility of TcpP, and we observe changes in TcpP-PAmCherry diffusion that we explain in the context of differences in the virulence pathway activity level.