The molecular function of T3SS

Despite years of dedicated research, the molecular events that lead to effector export through the T3SS are still unknown. The components that govern these events are located at the cytosolic interface of the injectisome.  Using single-molecule tracking to monitor these components in the working T3SS in real time, we have recently discovered that parts of the T3SS are dynamic, exchanging subunits between a cytosolic pool and the injectisome, while secreting effectors (Diepold et al., 2015). Strikingly, the exchange correlates with the function of the T3SS. We found that the reason for this unexpected behavior is a switch in the interactions between the cytosolic T3SS components upon activation of the system (Diepold et al., 2017).

But how exactly does this dynamic behavior enable secretion? Does it directly allow to activate the system in response to external signals? And could this be an Achilles heel we can exploit to find a generally applicable treatment for T3SS-dependent bacterial infections?

The injectisome is a dynamic protein complex, regulated by external cues at the molecular and cellular level. (A) Schematic depiction of of the dynamic turnover of the cytosolic T3SS components. The location of the soluble components at the injectisome is indicated. (B) Cellular distribution of motile (blue) and stationary (red) PAmCherry-YscQ molecules in PALM. (C) Upon induction of secretion (filled bars and circles), the cytosolic components SctK, SctQ, and SctL - but not the ATPase SctN - diffuse significantly faster within live bacteria (boxes, standard error of the mean; whiskers, standard deviation; ***, p<1E-6; n.s., no statistically significant difference). Adapted from Diepold et al., 2015 and Diepold et al., 2017.

To answer these questions, our group visualizes the T3SS and its substrates during its function in live bacteria, from the cellular to the single-molecule level.

Working with live bacteria, we can directly compare the molecular events under different conditions. This allows us to determine the changes that occur upon external cues, such as the activation of the system in vitro or by direct host cell contact.

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