Distinct Cytoso Complexes Containing the Type III Secretion System ATPase Resolved by Three-Dimensional Single-Molecule Tracking in Live Yersinia enterocolitica

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 are required for export of effectors and display both stationary, injectisome-bound populations and freely diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilized three-dimensional (3D) singlemolecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the enhanced yellow fluorescent protein (eYFP)-labeled ATPase YeSctN and its regulator, YeSctL, revealed that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed both in wild-type cells and in DsctD mutants, which cannot assemble injectisomes. In DsctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding, which has important implications for how injectisomes are functionally regulated.

Automated summary

This study used three-dimensional single-molecule tracking to elucidate the interaction of injectisome proteins in the cytosol and found that different cytosolic complexes of these proteins can form prior to injectisome binding, which has significant implications for the functional regulation of injectisomes.