Distinct Cytosolic Complexes Containing the Type III Secretion System ATPase Resolved by Three-Dimensional...

Area of Science:

• Microbiology
• Molecular Biology
• Cell Biology

Background:

• Type III secretion systems (T3SS) utilize injectisomes to deliver effector proteins into host cells.
• Cytosolic injectisome proteins are crucial for effector export but their interactions and assembly remain unclear.
• Understanding these interactions is key to deciphering bacterial virulence mechanisms.

Purpose of the Study:

• To investigate the in vivo interactions and complex formation of cytosolic injectisome proteins.
• To determine the timing of cytosolic complex assembly relative to injectisome binding.
• To elucidate the regulatory mechanisms governing injectisome function.

Main Methods:

• Employed three-dimensional (3D) single-molecule tracking in living Yersinia enterocolitica.
• Utilized enhanced yellow fluorescent protein (eYFP) tagging of injectisome proteins (YeSctN, YeSctL, YeSctQ).
• Analyzed diffusion rates and complex formation in wild-type and mutant strains (ΔsctD, ΔsctQ).

Main Results:

• Identified distinct cytosolic complexes of injectisome proteins, including YeSctNL and YeSctNLQ.
• Demonstrated that these complexes form independently of injectisome assembly (observed in ΔsctD mutants).
• Quantified increased YeSctNL complex abundance in ΔsctQ mutants, indicating SctQ's role in complex regulation.

Conclusions:

• Cytosolic injectisome protein complexes assemble prior to their association with the membrane-embedded injectisome.
• These pre-assembled complexes play a significant role in the functional regulation of the type III secretion system.
• The findings provide insights into the dynamic assembly and regulation of large molecular machines like injectisomes and flagellar motors.