Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes...

Area of Science:

• Microbiology
• Structural Biology
• Cellular Biology

Background:

• Type IV secretion systems (T4SSs) are crucial for bacterial pathogenesis, enabling translocation of proteins and DNA into host cells.
• The Dot/Icm T4SS of *Legionella pneumophila* is a complex machine responsible for delivering over 300 effector proteins into host cells.
• Understanding the biogenesis and mechanism of T4SSs is essential for developing strategies against bacterial infections.

Purpose of the Study:

• To visualize the *Legionella pneumophila* Dot/Icm secretion machine in its native cellular context.
• To identify and structurally characterize intermediate assembly states of the Dot/Icm machine.
• To elucidate the mechanism of Dot/Icm machine assembly and substrate translocation initiation.

Main Methods:

• High-throughput cryoelectron tomography (cryo-ET) was employed to image Dot/Icm machines within whole *L. pneumophila* cells.
• Subtomogram averaging was used to determine high-resolution structures of the Dot/Icm machine and its subassemblies.
• Comparative analysis of wild-type and mutant strains provided insights into assembly pathways.

Main Results:

• The distribution of Dot/Icm machines within the bacterial cell was mapped.
• Five distinct subassembled intermediates of the Dot/Icm machine were identified.
• Structural analysis revealed that DotB ATPase docking onto DotO ATPase induces a conformational change, opening an inner membrane channel.

Conclusions:

• A model for the assembly and activation of the *L. pneumophila* Dot/Icm machine has been proposed.
• The findings provide a structural basis for understanding T4SS biogenesis and function.
• This research advances our knowledge of bacterial secretion systems and their role in infection.