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

Abstract

ABSTRACT Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane. IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

Keywords

• secretion system
• whole-cell tomography
• nanomachine
• protein transport
• Dot/Icm system
• effector proteins