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

Donghyun Park,David Chetrit,Craig R Roy,Bo Hu,Jun Liu,Carmen Buchrieser

doi:10.1128/mbio.03328-19

Donghyun Park, David Chetrit + Show 4 more

Open Access

https://doi.org/10.1128/mbio.03328-19

Copy DOI

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.

Highlights

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
Xanthomonas citri VirB/D, Helicobacter pylori Cag, and E. coli R388-encoded IVA secretion machine structures resolved by single-particle cryoelectron microscopy and X-ray crystallography revealed that the IVA secretion machines are composed of an outer membrane-associated core complex (OMCC) that includes a large periplasmic assembly of proteins that connects to the secretion channel in the outer membrane [9,10,11,12,13]
Unlike the E. coli R388-encoded IVA secretion machine, the cytoplasmic complex of the L. pneumophila Dot/Icm machine forms a central secretion channel made of an inner membrane (IM)-anchored DotO ATPase (VirB4-related) complex that consists of a hexamer of DotO dimers

Summary

Introduction

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. Type IV secretion systems (T4SSs) are used by many bacteria to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. Xanthomonas citri VirB/D-, Helicobacter pylori Cag-, and E. coli R388-encoded IVA secretion machine structures resolved by single-particle cryoelectron microscopy (cryo-EM) and X-ray crystallography revealed that the IVA secretion machines are composed of an outer membrane-associated core complex (OMCC) that includes a large periplasmic assembly of proteins that connects to the secretion channel in the outer membrane [9,10,11,12,13]. In situ cryo-ET structures of the Cag secretion system in H. pylori and the F plasmid conjugation system in E. coli [19, 20] provided evidence that the VirB4-related ATPases in these systems assemble a structure that is similar to that of the DotO complex

Methods

Results

Conclusion