T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly

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The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the Salmonella enterica serovar Typhimurium Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR ‘export apparatus’ complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.

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Fig. 1: Cryo-EM snapshots capture the molecular basis of needle complex assembly.
Fig. 2: Recruitment of an extra secretin monomer in the periplasm locally alters ring stoichiometry and facilitates multivalent symmetrical coupling of the OM and IM T3S components.
Fig. 3: Molecular interactions that govern NC assembly.
Fig. 4: The SpaPQR complex forms an assembly platform for PrgJ and PrgI needle polymerization.
Fig. 5: The structural snapshots capture the transitions associated with needle assembly and secretin gating.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Cryo-EM maps and atomic coordinates have been deposited with the EMDB and PDB with the following accession codes EMDB ID: EMD-20310, EMD-20311, EMD-20312, EMD-20313, EMD-20314, EMD-20315, EMD-20316, EMD-20317, EMD-20556 and PDB ID: 6PEE, 6PEM, 6PEP, 6Q14, 6Q15 and 6Q16.


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We thank S. Miller for providing S. Typhimurium deletion strains and plasmids, as well as the InvG antibody. This work was funded by operating grants from CIHR, to N.C.J.S. and B.B.F. and the Howard Hughes International Senior Scholar Program, to N.C.J.S. B.B.F. is the UBC Peter Wall Distinguished Professor. N.C.J.S. is a Tier I Canada Research Chair in Antibiotic Discovery.

Author information

M.V. performed all cloning and protein purification. C.H. performed single-particle cryo-EM grid preparation and data collection with assistance from Z.Y. J.H. performed data processing and map generation with assistance from L.J.W. and C.E.A. L.J.W. performed model building, refinement and structural analysis with help from J.H. W.D. made the Salmonella deletion mutants and expression strains and carried out secretion assays with assistance from B.B.F. L.J.W., J.H. and N.C.J.S. wrote the manuscript with input from all.

Correspondence to Natalie C. J. Strynadka.

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Supplementary Information

Supplementary Table 1, Supplementary Figs. 1–10, Supplementary Video Legends and Supplementary References.

Reporting Summary

Supplementary Video 1

Allosteric step of InvG periplasmic gate opening.

Supplementary Video 2

Steric step of InvG periplasmic gate opening.

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