Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System

Abstract

Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P. asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing a hexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.

The extracellular contractile injection system is enriched in environmental microbes and associates with numerous toxins

“…Some of the toxins were reported to be located inside the hollow tube. The illustra on is based on Jiang et al 3 cultures of the strains harboring the vectors of interest were grown in SD -leu media. The cultures OD was normalized and then washed once with water and split into two: one part was grown overnight in repressive condi ons (SD -leu + 2% glucose) and the other part was grown in induc ve condi ons (SD -leu + 2% galactose).…”

“…and tail fibers that likely serve to adhere to target cells ( Figure S1) 2,3 . eCIS contrac on propels the tube out of the sheath, likely enabling the sharp p to perforate the target cell membrane.…”

“…, and SepC-like 5 . The high-resolu on structures of Afp and PVC par cles were recently solved using cryo-EM and provided excellent maps of the protein interac ons within these par cles 2,3 . Unlike the insec cidal ac vi es of Afp, AfpX, and PVC, the metamorphosis-associated contrac le structures (MACs) encoded by Pseudoalteromonas luteoviolacea plays a mutualis c role.…”

Photorhabdus: a tale of contrasting interactions

“…Heterologous expression of PVC loci from P. asymbiotica in E. coli established that toxicity was associated with the appearance of particles resembling R-type pyocins in the culture supernatant [65]. Recent cryo-EM studies have shown the PVCs to be a >10 MDa particle that resembles a simplified T4 phage tail [66]. The PVC particle appears to act like a primed syringe that, when triggered, delivers a payload directly through the membrane of the target cell [67] (see Fig.…”

Architecture of the flexible tail tube of bacteriophage SPP1

“…Also, cryo-EM models of the tails of the flagellotropic tailed bacteriophage YSD1 11 and the Siphoviridae-like gene transfer agent of Rhodobacter capsulatus 12 were reported recently. All models show a striking structural homology between TTPs from Myoviridae and Siphoviridae; as well as between these phage TTPs and the tube-forming proteins from other injection systems, like the bacterial type VI secretion system 13 and the extracellular injection system from bacteria and archeae 14 . The tube-forming proteins share a common fold composed of two orthogonally packed β-sheets that hexamerize through the formation of an inner β-barrel that defines the lumen of the tube.…”