In Situ Molecular Architecture of the Helicobacter pylori Cag Type IV Secretion System

Abstract

Helicobacter pylori colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. H. pylori strains carrying the cag pathogenicity island (cagPAI) are associated with increased risk of disease progression. The cagPAI encodes the Cag type IV secretion system (Cag_T4SS), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant Cag_T4SS machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple Cag_T4SS nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cagα, docked at the base of the CagE hexamer; and (iii) VirD4-like Cagβ and other unspecified Cag subunits, associated with the stacked CagE/Cagα complex and forming the outermost rings. The Cag_T4SS and recently solved Legionella pneumophila Dot/Icm system comprise new structural prototypes for the T4SS superfamily.IMPORTANCE Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciens VirB/VirD4_T4SS, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori Cag_T4SS T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/Icm_T4SS Here, we present structures of native and mutant H. pylori Cag machines determined by in situ cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the Cag_T4SS aligns structurally much more closely to the Dot/Icm_T4SS than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.

Keywords: Helicobacter pylori; cryoelectron tomography; nanomachine; pathogenesis; protein translocation; type IV secretion.

FIG 1

H. pylori cell with Cag T4SSs visualized by cryoelectron tomography. (A and B) Slice of a tomographic reconstruction of a typical H. pylori cell (A) and its surface rendering (B) showing multiple T4SSs (arrows) embedded in the cell envelope. OM, outer membrane; IM, inner membrane. (C) A central section of the subtomogram average structure of the intact T4SS showing the OM, IM, outer membrane complex (OMC), and central cylinder in detail. O, outer layer; I, inner layer. (D) Cross sections viewed from the top of the OMC at the positions indicated in panel C reveal the 14-fold symmetry as well as the change in chirality of the OMC across its height (see Movie S3). Colored dots denote diameters of central cylinder (19 nm, orange), central channel (14 nm, light blue), and plug domain (7 nm, white). (E and F) A central section of the subtomogram average structure of the Δcag3 mutant machine reveals a truncated OMC (E); cross-sectional views (F) show knobbed projections from the central cylinder with 14-fold symmetry present in the I-layer but not in the O-layer. (G, H, and I) 3D surface renderings of the Cag_T4SS show the O/I-layered spoked wheel and the cylinder, plug, and collar domains of the OMC in side, central cut, and top-down views. (J) A cartoon model of the OMC with proposed contributions of CagX, CagY, and CagM to the central cylinder, of CagT to the cylinder, and of Cag3 to the spoked wheel. The local refinements of the OMC did not resolve the IMC (gray box).

FIG 2

The Cag_T4SS IMC. (A) A local refinement of the cytoplasmic portion of the Cag_T4SS (boxed) revealed a detailed structure of the IMC. (B) Central section of the subtomogram average structure of the IMC. IM, inner membrane. The central Chi (X) structure and flanking tiers are identified (dashed lines). (C) The section shown in panel B presented in a different view as indicated by the vertical and curved arrows shown between panels B and C. (D) Cross section of the IMC at the position indicated in panel C revealing the 3-ring architecture and 6-fold symmetry of the IMC. (E, F, and G) 3D surface renderings of the T4SS machine IMC presented in side (E), central cut (F), and bottom (G) views. I, inner ring; M, middle ring; O, outer ring. Numbers correspond to the diameters (in nanometers) of the upper, middle, and lower regions of the central Chi and of the M and O rings. Panel G shows the hexameric arrangements of the (i) bottom portion of the central Chi (I-ring), (ii) M ring with the 6 tiers joined at their bases, and (iii) O ring composed of 12 tiers arranged in 6 pairs.

FIG 3

Contributions of the Cag ATPases to the Cag_T4SS IMC. Columns A to D present structures of the IMCs from the native (WT) and Δcagβ, Δcagα, and ΔcagE mutant machines. (Row I) Central slices of the averaged IMC structures. IM, inner membrane. (Row II) 3D surface renderings, cutaway side views. Numbers correspond to diameters (in nanometers) of the regions shown. (Row III) 3D surface renderings, bottom views. The lower regions of the I and M rings are shaded in yellow and, together with the yellow-shaded O-ring, reflect density contributions to the IMC associated with production of Cagβ.

FIG 4

Model depicting an outside-to-inside assembly pathway for the Cag_T4SS. The central cylinder assembles first and serves as a scaffold for elaboration of the OMC and then the IMC. Our findings highlight the sequential order of assembly of the Cag ATPases as follows: CagE is initially recruited followed by Cagα and, finally, Cagβ. Cag subunits in addition to those shown are recruited to build out the OMC and IMC. In H. pylori cells exposed to human epithelial cells, the Cag_T4SS is activated (yellow lightning bolt) to assemble extracellular pili or sheathed tubes and translocate the CagA substrate. Structural changes accompanying Cag_T4SS activation are not yet defined. See text for further details.