Helicobacter pylori is a species of bacterium that can colonize the human stomach, causing changes that greatly increase the risk of ulcers and stomach cancer. Some strains of H. pylori produce a protein called CagA, which alters how stomach cells grow and divide. The bacterium injects CagA directly into stomach cells using a syringe-like structure called a type IV secretion system.

Type IV secretion systems are found in many species of bacteria and are involved in a variety of processes, including the exchange of genes between neighboring bacteria. The systems typically have at least 12 components. Previous studies have revealed how the components of some of these systems fit together to form working machines. However, the type IV secretion system that delivers CagA (called the Cag T4SS) contains additional components and it remains unclear how these components are organized in the structure.

A technique called cryo-electron microscopy uses electrons to visualize proteins that have been rapidly frozen so they can be captured and imaged in their natural shape and form. Chung, Sheedlo et al. extracted the Cag T4SS apparatus directly from H. pylori and used cryo-electron microscopy to determine its shape to a high level of detail. These images were then used to build a detailed model of the Cag T4SS that included many of its components.

The model shows that the Cag T4SS is larger and more complex than other type IV secretion systems that have been studied previously. Therefore, Chung, Sheedlo et al. propose that the Cag T4SS is specially adapted to work in the stomach.

These findings open the door for future research to define how individual components of the Cag T4SS help to inject CagA into stomach cells. In addition, future research will allow researchers to understand how the type IV secretion systems found in different bacterial species carry out a wide range of roles.

Bacterial pathogens are a threat to global health and have evolved elaborate strategies to infect their hosts. Many effects of bacteria on host cells require the actions of bacterial secretion systems. Bacterial type IV secretion systems (T4SS) are a diverse class of molecular machines that mediate interbacterial DNA transfer through conjugation as well as delivery of effector proteins into host cells. T4SSs are found in a wide range of bacterial species, including many species that cause human disease, such as Helicobacter pylori, Legionella pneumophila, Bordetella pertussis, Brucella, and Bartonella (Christie et al., 2014; Grohmann et al., 2018).

T4SSs in Gram-negative bacteria contain a minimum of 12 components (designated VirB1-VirB11 and VirD4 in prototype systems), organized into an outer membrane core complex (OMCC), an inner membrane complex (IMC), and in some species an extracellular pilus (Christie et al., 2014; Grohmann et al., 2018; Waksman, 2019). High-resolution structures have been determined for OMCCs from two minimized T4SSs (Xanthomonas citri T4SS and a portion of the OMCC from the pKM101 conjugation system) (Sgro et al., 2018; Chandran et al., 2009). T4SSs in several bacterial species, including Helicobacter pylori and Legionella pneumophila, contain additional components, which are not present in the minimized systems (Grohmann et al., 2018; Frick-Cheng et al., 2016; Schroeder, 2017; Ghosal et al., 2017; Chang et al., 2018; Chetrit et al., 2018; Ghosal et al., 2019; Hu et al., 2019). The H. pylori Cag T4SS is of particular interest because of its role in translocating CagA (a bacterial oncoprotein) into host cells, an important step in gastric cancer pathogenesis (Fischer, 2011; Backert et al., 2017). In addition to containing unique components, the H. pylori Cag T4SS has an OMCC much larger in size and more intricate than those in minimized systems, and contains a periplasmic sub-complex not seen in minimized T4SSs (Grohmann et al., 2018; Chang et al., 2018; Hu et al., 2019). Here we report the use of single particle cryo-EM to determine the structure of a transmembrane Cag T4SS complex extracted and purified from H. pylori. The structure can be divided into three major regions: the OMCC, a periplasmic ring complex (PRC), and a central stalk. The OMCC has a structural organization markedly different from that of T4SS OMCCs in other species, and there is an unexpected symmetry mismatch between the 14-fold-symmetric OMCC and a contiguous 17-fold-symmetric PRC. We propose that the observed structural differences between the Cag T4SS and previously described minimized T4SSs have important functional implications for how T4SSs secrete various kinds of effectors.

Cag T4SS complexes were purified from H. pylori as described (Frick-Cheng et al., 2016) and visualized by single particle cryo-EM (Figure 1 and Figure 1—figure supplement 1A,B). The resulting structure is a large mushroom-shaped complex,~410 Å wide by ~460 Å long, with features that closely match those of the Cag T4SS detected in intact H. pylori using cryo-electron tomography (Chang et al., 2018; Hu et al., 2019) (Figure 1A,B). The global resolution of the map is 5.4 Å when no symmetry is imposed, with the highest resolution regions found near the center of the complex (Figure 1A and Figure 1—figure supplement 1C–E). Although particles adopt a preferred orientation in vitrified ice, both en face and side views are present, which allowed for 3D reconstruction (Figure 1—figure supplement 1B,F). The map can be divided into three major regions: an intricately organized domed cap that is associated with the outer membrane (OMCC), a hollow ring-like mid-region localized within the periplasm (PRC), and a tapered density that extends from the PRC to the inner membrane (Stalk) (Figure 1A and Video 1). Blurry density around the PRC, also visible in cryo-ET images (Chang et al., 2018; Hu et al., 2019), may represent a dynamic or less-structured portion of the T4SS (Figure 1B). Using symmetry, focused refinement, and symmetry expansion, we determined a 3.7 Å resolution map of the OMCC and a 3.5 Å map of the PRC (Figure 1C, Figure 1—figure supplement 2, Videos 2 and 3). The resolution of the OMCC and PRC maps made it possible to trace the secondary structure in these regions (Figure 1D and Videos 2 and 3). Since only a small number of T4SS particles seen in vitrified ice contained the Stalk density, it was not possible to determine the symmetry or obtain high resolution of this part of the complex. An axial section through the map in Figure 1D shows a large cavity running through the Cag T4SS, starting from where the OMCC spans the outer membrane (OM) and extending to the bottom of the PRC (Figure 1E,F). The tapered end of the Stalk begins in the PRC channel and continues through the inner membrane (IM). Models developed from cryo-ET analyses of both the H. pylori and L. pneumophila T4SSs propose a channel in this region of the complex (Ghosal et al., 2017; Chang et al., 2018; Chetrit et al., 2018; Ghosal et al., 2019; Hu et al., 2019). A central section through the longitudinal plane of 3D density suggests there may be a channel that runs through the Stalk (Figure 1B,F). However, due to the low resolution of the Stalk, this channel cannot be clearly visualized in the 3D map.

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The OMCC, with 14-fold symmetry, makes up the ‘mushroom cap’ of the T4SS and is organized into a central and outer ring connected by ‘spokes’ (Figure 2). The outer ring of the OMCC surrounds a central chamber that is ~270 Å wide and tapers to a ~ 35 Å opening at the top (Figure 2B). As seen in OMCCs from minimized T4SSs (Sgro et al., 2018; Chandran et al., 2009), the H. pylori Cag T4SS OMCC contains both an outer layer (O-layer) and a thinner inner layer (I-layer) (Figure 3A). ‘Spokes’ and outer rings are visible in both the O-layer and I-layer of the Cag T4SS (Figure 3B and Figure 3—figure supplement 1), but are not present in OMCCs from minimized T4SSs. The resolution of the cap is high enough to begin mapping the molecular organization of individual OMCC components (Figure 4A,B). Previous mass spectrometry and Western blot analyses indicated that the isolated complexes contain CagY, CagX, CagM, CagT, and Cag3 (Frick-Cheng et al., 2016). In the map, we can trace and identify portions of CagY (residues 1677–1816 and 1850–1907), CagX (residues 349–510), and CagT (residues 26–269) (Figure 4C and Figure 4—figure supplement 1A–C, left panels).

Figure 2

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CagY forms the crest of the cap-like structure of the OMCC and is comprised of β-sheets intertwining to position two helices per protomer atop the cap (Figure 4A–C and Figure 4—figure supplement 1A, left panel). We predict that these helices breach the outer membrane, resulting in the formation of a channel (Figure 4B). The C-terminal portion of CagX is comprised of two β-sheets preceded by a long helix (Figure 4C and Figure 4—figure supplement 1B, left panel). This helix extends from the top of the O-layer through the central chamber into the I-layer of the OMCC (Figure 4B). Adjacent to CagX, we traced a continuous chain 243 residues long corresponding to CagT (Figure 4C). It includes a globular subdomain consisting of two β-sheets followed by a long C-terminal extension of three helices that contribute to the spoke and extend toward the edge of the map (Figure 4B and Figure 4—figure supplement 1C, left panel). A predicted lipidation site within the N-terminal tail of CagT (Fischer, 2011; Akopyants et al., 1998) was not observed in the cryo-EM map, but the N-terminus of CagT is positioned for this interaction with the outer membrane.

Extending to the edge of the O-layer, we have constructed poly-alanine models of protein(s) that we could not identify (Figure 4A,B and Figure 4—figure supplement 1D). These components fill the remaining density within the spokes and consist predominantly of a repetitive fold composed of repeating units of β-sheets flanked by helices (Figure 4A,B and Figure 4—figure supplement 1D). We predict that Cag3 is a component of the OMCC periphery based on a previous study showing that Cag T4SS complexes isolated from a Δcag3 mutant strain lacked peripheral components of the OMCC (Frick-Cheng et al., 2016; Hu et al., 2019), but we were unable to obtain a register. Within the I-layer we have observed at least two distinct bundles of helices of ~200 residues and ~300 residues (Figure 4A,B and Figure 4—figure supplement 1E). Based on structural studies of the X. citri T4SS (Grohmann et al., 2018), the I-layer of the Cag T4SS is predicted to contain portions of CagY and CagX, but none of the poly-alanine models allowed us to unambiguously attribute any portion of CagY, CagX, or other T4SS components to this region.

The PRC, with 17-fold symmetry (Figure 4D,E and Figure 4—figure supplement 2), is a short hollow tube, 90 Å tall and 185 Å wide with 96 Å internal diameter (Figure 4D,E), connecting the OMCC and the Stalk regions of the Cag T4SS (Figure 1A). Although PRCs have not been detected in structural studies of E. coli or X. citri T4SSs (Waksman, 2019), this region was identified in cryo-ET studies of both H. pylori and L. pneumophila T4SSs (Ghosal et al., 2017; Chang et al., 2018; Chetrit et al., 2018; Ghosal et al., 2019; Hu et al., 2019). The protein(s) in this region of the cryo-EM maps were modeled as three distinct polyalanine chains (Figure 4—figure supplement 1F–H). This portion of the H. pylori Cag T4SS structure is predicted to contain CagM (Waksman, 2019; Chang et al., 2018), but the three polyalanine chains could not be unambiguously attributed to CagM or other T4SS components. A symmetry mismatch occurs between the OMCC and PRC, going from 14-fold symmetry in the OMCC to 17-fold symmetry in the PRC. While the OMCC, PRC, and Stalk make physical contact in the lower resolution structure with no applied symmetry (Figure 5), in the refined structures these connections are lost because of the symmetry mismatch. Analysis of the 5.4 Å structure suggests that the connection between OMCC and PRC may occur by contacts between the 14 α-helices extending from the N-terminus of CagX and the 17 unidentified α-helices extending up from the PRC (Figure 1A,E, and Figure 5). The tips of these α-helices are presumed to be flexible, since the corresponding densities are not well defined in the higher resolution maps (Figure 4D).

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Overall, the H. pylori OMCC is much larger (410 Å diameter) than other structurally characterized OMCCs (225 Å in X. citri and 170 Å in pKM101 conjugation system) (Sgro et al., 2018; Chandran et al., 2009) (Figure 6), and the components are more intertwined. While the inner chamber of the H. pylori OMCC is larger than what is seen in the E. coli and X. citri structures, the dimension of the H. pylori outer membrane pore, at 35 Å, is smaller than the X. citri outer membrane pore (45 Å) but larger than the E. coli outer membrane pore (25 Å) (Figure 6B). The dimensions of the H. pylori OMCC resemble those of the L. pneumophila Dot/Icm T4SS (Ghosal et al., 2017; Chetrit et al., 2018; Ghosal et al., 2019), but there is very little sequence relatedness when comparing components of the H. pylori and L. pneumophila T4SSs, and the L. pneumophila T4SS OMCC has 13-fold symmetry instead of 14-fold symmetry (Chetrit et al., 2018; Ghosal et al., 2019). The C-terminal portion of CagY is structurally similar to VirB10 and TraF homologs (Figure 6 and Figure 4—figure supplement 1A), and the C-terminal portion of CagX is structurally similar to VirB9 and TraO homologs (Figure 6 and Figure 4—figure supplement 1B). Both CagY and CagX are much larger in size than characterized VirB/Tra homologs (Fischer, 2011; Backert et al., 2017), and correspondingly, CagX has a long α-helix that is absent in the structures of TraO or VirB9 (4, 5) (Figure 6 and Figure 4—figure supplement 1B). Consistent with the limited sequence relatedness of CagT to VirB7 homologs (Fischer, 2011; Backert et al., 2017), there is relatively little structural relatedness when comparing CagT with VirB7 except for an N-terminal portion that contains little secondary structure (Figure 6 and Figure 4—figure supplement 1C).

Figure 6

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In summary, these results provide the first high resolution structure of a transmembrane complex from a non-canonical T4SS and provide important new insights into a bacterial T4SS that contributes to the pathogenesis of gastric cancer. The Cag T4SS structure differs markedly from structures of previously described T4SSs, including the presence of an expanded OMCC with components that are more structurally intertwined and an unexpected symmetry mismatch between the OMCC and PRC. We predict that these differences will have important functional implications for the mechanism of CagA translocation.

The cryo-EM volumes have been deposited in the Electron Microscopy Data Bank under accession codes EMD-20023 (T4SS C1 reconstruction), EMD-20020 (Focused OMCC Reconstruction), EMD-20022 (OMCC Asymmetric Reconstruction), EMD-20021 (Focused PRC Reconstruction). Map coordinates have been deposited in the Protein Data Bank under accession numbers 6OEE (CagT), 6OEG (CagX), and 6ODI (CagY), 6OEF (O-layer), 6OEH (I-Layer), and 6ODJ (PRC).

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   2. Sheedlo MJ
   3. Campbell AM
   4. Sawhney N
   5. Frick-Cheng AE
   6. Lacy DB
   7. Cover TL
   8. Ohi MD

   (2019) Electron Microscopy Data Bank

   ID EMD-20023. T4SS C1 reconstruction.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20023
2. 1. Chung JM
   2. Sheedlo MJ
   3. Campbell AM
   4. Sawhney N
   5. Frick-Cheng AE
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   (2019) Electron Microscopy Data Bank

   ID EMD-20020. Focused OMCC Reconstruction.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20020
3. 1. Chung JM
   2. Sheedlo MJ
   3. Campbell AM
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   ID EMD-20022. OMCC Asymmetric Reconstruction.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20022
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   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20021
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   ID 6OEE. CagT.

   https://www.rcsb.org/structure/6OEE
6. 1. Chung JM
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   ID 6OEG. CagX.

   https://www.rcsb.org/structure/6OEG
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   ID 6ODI. CagY.

   https://www.rcsb.org/structure/6ODI
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Author details

1. Jeong Min Chung

   Life Sciences Institute, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Michael J Sheedlo

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4285-8764

2. Michael J Sheedlo

   Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, United States

   Contribution

   Data curation, Formal analysis, Validation, Visualization, Writing—original draft, Writing—review and editing

   Contributed equally with

   Jeong Min Chung

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3185-1727

3. Anne M Campbell

   Department of Medicine, Vanderbilt University School of Medicine, Nashville, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Neha Sawhney

   Department of Medicine, Vanderbilt University School of Medicine, Nashville, United States

   Contribution

   Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4943-1018

5. Arwen E Frick-Cheng

   1. Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, United States
   2. Department of Immunology and Microbiology, University of Michigan School of Medicine, Ann Arbor, United States

   Contribution

   Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

6. Dana Borden Lacy

   1. Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, United States
   2. Veterans Affairs Tennessee Valley Healthcare System, Nashville, United States

   Contribution

   Resources, Formal analysis, Supervision, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   For correspondence

   borden.lacy@vumc.org

   Competing interests

   No competing interests declared

7. Timothy L Cover

   1. Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, United States
   2. Department of Medicine, Vanderbilt University School of Medicine, Nashville, United States
   3. Veterans Affairs Tennessee Valley Healthcare System, Nashville, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   For correspondence

   timothy.l.cover@vumc.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8503-002X

8. Melanie D Ohi

   1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
   2. Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   mohi@umich.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1750-4793

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