H. pylori colonizes the stomach in about half of the world’s human population, and its presence in the stomach is the strongest known risk factor for gastric cancer (Uemura et al., 2001). H. pylori strains containing a pathogenicity island (cag PAI), encoding CagA (a secreted effector protein) and the Cag T4SS, are associated with a significantly higher incidence of gastric cancer than that associated with strains lacking the cag PAI (Blaser et al., 1995; Cover, 2016). Gastric cancer is the third leading cause of cancer-related death worldwide, and there are nearly one million new gastric carcinoma cases annually (Plummer et al., 2016). Therefore, understanding the molecular organization of the Cag T4SS is an important goal.

Bacterial T4SSs are versatile molecular machines that can mediate a wide variety of functions, including horizontal transfer of DNA among bacteria (conjugation) and delivery of effector proteins into eukaryotic cells (Galán and Waksman, 2018; Grohmann et al., 2018; Waksman, 2019). T4SSs in Gram-negative bacteria are comprised of an inner membrane complex, a membrane-spanning core complex that extends from the inner membrane to the outer membrane, and in some systems, an extracellular pilus (Galán and Waksman, 2018; Grohmann et al., 2018; Waksman, 2019). T4SSs vary in complexity, ranging from ‘minimized’ or ‘prototype’ systems that contain about 12 components (VirB1-11 and VirD4), as found in Agrobacterium tumefaciens, Xanthomonas citri, or conjugation systems, to expanded systems containing more than 20 components (Bhatty et al., 2013). Expanded T4SSs contain unique species-specific components and are much larger in size than their minimized counterparts. Examples of the larger, ‘expanded’ T4SSs include the Dot/Icm T4SSs of Legionella pneumophila and Coxiella and the Cag T4SS of H. pylori, each of which transports effector proteins into host cells (Chetrit et al., 2018; Chung et al., 2019; Frick-Cheng et al., 2016; Ghosal et al., 2019). The Legionella Dot/Icm translocates more than 300 effector proteins, thereby allowing intracellular bacterial replication (Qiu and Luo, 2017). In contrast, the H. pylori Cag T4SS is only known to deliver one effector protein, CagA (a bacterial oncoprotein), into gastric cells, resulting in altered cell signaling (Cover et al., 2020).

Structural studies of minimized T4SSs have revealed a core complex (typically ~250 Å in width and ~150 Å in height) that contains three proteins (VirB7, VirB9, and VirB10) (Chandran et al., 2009; Fronzes et al., 2009; Low et al., 2014; Sgro et al., 2018). The H. pylori Cag T4SS core complex is a much larger mushroom-shaped assembly (~400 Å in width and ~250 Å in height) that contains structural homologs of the proteins found in minimized systems [CagT (VirB7), CagX (VirB9), and CagY (VirB10)] along with additional proteins (Cag3 and CagM) that lack homologs in other bacterial species (Chung et al., 2019; Frick-Cheng et al., 2016). The Cag T4SS core complex has been described as consisting of three distinct structural features: the outer membrane cap (OMC) consisting of an outer-layer (O-layer) and inner-layer (I-layer), a periplasmic ring (PR), and a stalk (Chang et al., 2018; Chung et al., 2019; Hu et al., 2019). There is an apparent symmetry mismatch between the 14-fold-symmetric OMC and the 17-fold symmetric PR (Figure 1a; Chung et al., 2019). Our recent sub-4Å structure of the Cag T4SS revealed that central portions of the OMC are composed of CagT, CagX, and CagY (VirB7, VirB9, and VirB10 homologs), but the resolution of the map did not allow us to build molecular models for the entire complex (Chung et al., 2019). Specifically, we could not determine the molecular composition of the PR or define the locations of Cag3 or CagM. In the current study, we undertook a further analysis of single particle cryo-EM data for the wild-type Cag T4SS core complex as well as analysis of a mutant form of the core complex. These efforts allow us to define the locations of Cag3 and CagM within the core complex, determine the molecular composition of the PR, and determine the stoichiometry of components within the OMC.

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To build a more complete model of Cag T4SS organization and determine how the OMC and PR interact, we expanded our cryo-EM studies to analyze the core complex from a Cag3-deficient H. pylori strain (Δcag3) (Frick-Cheng et al., 2016). We also implemented new data analysis techniques for processing previously collected cryo-EM data for wild-type (WT) core complexes (including per-particle defocus refinement and beam tilt estimation), which resulted in higher resolution maps (Figure 1—figure supplements 1, 2 and 3, Appendix 1—table 1). When comparing the maps reconstructed from the WT and Δcag3 strains, peripheral components of the O-layer of the OMC are missing in the Δcag3 map. At least a portion of this peripheral density is likely composed of Cag3, since it is the component most notably absent in core complex preparations from the Δcag3 mutant [based on SDS-PAGE and mass spectrometry analyses (Figure 1—figure supplement 4)]. Notably, the PR remained intact in the Δcag3 map, and weak density was still observed within the I-layer (Figure 1b,c).

As described previously, CagT and the C-terminal portions of CagX (residues 349–514) and CagY (residues 1677–1909) are localized to central regions of the O-layer of the OMC (Chung et al., 2019). The increased resolution of the new maps allowed us to define the OMC asymmetric unit and determine the molecular composition of regions that were previously undefined (Figure 1—figure supplement 2d, Figure 2a, Figure 2—figure supplements 1–6, Video 1). Positioned adjacent and peripheral to the previously described CagT (CagT-1), we identified another copy of CagT which we call CagT-2 (Figures 2a,b and 3a). We found that the two copies of CagT differ significantly in the conformations of both their N- and C-termini (Figure 4a). In CagT-1, the N-terminus is an extended loop that is nestled against a portion of CagT-1 and CagX from the neighboring asymmetric unit, resulting in an interface similar to what was described for X. citri VirB7 and VirB9 (Sgro et al., 2018). Within CagT-2, the N-terminus adopts a different conformation, one in which it is wrapped back around the central, globular VirB7-like fold, forming a β-strand that completes a β-sheet within an adjacent protein that we designate as Cag3-1 (Figure 4c–f, Video 2). This rearrangement likely occurs due to changes in a stretch of amino acids (I44-I50) within the center of this loop (Figure 4—figure supplement 1a–c). While it is currently not clear what drives these conformational differences, it is possible that this rearrangement results from the different repertoire of binding partners mediated by this loop (Figure 4—figure supplement 1d–f). Despite the structural differences between CagT-1 and CagT-2, a putative lipidation site (C21) in both proteins (McClain et al., 2020) is placed in close proximity to the outer membrane (Figure 4g). The C-termini of both CagT molecules adopt extended conformations that differ in their overall direction and organization. In CagT-1, the C-terminal α-helices extend outward from the center of the map, forming contacts with CagT-1 Cag3-1, Cag3-2, and Cag3-4 within the OMC (Figure 4h; Chung et al., 2019). Conversely, the C-terminal α-helices of CagT-2 are connected by an apparently dynamic linker and are arranged such that they contact a distinct repertoire of OMC proteins, including Cag3-1, Cag3-2, and CagM-1 (Figure 4i). In both cases, the arrangement of these helical extensions deviates significantly from corresponding regions in the X. citri VirB7 structure, where the domain terminates shortly after the core VirB7 fold (Sgro et al., 2018; Figure 4—figure supplement 2a). Because of this, we suggest that the addition of these C-terminal α-helices is critical to maintaining the expanded architecture of the Cag T4SS OMC, as it has been previously shown that Cag3 cannot be effectively incorporated into core complexes lacking CagT (Frick-Cheng et al., 2016).

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The orientation of the CagT-1 C-terminal α-helices differs significantly in the Δcag3 OMC reconstruction compared to their orientation in the wild-type OMC reconstruction. In contrast to the extended conformation of the CagT-1 α-helices in the WT OMC, in the Δcag3 OMC these helices are folded inward and pack against α-helices of CagX and CagY within the central chamber (Figures 2c,d and 5a, Figure 4—figure supplement 2b). Notably, the second α-helix of the C-terminal extension is not observed within the Δcag3 OMC map and may be flexible. We suspect that the C-terminal α-helices of CagT-1 adopt multiple conformations in the Δcag3 OMC, since additional components that would otherwise lock them into a single conformation are not present. In addition to the loss of peripheral density corresponding to Cag3 within the Δcag3 map, we observe no density for CagT-2 in the Δcag3 map. One possible explanation for the loss of CagT-2 is that it cannot effectively fold when Cag3 is absent, leading to dissociation of the complex or unfolding of the protein. These results are largely in agreement with previous cryo-electron tomography (Cryo-ET) studies, which noted the loss of most of the O-layer of the OMC in the Δcag3 strain (Hu et al., 2019). CagX and CagY adopt nearly identical conformations in the ∆Cag3 mutant core complex when compared to the WT structure (RMSDs of 0.5 Å and 0.3 Å, respectively) (Figure 5b,c). This is consistent with a proposal that assembly of the Cag T4SS is initiated by positioning CagX and CagY in association with the outer membrane (Hu et al., 2019).

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Further analysis revealed five copies of Cag3 within each asymmetric unit (named Cag3-1 through Cag3-5, based on their proximity to the central channel) (Figure 2a,b Figure 3b). All five copies of Cag3 are found within the periphery of the OMC and adopt similar core folds (with RMSDs of 0.5–1.3 Å) that are connected via a highly interwoven architecture (Figure 6a–c). Although the resolution is lower towards the periphery of the map than near the center, we note that several Cag3 structural features are recognizable in all five copies of Cag3 within the cryo-EM density map (Figure 6d–f, Figure 6—figure supplement 1). The first copy of Cag3 (Cag3-1) contains the longest uninterrupted span of density (corresponding to residues 62–308) and consists of two globular domains (proximal and distal) connected by a long loop, without an intramolecular interface (Figure 6a,b). The proximal domain of Cag3-1 is positioned adjacent to CagT-2 within the center of the map and contains a loop (residues 181–204) that interacts with adjacent asymmetric units (Figure 6g). We predict that this loop may act as a lynchpin during assembly, since it forms the most extensive network of contacts within the center of the complex. Notably, the proximal domain of Cag3-1 consists of a ‘core’ fold that is similar to a corresponding fold in CagT and the related VirB7 homolog from X. citri (Figure 6h; Sgro et al., 2018). The distal domain of Cag3-1 is linked to the proximal domain through a linker that runs along a second copy of Cag3 (Cag3-2) and resides within the periphery of the map,~50 Å away from the proximal domain, tucked up next to another copy of Cag3 (Cag3-3) (Figure 2a,b). A distal domain was not seen in any of the other copies of Cag3, possibly due to the lower resolution at the periphery of the map or to inherent heterogeneity associated with this domain.

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While the local resolution of the I-layer is lower than that of the O-layer, we observe several key features of the I-layer that are consistent with the sequence of CagM (Figures 3c and 7a). We modeled a portion of CagM (residues 187–366) into peripheral density within the I-layer and observe an α-helical fold with two sub-domains: a 3-helix bundle at the N-terminus connected by a short loop to a 5-helix fold at the C-terminus. We also observe a second helical fold containing 9 α-helices, arranged in a nearly identical fashion as in CagM-1 within the center of the I-layer, albeit at lower resolution (Figure 7b). We fit the CagM sequence into this density and note that the refined structure (designated CagM-2) again contains two subdomains connected via a flexible hinge (Figure 7c). When aligning each subdomain of CagM-1 with the corresponding subdomain of CagM-2, the RMSD for each is ~1.1 Å, supporting our assignment of this portion of the map as a second copy of CagM (Figure 7d,e). The difference in the orientation of the subdomains in CagM-1 and CagM-2 is the result of unique contacts mediated by the C-terminal subdomains (Figure 7f,g).

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Based on these analyses, we conclude that the OMC proteins exist at a stoichiometry of 1:1:2:2:5 (CagX:CagY:CagM:CagT:Cag3). This stoichiometry helps explain the large size of the Cag T4SS core complex, since core complexes in characterized minimized systems contain only single copies of each component within the asymmetric unit (Fronzes et al., 2009; Sgro et al., 2018). Illustrating this point, we note that the inclusion of 5 copies of Cag3 within the asymmetric unit results in 70 copies of Cag3 incorporated into the fully assembled Cag T4SS OMC. In total, the Cag T4SS OMC contains 154 different polypeptide chains. In comparison, prototype core complexes contain a total of 42 different polypeptide chains (14 copies each of VirB7, VirB9 and VirB10).

The resolution of the PR within the Δcag3 map is higher than what was previously determined for the wild-type complex (Figure 1—figure supplement 2j,k; Chung et al., 2019). Because of this advance, it is possible to identify the two components of the PR as portions of CagX (residues 32–130, 261–323) and CagY (residues 1469–1603). The PR is mostly comprised of a globular domain formed by an N-terminal portion of CagX, which starts from the cytoplasm-facing side of the PR, wraps back and forth to form two β-sheets, and ends in an α-helix pointed towards the OMC (Figure 8a, Video 3). The N-terminal domain of CagX is homologous to the N-terminal domain of X. citri VirB9 (RMSD of 0.8 Å, PDB 6GYB) (Figure 8b–d; Sgro et al., 2018). The C-terminal domain of CagX is also highly similar to its X. citri counterpart (RMSD of 0.6 Å, PDB 6GYB) (Figure 8e,f). The major difference between the two proteins is the presence of an insertion within CagX that forms a long α-helix in the center of the molecule (Figure 8d,g). Because of this feature, the N- and C-terminal domains of H. pylori CagX are positioned ~200 Å apart (within the PR and OMC, respectively), whereas the corresponding domains of X. citri VirB9 are ~80 Å apart (Figure 8h).

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Wrapped around the PR portion of CagX is a segment of CagY that starts from the cytoplasm-facing side of the PR and forms four α-helices that are connected by loops that vary in length (ranging from 3 to 41 residues) (Figure 8i). This is similar to a described interaction between a corresponding part of VirB10 and the N-terminal domain of VirB9 in the X. citri T4SS (Sgro et al., 2018). In both cases, a segment of CagY/VirB10 in this region is linked to the C-terminal portion of the protein through a linker which is not directly observed in the data. Both CagY and X. citri VirB10 start with the N-terminus of this portion positioned near the periplasmic space and pass ‘below’ two protomers of CagX/VirB9. Both proteins then extend upward toward the outer membrane through the interior of the ring formed by the N-terminal domain of CagX/VirB9. The CagX-CagY interface in H. pylori is much larger than the corresponding VirB9-VirB10 interface, likely due to an expansion of CagY compared to VirB10 homologs in minimized systems (Figure 8j). The position of CagY within the PR could potentially allow the unresolved segment (residues 1604–1676) to span the inner chamber of the OMC to connect with a C-terminal domain of CagY in the OMC. We note that the portion of CagY visible in the PR is positioned so that the remaining N-terminal 1468 residues (not observed) likely extend into the periplasm, possibly contributing to the low resolution ‘collar’ and stalk densities (Figure 8k; Hu et al., 2019).

The identification of CagX and CagY within the PR was unexpected, as it suggests that each protein exists within both the OMC and PR, two distinct regions of the T4SS that differ in overall symmetry (Figure 9a). Although we cannot specifically trace either component across the symmetry mismatch, we note that the long helical expansion within CagX appears to bridge the OMC and PR. This model is supported by focused refinement of the connecting region between the OMC and PR in the WT map without imposition of symmetry, which shows 14 tubes of helical density connecting the OMC and PR (Figure 9b). Notably, only 14 copies of CagX within the PR appear to traverse the symmetry mismatch, leaving three domains within the PR without an obvious connection to the OMC (Figure 9c, Video 4). It is not clear if the density corresponding to these copies of CagX and CagY cannot be traced due to inherent flexibility within the respective C-terminal domains, if these additional protomers represent truncated versions of CagX and CagY, or if they represent uncharacterized structural homologs.

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The Cag T4SS structure reported here represents the most comprehensive high-resolution analysis of the unique architectural features of an expanded T4SS to date. We have identified the positions of all five core complex proteins, including the previously uncharacterized Cag3 and CagM. A notable discovery is the structural conservation between central domains of CagT and Cag3, a result which could not have been predicted based on sequence comparisons alone. Our finding that the Cag T4SS incorporates multiple copies of Cag3, CagM and CagT into the OMC allows for a refined understanding of how this very large complex is assembled from only a few components. The current analysis also provides a better understanding of how the components of the Cag T4SS are arranged, highlighting important interactions among the newly described components. Specifically, interactions between CagT-1-CagT-2, CagT-1-Cag3-1, Cag3-Cag3, Cag3-CagM, and CagM-CagX have all been localized within this map. Although this study presents the first structural description of these interactions, it is interesting to note that many of these interactions have been previously detected by various biochemical methods. Specifically, interactions between Cag3-Cag3, Cag3-CagT, Cag3-CagM, CagX-CagM, and CagT-CagM were previous detected using a yeast two-hybrid screening approach or by studies of recombinant proteins produced in E. coli, and are consistent with the structural observations reported here (Busler et al., 2006; Kutter et al., 2008; Smart et al., 2017). Importantly, the structural detail obtained from single particle cryo-EM analysis reveals the context of these interactions, allowing us to define which interactions occur within one asymmetric unit and which interactions occur between adjacent asymmetric units.

The structure that we present here provides an improved understanding of how the PR is assembled and highlights the presence of portions of CagX and CagY within the PR. We show that CagX and CagY assemble to form portions of both the OMC and PR, bridging the previously described symmetry mismatch within the core complex (Chung et al., 2019). Although the existence of CagX and CagY in both the OMC and PR seems inconsistent with the observed symmetry mismatch, it should be noted that this is not the first example of such a phenomenon. Indeed, a similar phenomenon has been detected in the Type Three Secretion System (T3SS) of Salmonella, in which one molecule (InvG) exists on both sides of a C15-C16 symmetry mismatch (Hu et al., 2018). Symmetry mismatch has been observed in multiple types of bacterial secretion systems or other large bacterial machines, including T2SSs, T3SSs, the L. pneumophila Dot/Icm T4SS, T6SSs, and the Salmonella flagellar motor (Chernyatina and Low, 2019; Dix et al., 2018; Hu et al., 2018; Johnson et al., 2020; Park et al., 2020), suggesting an important physiological function for this architecture. The mechanisms by which symmetry mismatch arises in these systems have not yet been determined, and the functional consequences of symmetry mismatch in these systems remain unclear. Interestingly, no symmetry mismatch has been observed in core complexes of minimized T4SS systems such as the X. citri T4SS, which contain 14 copies of both the N-terminal and C-terminal domains of VirB9 (Sgro et al., 2018).

When comparing the H. pylori Cag T4SS core complex to corresponding T4SS subassemblies in other species, there are striking differences. Most notably, the Cag T4SS core complex is much larger in size than core complexes in prototype systems, and it contains two species-specific components (Cag3 and CagM). The CagT, CagX and CagY components of the Cag T4SS are homologs of VirB7, VirB9 and VirB10 components of prototype systems, but there are marked differences in the sequences and structures. Finally, the Cag T4SS core complex includes a PR feature, which has not been detected in prototype systems. In the current study, we discovered that the portions of CagX and CagY within the PR are similar to the parts of VirB9 and VirB10 which reside in the I-layer of the X. citri T4SS (Sgro et al., 2018). Because of this relationship, it is perhaps appropriate to consider the PR of the Cag T4SS and the I-layer of the X. citri T4SS as analogous structures. However, there are several important distinctions. First, the difference in symmetry between the OMC and the PR of the H. pylori Cag T4SS contrasts the C14 symmetry observed throughout the X. citri T4SS core complex. Second, the position of the X. citri T4SS I-layer within the OMC (~115 Å below the outer membrane, as determined by the position of the membrane spanning helices) is similar to that of the I-layer of the Cag T4SS (positioned ~120 Å beneath the outer membrane, and composed of CagM). Therefore, in comparison to the Cag T4SS PR, the I-layer within the X. citri T4SS is positioned much closer to the OMC. The functional roles of these structures are currently unclear, but we speculate that the OMC I-layer and the PR represent structural expansions within the Cag T4SS that are associated with specific functions. Future investigations building on the current structural studies will be needed to develop a more complete understanding of the similarities and differences that exist among T4SSs.

All cryo-EM data included in this manuscript are available through the Electron Microscopy Data Bank (EMD-20021, EMD-22081, EMD-22076, and EMD-22077). All models that were constructed from these data are available via the Protein Data Bank (PDB 6X6S, 6X6J, 6X6K, and 6X6L).

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   3. Sawhney N
   4. Durie CL
   5. Cover TL
   6. Ohi MD
   7. Lacy DB

   (2020) RCSB Protein Data Bank

   ID 6X6S. Cryo-EM Structure of the Helicobacter pylori OMC.

   http://www.rcsb.org/structure/6X6S
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   7. Lacy DB

   (2020) RCSB Protein Data Bank

   ID 6X6J. Cryo-EM Structure.

   http://www.rcsb.org/structure/6X6J
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   (2020) RCSB Protein Data Bank

   ID 6X6K. Cryo-EM Structure.

   http://www.rcsb.org/structure/6X6K
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   (2020) RCSB Protein Data Bank

   ID 6X6L. Cryo-EM Structure.

   http://www.rcsb.org/structure/6X6L
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   ID EMD-22081. HpT4SS OMC WT.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22081
6. 1. Sheedlo MJ
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   (2020) Electron Microscopy Data Bank

   ID EMD-20021. HpT4SS PC WT.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20021
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   (2020) Electron Microscopy Data Bank

   ID EMD-22076. HpT4SS OMC Δcag3.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22076
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   (2020) Electron Microscopy Data Bank

   ID EMD-22077. HpT4SS PR Δcag3.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22077

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Author details

1. Michael J Sheedlo

   Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, United States

   Contribution

   Formal analysis, Validation, Investigation, 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

2. Jeong Min Chung

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

   Contribution

   Formal analysis, Validation, Investigation, Visualization, 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

3. Neha Sawhney

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

   Contribution

   Resources, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

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

4. Clarissa L Durie

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

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4027-4386

5. Timothy L Cover

   1. Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, 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, Supervision, Funding acquisition, Project administration, 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

6. Melanie D Ohi

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

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Project administration, 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

7. D Borden Lacy

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

   Contribution

   Supervision, Validation, Investigation, Writing - review and editing

   For correspondence

   borden.lacy@vanderbilt.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2273-8121

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