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 (CagT4SS), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant CagT4SS machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple CagT4SS 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 CagT4SS 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/VirD4T4SS, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori CagT4SS T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/IcmT4SS 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 CagT4SS aligns structurally much more closely to the Dot/IcmT4SS 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.

中文翻译：

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幽门螺杆菌Cag IV型分泌系统的原位分子结构。

幽门螺杆菌在全世界约一半的人口中定居，其在胃粘膜中的存在与胃腺癌，胃淋巴瘤和消化性溃疡疾病的风险增加有关。携带cag致病岛（cagPAI）的幽门螺杆菌菌株与疾病进展的风险增加相关。cagPAI编码Cag IV型分泌系统（CagT4SS），该系统将CagA癌蛋白和其他效应分子传递到人胃上皮细胞中。我们通过冷冻电子断层扫描可视化了幽门螺杆菌细胞膜上的天然和突变体CagT4SS机器的结构。单个幽门螺杆菌细胞包含多个CagT4SS纳米机器，每个由具有14倍对称性的轮状外膜复合物（OMC）和具有6倍对称性的内膜复合物（IMC）组成。CagX，CagY，OMC的组装需要CagM和CagM，而缺少Cag3和CagT的菌株会产生缺乏外围成分的外膜复合物。从未详细显示过的IMC在横截面视图中配置为六层，在端视图中配置为围绕中心通道的三个同心环。IMC包含三个T4SS ATPase：（i）VirB4样CagE，在通道入口处以二聚体的六聚体形式排列；（ii）VirB11样Cagα的六聚体，对接在CagE六聚体的底部；（iii）与堆叠的CagE /Cagα复合物相关并形成最外环的VirD4样Cagβ和其他未指定的Cag亚基。CagT4SS和最近解决的肺炎军团菌Dot / Icm系统包括T4SS超家族的新结构原型。重要事项IV型细菌分泌系统（T4SS）已在系统发育上分为两个亚科。由根癌农杆菌VirB / VirD4T4SS代表的T4ASS包括由12个VirB和VirD4样亚基组装而成的“最小化”机器，以及组成更大的系统，例如幽门螺杆菌CagT4SS T4BSS涵盖了与嗜肺军团菌亚基组成密切相关的系统/ IcmT4SS在这里，我们介绍通过原位低温电子断层扫描确定的天然和突变幽门螺杆菌Cag机的结构。我们确定了独特的外膜和内膜复合物，并首次使T4SSs的三个“签名” ATPases在转运通道的胞质入口处的结构贡献可视化。尽管在进化上存在分歧，与可用的VirB / VirD4亚复合物相比，CagT4SS在结构上与Dot / IcmT4SS的排列更加紧密。我们的发现突出了T4SS的多样性，并提出了一种结构分类方案，其中T4SS被归类为最小化的VirB / VirD4类或较大的Cag类和Dot / Icm类系统。