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ABSTRACTHelicobacter pyloricolonizes the human stomach and confers an increased risk for the development of peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. A secreted H. pyloritoxin, VacA, can cause multiple alterations in gastric epithelial cells, including cell death. In this study, we sought to identify host cell factors that are required for VacA-induced cell death. To do this, we analyzed gene trap and short hairpin RNA (shRNA) libraries in AZ-521 human gastric epithelial cells and selected for VacA-resistant clones. Among the VacA-resistant clones, we identified multiple gene trap library clones and an shRNA library clone with disrupted expression of connexin 43 (Cx43) (also known as gap junction protein alpha 1 [GJA1]). Further experiments with Cx43-specific shRNAs confirmed that a reduction in Cx43expression results in resistance to VacA-induced cell death. Immunofluorescence microscopy experiments indicated that VacA did not colocalize with Cx43. We detected production of the Cx43 protein in AZ-521 cells but not in AGS, HeLa, or RK-13 cells, and correspondingly, AZ-521 cells were the most susceptible to VacA-induced cell death. When Cx43 was expressed in HeLa cells, the cells became more susceptible to VacA. These results indicate that Cx43 is a host cell constituent that contributes to VacA-induced cell death and that variation among cell types in susceptibility to VacA-induced cell death is attributable at least in part to cell type-specific differences in Cx43 production.

Different experimental models have been used to study UPEC pathogenesis, including in vitrocultures in different media, tissue culture, and mouse models of infection. The last is especially important since it allows evaluation of mechanisms of pathogenesis and potential therapeutic strategies against UPEC. Bacterial physiology is greatly shaped by environment, and it is therefore critical to understand how closely bacterial physiology in any experimental model relates to human infection. In this study, we found strong correlation in bacterial gene expression between the mouse model and human UTI using identical strains, suggesting that the mouse model accurately mimics human infection, definitively supporting its continued use in UTI research.

Helicobacter pyloricolonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated H. pyloristrains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells.

Helicobacter pyloricolonizes the stomach in about half of the world’s population. H. pyloristrains containing the cagpathogenicity island (cagPAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cagPAI-negative strains. The cagPAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells.

Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciensVirB/VirD4T4SS, include “minimized” machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pyloriCagT4SS. T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophilaDot/IcmT4SS. Here, we present structures of native and mutant H. pyloriCag machines determined by in situcryoelectron 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 CagT4SSaligns structurally much more closely to the Dot/IcmT4SSthan 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.

ABSTRACTBacterial type IV secretion systems (T4SSs) can function to export or import DNA, and can deliver effector proteins into a wide range of target cells. Relatively little is known about the structural organization of T4SSs that secrete effector proteins. In this report, we describe the isolation and analysis of a membrane-spanning core complex from the Helicobacter pyloricagT4SS, which has an important role in the pathogenesis of gastric cancer. We show that this complex contains five H. pyloriproteins, CagM, CagT, Cag3, CagX, and CagY, each of which is required for cagT4SS activity. CagX and CagY are orthologous to the VirB9 and VirB10 components of T4SSs in other bacterial species, and the other three Cag proteins are unique to H. pylori. Negative stain single-particle electron microscopy revealed complexes 41 nm in diameter, characterized by a 19-nm-diameter central ring linked to an outer ring by spoke-like linkers. Incomplete complexes formed by Δcag3or ΔcagTmutants retain the 19-nm-diameter ring but lack an organized outer ring. Immunogold labeling studies confirm that Cag3 is a peripheral component of the complex. The cagT4SS core complex has an overall diameter and structural organization that differ considerably from the corresponding features of conjugative T4SSs. These results highlight specialized features of the H. pylori cagT4SS that are optimized for function in the human gastric mucosal environment.IMPORTANCEType IV secretion systems (T4SSs) are versatile macromolecular machines that are present in many bacterial species. In this study, we investigated a T4SS found in the bacterium Helicobacter pylori. H. pyloriis an important cause of stomach cancer, and the H. pyloriT4SS contributes to cancer pathogenesis by mediating entry of CagA (an effector protein regarded as a “bacterial oncoprotein”) into gastric epithelial cells. We isolated and analyzed the membrane-spanning core complex of the H. pyloriT4SS and showed that it contains unique proteins unrelated to components of T4SSs in other bacterial species. These results constitute the first structural analysis of the core complex from this important secretion system.