scholarly journals Bacterial Energetic Requirements for Helicobacter pylori Cag Type IV Secretion System-Dependent Alterations in Gastric Epithelial Cells

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Aung Soe Lin ◽  
Samuel D. R. Dooyema ◽  
Arwen E. Frick-Cheng ◽  
M. Lorena Harvey ◽  
Giovanni Suarez ◽  
...  
Keyword(s):  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
Type Iv ◽  
Mutant Strains ◽  
H Pylori ◽  
Tlr9 Activation ◽  
Bacterial Constituents

ABSTRACT Helicobacter pylori colonizes the stomach in about half of the world’s population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI 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. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagβ, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagβ was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.

scholarly journals Genes Required for Assembly of Pili Associated with the Helicobacter pylori cag Type IV Secretion System

2014 ◽  
Vol 82 (8) ◽  
pp. 3457-3470 ◽  
Author(s):  
Elizabeth M. Johnson ◽  
Jennifer A. Gaddy ◽  
Bradley J. Voss ◽  
Ewa E. Hennig ◽  
Timothy L. Cover
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
Type Iv ◽  
Pilus Biogenesis ◽  
H Pylori

ABSTRACTHelicobacter pyloricauses numerous alterations in gastric epithelial cells through processes that are dependent on activity of thecagtype IV secretion system (T4SS). Filamentous structures termed “pili” have been visualized at the interface betweenH. pyloriand gastric epithelial cells, and previous studies suggested that pilus formation is dependent on the presence of thecagpathogenicity island (PAI). Thus far, there has been relatively little effort to identify specific genes that are required for pilus formation, and the role of pili in T4SS function is unclear. In this study, we selected 7 genes in thecagPAI that are known to be required for T4SS function and investigated whether these genes were required for pilus formation.cagT,cagX,cagV,cagM, andcag3mutants were defective in both T4SS function and pilus formation; complemented mutants regained T4SS function and the capacity for pilus formation.cagYandcagCmutants were defective in T4SS function but retained the capacity for pilus formation. These results define a set ofcagPAI genes that are required for both pilus biogenesis and T4SS function and reveal that these processes can be uncoupled in specific mutant strains.

scholarly journals The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
William E. Sause ◽  
Daniela Keilberg ◽  
Soufiane Aboulhouda ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
H Pylori ◽  
Α5β1 Integrin ◽  
Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

scholarly journals Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Julieta Aguilar ◽  
Todd A. Cameron ◽  
John Zupan ◽  
Patricia Zambryski
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Content Type ◽  
Protein Substrates ◽  
Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

scholarly journals In vivostructures of theHelicobacter pylori cagtype IV secretion system

2017 ◽  
Author(s):  
Yi-Wei Chang ◽  
Carrie L. Shaffer ◽  
Lee A. Rettberg ◽  
Debnath Ghosal ◽  
Grant J. Jensen
Keyword(s):  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Molecular Architecture ◽  
Type Iv ◽  
H Pylori ◽  
Electron Cryotomography ◽  
Rod Structures ◽  
Bacterial Type

SummaryThe bacterial type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Using electron cryotomography, here we reveal the molecular architecture of the cancer-associatedHelicobacter pylori cagT4SS. Although most components are unique toH. pylori, thecagT4SS exhibits remarkable architectural similarity to previously studied T4SSs. WhenH. pyloriencounters host cells, however, the bacterium elaborates rigid, membranous tubes perforated by lateral ports. Dense, pilus-like rod structures extending from the inner membrane were also observed. We propose that the membrane tubes assemble out of the T4SS and are the delivery system forcagT4SS cargo. These studies reveal the architecture of a dynamic molecular machine that evolved to function in the human gastric niche.

scholarly journals ALPK1 and TIFA dependent innate immune response triggered by the Helicobacter pylori type IV secretion system

2017 ◽  
Author(s):  
Stephanie Zimmermann ◽  
Lennart Pfannkuch ◽  
Munir A. Al-Zeer ◽  
Sina Bartfeld ◽  
Manuel Koch ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protein Translocation ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Innate Immune ◽  
Host Cells ◽  
Cell Contact ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
H Pylori

SummaryActivation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori and associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous hits involved in H. pylori-, but not IL-1β- and TNF-α- dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry and mutant H. pylori strains, identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation as well as TIFAsome formation depended on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as core of a novel innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen’s T4SS.

scholarly journals Maintenance of Type IV Secretion Function During Helicobacter pylori Infection in Mice

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e03147-20
Author(s):  
Emma C. Skoog ◽  
Miriam E. Martin ◽  
Roberto M. Barrozo ◽  
Lori M. Hansen ◽  
Lucy P. Cai ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Murine Model ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Biologically Relevant ◽  
Type Iv ◽  
H Pylori

ABSTRACTThe Helicobacter pylori type IV secretion system (T4SS) encoded on the cag pathogenicity island (cagPAI) secretes the CagA oncoprotein and other effectors into the gastric epithelium. During murine infection, T4SS function is lost in an immune-dependent manner, typically as a result of in-frame recombination in the middle repeat region of cagY, though single nucleotide polymorphisms (SNPs) in cagY or in other essential genes may also occur. Loss of T4SS function also occurs in gerbils, nonhuman primates, and humans, suggesting that it is biologically relevant and not simply an artifact of the murine model. Here, we sought to identify physiologically relevant conditions under which T4SS function is maintained in the murine model. We found that loss of H. pylori T4SS function in mice was blunted by systemic Salmonella coinfection and completely eliminated by dietary iron restriction. Both have epidemiologic parallels in humans, since H. pylori strains from individuals in developing countries, where iron deficiency and systemic infections are common, are also more often cagPAI+ than strains from developed countries. These results have implications for our fundamental understanding of the cagPAI and also provide experimental tools that permit the study of T4SS function in the murine model.IMPORTANCE The type IV secretion system (T4SS) is the major Helicobacter pylori virulence factor, though its function is lost during murine infection. Loss of function also occurs in gerbils and in humans, suggesting that it is biologically relevant, but the conditions under which T4SS regulation occurs are unknown. Here, we found that systemic coinfection with Salmonella and iron deprivation each promote retention of T4SS function. These results improve our understanding of the cag pathogenicity island (cagPAI) and provide experimental tools that permit the study of T4SS function in the murine model.

scholarly journals CagY-Dependent Regulation of Type IV Secretion inHelicobacter pyloriIs Associated with Alterations in Integrin Binding

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Emma C. Skoog ◽  
Vasilios A. Morikis ◽  
Miriam E. Martin ◽  
Greg A. Foster ◽  
Lucy P. Cai ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Inflammatory Response ◽  
Persistent Infection ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv ◽  
H Pylori ◽  
Host Inflammatory Response

ABSTRACTStrains ofHelicobacter pylorithat cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by thecagpathogenicity island (cagPAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4+T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human α5β1integrin, we tested the hypothesis that recombination in the CagY MRR regulates T4SS function by modulating binding to α5β1integrin. Using a cell-free microfluidic assay, we found thatH. pyloribinding to α5β1integrin under shear flow is dependent on the CagY MRR, but independent of the presence of the T4SS pili, which are only formed whenH. pyloriis in contact with host cells. Similarly, expression of CagY in the absence of other T4SS genes was necessary and sufficient for whole bacterial cell binding to α5β1integrin. Bacteria with variantcagYalleles that reduced T4SS function showed comparable reduction in binding to α5β1integrin, although CagY was still expressed on the bacterial surface. We speculate thatcagY-dependent modulation ofH. pyloriT4SS function is mediated by alterations in binding to α5β1integrin, which in turn regulates the host inflammatory response so as to maximize persistent infection.IMPORTANCEInfection withH. pylorican cause peptic ulcers and is the most important risk factor for gastric cancer, the third most common cause of cancer death worldwide. The majorH. pylorivirulence factor that determines whether infection causes disease or asymptomatic colonization is the type IV secretion system (T4SS), a sort of molecular syringe that injects bacterial products into gastric epithelial cells and alters host cell physiology. We previously showed that recombination in CagY, an essential T4SS component, modulates the function of the T4SS. Here we found that these recombination events produce parallel changes in specific binding to α5β1integrin, a host cell receptor that is essential for T4SS-dependent translocation of bacterial effectors. We propose that CagY-dependent binding to α5β1integrin acts like a molecular rheostat that alters T4SS function and modulates the host immune response to promote persistent infection.

scholarly journals Sensing of Bacterial Type IV Secretion via the Unfolded Protein Response

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Maarten F. de Jong ◽  
Tregei Starr ◽  
Maria G. Winter ◽  
Andreas B. den Hartigh ◽  
Robert Child ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Unfolded Protein ◽  
Type Iv ◽  
Er Chaperone ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACTHost cytokine responses toBrucella abortusinfection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces proinflammatory responses. Ectopically expressed VceC interacted with the endoplasmic reticulum (ER) chaperone BiP/Grp78 and localized to the ER of HeLa cells. ER localization of VceC required a transmembrane domain in its N terminus. Notably, the expression of VceC resulted in reorganization of ER structures. In macrophages, VceC was required forB. abortus-induced inflammation by induction of the unfolded protein response by a process requiring inositol-requiring transmembrane kinase/endonuclease 1. Altogether, these findings suggest that translocation of the T4SS effector VceC induces ER stress, which results in the induction of proinflammatory host cell responses duringB. abortusinfection.IMPORTANCEBrucellaspecies are pathogens that require a type IV secretion system (T4SS) to survive in host cells and to maintain chronic infection. By as-yet-unknown pathways, the T4SS also elicits inflammatory responses in infected cells. Here we show that inflammation caused by the T4SS results in part from the sensing of a T4SS substrate, VceC, that localizes to the endoplasmic reticulum (ER), an intracellular site ofBrucellareplication. Possibly via binding of the ER chaperone BiP, VceC causes ER stress with concomitant expression of proinflammatory cytokines. Thus, induction of the unfolded protein response may represent a novel pathway by which host cells can detect pathogens deploying a T4SS.

scholarly journals Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Michael J Sheedlo ◽  
Jeong Min Chung ◽  
Neha Sawhney ◽  
Clarissa L Durie ◽  
Timothy L Cover ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Molar Ratio ◽  
Type Iv Secretion System ◽  
Core Complex ◽  
Type Iv ◽  
H Pylori ◽  
Species Specific

The pathogenesis of Helicobacter pylori-associated gastric cancer is dependent on delivery of CagA into host cells through a type IV secretion system (T4SS). The H. pylori Cag T4SS includes a large membrane-spanning core complex containing five proteins, organized into an outer membrane cap (OMC), a periplasmic ring (PR) and a stalk. Here, we report cryo-EM reconstructions of a core complex lacking Cag3 and an improved map of the wild-type complex. We define the structures of two unique species-specific components (Cag3 and CagM) and show that Cag3 is structurally similar to CagT. Unexpectedly, components of the OMC are organized in a 1:1:2:2:5 molar ratio (CagY:CagX:CagT:CagM:Cag3). CagX and CagY are components of both the OMC and the PR and bridge the symmetry mismatch between these regions. These results reveal that assembly of the H. pylori T4SS core complex is dependent on incorporation of interwoven species-specific components.

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

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Bo Hu ◽  
Pratick Khara ◽  
Liqiang Song ◽  
Aung Soe Lin ◽  
Arwen E. Frick-Cheng ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Legionella Pneumophila ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Membrane Complex ◽  
Type Iv ◽  
Increased Risk ◽  
H Pylori

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 (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.

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