Functional Analysis of the Helicobacter pylori cag Pathogenicity Island Reveals Both VirD4-CagA-Dependent and VirD4-CagA-Independent Mechanisms

ABSTRACT The type IV secretion machinery encoded by the cag pathogenicity island (PAI) of Helicobacter pylori has been implicated in a series of host responses during infection. Here, we analyzed the function of 12 cag PAI genes from both cag I and cag II loci, including the complete virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, and virD4). We monitored interleukin-8 (IL-8) secretion, CagA translocation and tyrosine phosphorylation, and induction of a scattering (“hummingbird”) phenotype upon H. pylori infection of AGS gastric epithelial cells. For the first time, we have complemented individual cag PAI gene knockout mutants with their intact genes expressed from a shuttle vector and showed that complemented CagA and VirD4 restored wild-type function. Our results demonstrate that phenotypic changes and phosphorylation of CagA depended on all virB/D genes and several other genes of the cag PAI. Induction of IL-8 secretion depended largely on the same set of genes but was independent of CagA and VirD4. Thus, CagA translocation and induction of IL-8 secretion are regulated by VirD4-CagA-dependent and VirD4-CagA-independent mechanisms, respectively. The function of VirD4 as a possible adapter protein which guides CagA into the type IV secretion channel is presented in a model.

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The organization of Helicobacter pylori cag-pathogenicity island (cagPAI) genes in multiracial population with histopathological changes of gastric mucosa

10.21203/rs.2.9601/v1 ◽

2019 ◽

Author(s):

Alfizah Hanafiah ◽

Shaza Azlin Razak ◽

Hui-min Neoh ◽

Noraziah Mohamad Zin ◽

Bruno S. Lopes

Keyword(s):

Helicobacter Pylori ◽

Gastric Mucosa ◽

Pathogenicity Island ◽

Type Iv Secretion ◽

Virulence Determinants ◽

Gastric Diseases ◽

Cag Pathogenicity Island ◽

Type Iv ◽

Inflammatory Score ◽

H Pylori

Abstract Background: Helicobacter pylori is a Gram-negative bacillus that colonises only the mucus layer of the human stomach and is implicated in gastric diseases. Virulent H. pylori harbouring cag-pathogenicity island (cagPAI) which encodes genes for type IV secretion system (T4SS) and CagA protein is one of the major virulence determinants involved in disease development. We examined the entire cagPAI genes in 95 H. pylori isolates from a multiracial population and examined the intactness of cagPAI region with histopathological scores of the gastric mucosa. Results: 95.8% of H. pylori isolates were cagPAI-positive with 23.2% having an intact cagPAI, whereas 72.6% had a partial/rearranged cagPAI. In our study, cag2 and cag4 were found to be significantly higher in H. pylori isolated from Malays, whereas cag4 was predominant in Chinese isolates. We also detected cag24 in significantly high proportion in isolates from the Malays and the Indians compared to the Chinese isolates. The intactness of cagPAI region showed an association with histopathological scores of the gastric mucosa. Significant association was observed between H. pylori harbouring partial cagPAI and higher density of H. pylori and neutrophil activity, whereas strains which lacked cagPAI was associated with higher inflammatory score. Conclusions: The screening of the entire cagPAI genes provides an accurate overview of the cagPAI organisation in H. pylori isolates in a multiracial population. The genotypes of H. pylori strains with various cagPAI rearrangement associated with patients’ ethnicities and histopathological scores might contribute to the pathogenesis of H. pylori infection in a multi-ethnic population.

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

mBio ◽

10.1128/mbio.03147-20 ◽

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.

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Role of amoebae for survival and recovery of ‘non-culturable’ Helicobacter pylori cells in aquatic environments

FEMS Microbiology Ecology ◽

10.1093/femsec/fiaa182 ◽

2020 ◽

Vol 96 (10) ◽

Author(s):

Rafik Dey ◽

Aja Rieger ◽

Graham Banting ◽

Nicholas J Ashbolt

Keyword(s):

Helicobacter Pylori ◽

Type Iv Secretion ◽

Aquatic Environments ◽

Rural Regions ◽

Free Living ◽

Type Iv ◽

Waterborne Transmission ◽

H Pylori ◽

First Time

ABSTRACT Helicobacter pylori is a fastidious Gram-negative bacterium that infects over half of the world's population, causing chronic gastritis and is a risk factor for stomach cancer. In developing and rural regions where prevalence rate exceeds 60%, persistence and waterborne transmission are often linked to poor sanitation conditions. Here we demonstrate that H. pylori not only survives but also replicates within acidified free-living amoebal phagosomes. Bacterial counts of the clinical isolate H. pylori G27 increased over 50-fold after three days in co-culture with amoebae. In contrast, a H. pylori mutant deficient in a cagPAI gene (cagE) showed little growth within amoebae, demonstrating the likely importance of a type IV secretion system in H. pylori for amoebal infection. We also demonstrate that H. pylori can be packaged by amoebae and released in extracellular vesicles. Furthermore, and for the first time, we successfully demonstrate the ability of two free-living amoebae to revert and recover viable but non-cultivable coccoid (VBNC)-H. pylori to a culturable state. Our studies provide evidence to support the hypothesis that amoebae and perhaps other free-living protozoa contribute to the replication and persistence of human-pathogenic H. pylori by providing a protected intracellular microenvironment for this pathogen to persist in natural aquatic environments and engineered water systems, thereby H. pylori potentially uses amoeba as a carrier and a vector of transmission.

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Identification of Pathogenicity Island Genes Associated with Loss of Type IV Secretion Function during Murine Infection with Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.00801-19 ◽

2020 ◽

Vol 88 (6) ◽

Cited By ~ 2

Author(s):

Lori M. Hansen ◽

Dylan J. Dekalb ◽

Lucy P. Cai ◽

Jay V. Solnick

Keyword(s):

Helicobacter Pylori ◽

Single Molecule ◽

Pathogenicity Island ◽

Type Iv Secretion ◽

Common Mechanism ◽

Loss Of Function ◽

Genetic Changes ◽

Content Type ◽

Type Iv ◽

H Pylori

ABSTRACT Chronic Helicobacter pylori colonization in animal models often leads to downregulation of the type IV secretion system (T4SS), typically by recombination in cagY, which is an essential T4SS gene. However, 17 other cag pathogenicity island (cagPAI) genes, as well as some non-cagPAI genes, are also essential for T4SS function. To get a more complete picture of how H. pylori regulates the T4SS during animal colonization, we examined cagY in 534 mouse-passaged isolates that lost T4SS function, defined as a normalized interleukin-8 (IL-8) value of <0.3 relative to the input H. pylori strain PMSS1. In order to analyze the genetic changes in the strains with unchanged cagY, we sequenced the entire pathogenicity island of 60 such isolates using single-molecule, real-time (SMRT) sequencing technology (PacBio, Menlo Park, CA), and we compared the results to the PMSS1 wild type (WT). Of the 534 strains, 271 (51%) showed evidence of recombination in cagY, but we also found indels or nonsynonymous changes in 13 other essential cagPAI genes implicated in H. pylori T4SS function, most commonly cag5, cag10, and cagA. While cagY recombination is the most common mechanism by which H. pylori downregulates T4SS function during murine infection, loss of function is also associated with changes in other essential cagPAI genes.

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

Infection and Immunity ◽

10.1128/iai.00450-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 5

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.

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Protein-Protein Interactions among Helicobacter pylori Cag Proteins

Journal of Bacteriology ◽

10.1128/jb.00066-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4787-4800 ◽

Cited By ~ 53

Author(s):

Valerie J. Busler ◽

Victor J. Torres ◽

Mark S. McClain ◽

Oscar Tirado ◽

David B. Friedman ◽

...

Keyword(s):

Helicobacter Pylori ◽

Protein Interactions ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Protein Protein Interactions ◽

Chromosomal Dna ◽

2D Dige ◽

Type Iv ◽

H Pylori

ABSTRACT Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.

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Molecular and Structural Analysis of the Helicobacter pylori cag Type IV Secretion System Core Complex

mBio ◽

10.1128/mbio.02001-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 67

Author(s):

Arwen E. Frick-Cheng ◽

Tasia M. Pyburn ◽

Bradley J. Voss ◽

W. Hayes McDonald ◽

Melanie D. Ohi ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Species ◽

Secretion System ◽

Effector Proteins ◽

Type Iv Secretion ◽

Secretion Systems ◽

Core Complex ◽

Type Iv ◽

Membrane Spanning ◽

H Pylori

ABSTRACT Bacterial 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 pylori cag T4SS, which has an important role in the pathogenesis of gastric cancer. We show that this complex contains five H. pylori proteins, CagM, CagT, Cag3, CagX, and CagY, each of which is required for cag T4SS 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 Δ cag3 or Δ cagT mutants 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 cag T4SS 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 cag T4SS that are optimized for function in the human gastric mucosal environment. IMPORTANCE Type 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. pylori is an important cause of stomach cancer, and the H. pylori T4SS 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. pylori T4SS 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.

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Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.73.8.4643-4652.2005 ◽

2005 ◽

Vol 73 (8) ◽

pp. 4643-4652 ◽

Cited By ~ 29

Author(s):

Bianca Bauer ◽

Stefan Moese ◽

Sina Bartfeld ◽

Thomas F. Meyer ◽

Matthias Selbach

Keyword(s):

Helicobacter Pylori ◽

Host Cell ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Type Iv ◽

Host Cell Proteins ◽

Mammalian Cell Lines ◽

Host Cell Factors ◽

H Pylori

ABSTRACT Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.

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T4SS-dependent TLR5 activation by Helicobacter pylori infection

Nature Communications ◽

10.1038/s41467-019-13506-6 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Suneesh Kumar Pachathundikandi ◽

Nicole Tegtmeyer ◽

Isabelle Catherine Arnold ◽

Judith Lind ◽

Matthias Neddermann ◽

...

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Pathogenic Bacteria ◽

Type Iv Secretion ◽

Downstream Signaling ◽

Type Iv ◽

Efficient Control ◽

H Pylori ◽

Highly Virulent

AbstractToll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5+ epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.

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