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 The Coupling Protein Cagβ and Its Interaction Partner CagZ Are Required for Type IV Secretion of the Helicobacter pylori CagA Protein

2010 ◽  
Vol 78 (12) ◽  
pp. 5244-5251 ◽  
Author(s):  
Angela Jurik ◽  
Elisabeth Haußer ◽  
Stefan Kutter ◽  
Isabelle Pattis ◽  
Sandra Praßl ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Protein Interactions ◽  
Translocation Factor ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Protein Protein Interactions ◽  
Type Iv ◽  
Coupling Protein ◽  
Translocation Factors

ABSTRACT Bacterial type IV secretion systems are macromolecule transporters with essential functions for horizontal gene transfer and for symbiotic and pathogenic interactions with eukaryotic host cells. Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject its effector protein CagA into gastric cells. This protein translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it has been linked to cancer development. Interactions of CagA with host cell proteins have been studied in great detail, but little is known about the molecular details of CagA recognition as a type IV secretion substrate or of the translocation process. Apart from components of the secretion apparatus, we previously identified several CagA translocation factors that are either required for or support CagA translocation. To identify protein-protein interactions between these translocation factors, we used a yeast two-hybrid approach comprising all cag pathogenicity island genes. Among several other interactions involving translocation factors, we found a strong interaction between the coupling protein homologue Cagβ (HP0524) and the Cag-specific translocation factor CagZ (HP0526). We show that CagZ has a stabilizing effect on Cagβ, and we demonstrate protein-protein interactions between the cytoplasmic part of Cagβ and CagA and between CagZ and Cagβ, using immunoprecipitation and pull-down assays. Together, our data suggest that these interactions represent a substrate-translocation factor complex at the bacterial cytoplasmic membrane.

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

2005 ◽  
Vol 73 (8) ◽  
pp. 4643-4652 ◽  
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.

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 Natural Competence Promotes Helicobacter pylori Chronic Infection

2012 ◽  
Vol 81 (1) ◽  
pp. 209-215 ◽  
Author(s):  
Marion S. Dorer ◽  
Ilana E. Cohen ◽  
Tate H. Sessler ◽  
Jutta Fero ◽  
Nina R. Salama
Keyword(s):  
Helicobacter Pylori ◽  
Chronic Infection ◽  
Acute Infection ◽  
Host Cells ◽  
High Rate ◽  
Natural Competence ◽  
Content Type ◽  
Type Iv ◽  
Competence Factor ◽  
H Pylori

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogenHelicobacter pylori. In most cases,H. pyloriinfects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, thecagYcomponent of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population ofcagY+andcagYmutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common inH. pyloristrains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneousH. pyloristrains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.

scholarly journals Helicobacter pylori Exploits a Unique Repertoire of Type IV Secretion System Components for Pilus Assembly at the Bacteria-Host Cell Interface

2011 ◽  
Vol 7 (9) ◽  
pp. e1002237 ◽  
Author(s):  
Carrie L. Shaffer ◽  
Jennifer A. Gaddy ◽  
John T. Loh ◽  
Elizabeth M. Johnson ◽  
Salisha Hill ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
System Components ◽  
Cell Interface ◽  
Pilus Assembly

scholarly journals Uptake of Helicobacter pylori Vesicles Is Facilitated by Clathrin-Dependent and Clathrin-Independent Endocytic Pathways

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Annelie Olofsson ◽  
Lars Nygård Skalman ◽  
Ikenna Obi ◽  
Richard Lundmark ◽  
Anna Arnqvist
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Host Cells ◽  
Gastric Epithelial Cells ◽  
Host Cell Membrane ◽  
Content Type ◽  
Effector Molecules ◽  
Chemical Inhibition ◽  
H Pylori ◽  
Endocytic Pathways

ABSTRACTBacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells.Helicobacter pyloriis a gastric pathogen that infects half of the world’s population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles fromH. pyloriare internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake ofH. pylorivesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed thatH. pylorivesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed thatH. pylorivesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization ofH. pylorivesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency ofH. pylorivesicle uptake.IMPORTANCEBacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake ofHelicobacter pylorivesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs theH. pylorivesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

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 TLR9 activation suppresses inflammation in response to Helicobacter pylori infection

2016 ◽  
Vol 311 (5) ◽  
pp. G852-G858 ◽  
Author(s):  
Matthew G. Varga ◽  
M. Blanca Piazuelo ◽  
Judith Romero-Gallo ◽  
Alberto G. Delgado ◽  
Giovanni Suarez ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Disease Risk ◽  
Host Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Host Genotype ◽  
Immunological Responses ◽  
Type Iv ◽  
H Pylori

Helicobacter pylori ( H. pylori) induces chronic gastritis in humans, and infection can persist for decades. One H. pylori strain-specific constituent that augments disease risk is the cag pathogenicity island. The cag island encodes a type IV secretion system (T4SS) that translocates DNA into host cells. Toll-like receptor 9 (TLR9) is an innate immune receptor that detects hypo-methylated CpG DNA motifs. In this study, we sought to define the role of the H. pylori cag T4SS on TLR9-mediated responses in vivo. H. pylori strain PMSS1 or its cagE − mutant, which fails to assemble a T4SS, were used to infect wild-type or Tlr9 −/− C57BL/6 mice. PMSS1-infected Tlr9 −/− mice developed significantly higher levels of inflammation, despite similar levels of colonization density, compared with PMSS1-infected wild-type mice. These changes were cag dependent, as both mouse genotypes infected with the cagE − mutant only developed minimal inflammation. Tlr9 −/− genotypes did not alter the microbial phenotypes of in vivo-adapted H. pylori strains; therefore, we examined host immunological responses. There were no differences in levels of TH1 or TH2 cytokines in infected mice when stratified by host genotype. However, gastric mucosal levels of IL-17 were significantly increased in infected Tlr9 −/− mice compared with infected wild-type mice, and H. pylori infection of IL-17A −/− mice concordantly led to significantly decreased levels of gastritis. Thus loss of Tlr9 selectively augments the intensity of IL-17-driven immune responses to H. pylori in a cag T4SS-dependent manner. These results suggest that H. pylori utilizes the cag T4SS to manipulate the intensity of the host immune response.

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