Helicobacter pylori interferes with leukocyte migration via the outer membrane protein HopQ and via CagA translocation

Busch, Benjamin; Weimer, Ramona; Woischke, Christine; Fischer, Wolfgang; Haas, Rainer

doi:10.1016/j.ijmm.2015.02.003

Cited by 9 publications

(7 citation statements)

References 66 publications

(75 reference statements)

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“…3A). The full-length tyrosine-phosphorylated CagA (ϳ135 kDa) was not detectable, suggesting its specific, rapid, and complete processing in human neutrophils, as described earlier (9,34). Surprisingly, in murine neutrophils CagA was not detectable as a phosphorylated protein (Fig.…”

Section: Resultssupporting

confidence: 56%

The HopQ-CEACAM Interaction Controls CagA Translocation, Phosphorylation, and Phagocytosis of Helicobacter pylori in Neutrophils

Behrens¹,

Busch²,

Ishikawa-Ankerhold

et al. 2020

mBio

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The cag type IV secretion system (cag-T4SS) of Helicobacter pylori exploits specific cellular carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), such as CEACAM1, -3, -5, and -6, as cellular receptors for CagA translocation into human gastric epithelial cells. We studied the interaction of H. pylori with human CEACAM1, CEACAM3, and CEACAM6 receptors (hCEACAMs) expressed on myeloid cells from CEACAM-humanized mice. Human and CEACAM-humanized mouse polymorphonuclear neutrophils (PMNs) allowed a specific HopQ-dependent interaction strongly enhancing CagA translocation. Translocated CagA was tyrosine phosphorylated, which was not seen in wild-type (wt) murine neutrophils. In contrast, human or murine bone marrow-derived macrophages and dendritic cells (DCs) revealed a low hCEACAM expression and bacterial binding. CagA translocation and tyrosine-phosphorylation was low and independent of the HopQ-CEACAM interaction. Neutrophils, but not macrophages or DCs, from CEACAM-humanized mice, significantly upregulated the proinflammatory chemokine MIP-1α. However, macrophages showed a significantly reduced amount of CXCL1 (KC) and CCL2 (MCP-1) secretion in CEACAM-humanized versus wt cells. Thus, H. pylori, via the HopQ-CEACAM interaction, controls the production and secretion of chemokines differently in PMNs, macrophages, and DCs. We further show that upon H. pylori contact the oxidative burst of neutrophils and phagocytosis of H. pylori was strongly enhanced, but hCEACAM3/6 expression on neutrophils allowed the extended survival of H. pylori within neutrophils in a HopQ-dependent manner. Finally, we demonstrate that during a chronic mouse infection, H. pylori is able to systemically downregulate hCEACAM1 and hCEACAM6 receptor expression on neutrophils, probably to limit CagA translocation efficiency and most likely gastric pathology. IMPORTANCE Helicobacter pylori is highly adapted to humans and evades host immunity to allow its lifelong colonization. However, the H. pylori mouse model is artificial for H. pylori, and few adapted strains allow gastric colonization. Here, we show that human or CEACAM-humanized, but not mouse neutrophils are manipulated by the H. pylori HopQ-CEACAM interaction. Human CEACAMs are responsible for CagA phosphorylation, activation, and processing in neutrophils, whereas CagA translocation and tyrosine phosphorylation in DCs and macrophages is independent of the HopQ-CEACAM interaction. H. pylori affects the secretion of distinct chemokines in CEACAM-humanized neutrophils and macrophages. Most importantly, human CEACAMs on neutrophils enhance binding, oxidative burst, and phagocytosis of H. pylori and enhance bacterial survival in the phagosome. The H. pylori-CEACAM interaction modulates PMNs to reduce the H. pylori CagA translocation efficiency in vivo and to fine-tune the expression of CEACAM receptors on neutrophils to limit translocation of CagA and gastric pathology.

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Section: Resultssupporting

confidence: 56%

The HopQ-CEACAM Interaction Controls CagA Translocation, Phosphorylation, and Phagocytosis of Helicobacter pylori in Neutrophils

Behrens¹,

Busch²,

Ishikawa-Ankerhold

et al. 2020

mBio

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“…A major role of the cag -T4SS is the translocation of the CagA protein into various types of host cells, where CagA interferes in a phosphorylation-dependent and phosphorylation-independent manner with signaling events to manipulate fundamental processes in the gastric epithelium [ 28 ]. Major outcomes include the suppression of innate defense mechanisms [ 29 ], changes in cell polarity and migration [ 30 , 31 ], and putatively oncogenic events [ 32 , 33 ]. The involvement of a host cell integrin heterodimer (α5β1 or any other β integrin heterodimer) acting as receptor for the Hp T4SS, especially for the pilus-associated RGD containing CagL protein, was considered as a major requirement for CagA translocation [ 13 , 14 ] [ 34 , 35 ].…”

Section: Discussionmentioning

confidence: 99%

Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation

Zhao¹,

Busch²,

Jiménez‐Soto³

et al. 2018

PLoS Pathog

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Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (cag-T4SS) into host cells is a hallmark of infection with Hp and a major risk factor for severe gastric diseases, including gastric cancer. To mediate the injection of CagA, Hp uses a membrane-embedded syringe-like molecular apparatus extended by an external pilus-like rod structure that binds host cell surface integrin heterodimers. It is still largely unclear how the interaction of the cag-T4SS finally mediates translocation of the CagA protein into the cell cytoplasm. Recently certain carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), acting as receptor for the Hp outer membrane adhesin HopQ, have been identified to be involved in the process of CagA host cell injection. Here, we applied the CRISPR/Cas9-knockout technology to generate defined human gastric AGS and KatoIII integrin knockout cell lines. Although confocal laser scanning microscopy revealed a co-localization of Hp and β1 integrin heterodimers on gastric epithelial cells, Hp infection studies using the quantitative and highly sensitive Hp β-lactamase reporter system clearly show that neither β1 integrin heterodimers (α1β1, α2β1 or α5β1), nor any other αβ integrin heterodimers on the cell surface are essential for CagA translocation. In contrast, deletion of the HopQ adhesin in Hp, or the simultaneous knockout of the receptors CEACAM1, CEACAM5 and CEACAM6 in KatoIII cells abolished CagA injection nearly completely, although bacterial binding was only reduced to 50%. These data provide genetic evidence that the cag-T4SS-mediated interaction of Hp with cell surface integrins on human gastric epithelial cells is not essential for CagA translocation, but interaction of Hp with CEACAM receptors is facilitating CagA translocation by the cag-T4SS of this important microbe.

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“…The cagA (strain P12) gene was inserted into the vector pETM30 (EMBL) between the Nco I and Xho I restriction sites. The sequence of cagA gene was mutated to remove the asparagine residues 880–885 (NNNNNN) by a single serine to reduce proteolysis occurring at this position during expression . The resulting CagA protein was therefore expressed in fusion with a N‐terminal His 6 ‐GST‐TEV‐ and a C‐terminal STREP‐tag.…”

Section: Methodsmentioning

confidence: 99%

Molecular dissection of protein–protein interactions between integrin α5β1 and the Helicobacter pylori Cag type IV secretion system

et al. 2017

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The more severe strains of the bacterial human pathogen Helicobacter pylori produce a type IV secretion system (cagT4SS) to inject the oncoprotein cytotoxin-associated gene A (CagA) into gastric cells. This syringe-like molecular apparatus is prolonged by an external pilus that exploits integrins as receptors to mediate the injection of CagA. The molecular determinants of the interaction of the cagT4SS pilus with the integrin ectodomain are still poorly understood. In this study, we have used surface plasmon resonance (SPR) to generate a comprehensive analysis of the protein-protein interactions between purified CagA, CagL, CagI, CagY repeat domain II (CagY ), CagY C-terminal domain (CagY ) and integrin α5β1 ectodomain (α5β1 ) or headpiece domain (α5β1 ). We found that CagI, CagA, CagL and CagY but not CagY were able to interact with α5β1 with affinities similar to the one observed for α5β1 interaction with its physiological ligand fibronectin. We further showed that integrin activation and its associated conformational change increased CagA, CagL and CagY affinities for the receptor. Furthermore, CagI did not interact with integrin unless the receptor was in open conformation. CagI, CagA but not CagL and CagY interacted with the α5β1 . Our SPR study also revealed novel interactions between CagA and CagL, CagA and CagY , and CagA and CagI. Altogether, our data map the network of interactions between host-cell α5β1 integrin and the cagT4SS proteins and suggest that activation of the receptor promotes interactions with the secretion apparatus and possibly CagA injection.

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Helicobacter pylori interferes with leukocyte migration via the outer membrane protein HopQ and via CagA translocation

Cited by 9 publications

References 66 publications

The HopQ-CEACAM Interaction Controls CagA Translocation, Phosphorylation, and Phagocytosis of Helicobacter pylori in Neutrophils

The HopQ-CEACAM Interaction Controls CagA Translocation, Phosphorylation, and Phagocytosis of Helicobacter pylori in Neutrophils

Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation

Molecular dissection of protein–protein interactions between integrin α5β1 and the Helicobacter pylori Cag type IV secretion system

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