Identification of novel Helicobacter pylori adhesins and host cell receptors facilitating bacterial access to beta1 integrin and translocation of CagA
Final Report Abstract
A major focus of this research project was a better understanding of the interaction of H. pylori with human host cells, especially in the context of the ability of H. pylori to translocate its major virulence factor, the CagA protein, into these cells to interfere with cellular signaling cascades and to manipulate these cells. We therefore concentrated our work first on the bacterial side, by trying to identify additional bacterial adhesins, which might contribute to the CagA translocation event. We generated a set of novel outer membrane protein deletion mutants in different H. pylori strains and studied their interaction and CagA translocation capacity with different gastric cell lines. Some of these OMPs with close homology to already known adhesins from the adhesin branch (HopM, HopN, HopA) were functionally inactivated and binding assays using different cell lines showed that they did not contribute adhesive functions for the host cells that we studied. Some other OMPs not allocated to the adhesin branch are still under investigation. Thus, novel adhesins could not be identified yet. We next concentrated on the host cell side to identify novel ligands or receptors for H. pylori, which might be involved in the function of the cag-T4SS. We thereby discovered human annexins as binding partners for H. pylori, especially annexin A1, annexin A2, and annexin A5, suggesting that annexins act as soluble protein ligands or receptors for H. pylori. The binding was calcium-dependent and independent of intact bacterial proteins. Further studies revealed the binding of ANXA5 to Lipopolysaccharide (LPS) of H. pylori. Binding of ANXA5 resulted in a significantly reduced capacity of H. pylori to translocate CagA into eukaryotic host cells. In a collaboration with the structural biology lab of Eric Sundberg, we generated and analyzed high resolution structures of several HopQ-CEACAM complexes and CEACAMs in their monomeric and dimeric forms and identified the molecular details of the tight HopQ-CEACAM interaction. We show that HopQ binds to the CEACAM dimerization interface and due to its high binding efficiency HopQ can open up CEACAM dimers to efficiently recruit CEACAM monomers for H. pylori binding. A major goal of this research project was to study how H. pylori uses integrins for CagA translocation. For the first time we provided genetic evidence that the cag-T4SS-mediated interaction of H pylori with cell surface integrins on human gastric epithelial cells is not essential for CagA translocation, but interaction of H pylori with CEACAM receptors is facilitating CagA translocation by the cag-T4SS. Thus integrin but not CEACAM receptors are dispensable for H. pylori CagA translocation. CRISPR/Cas9-mediated knockout of CEACAM receptors (CEACAM1, CEACAM5 and CEACAM6 simultaneously) generated in KatoIII cells resulted in a complete loss of CagA translocation capacity by H. pylori, suggesting that β-integrin receptors play a minor role in the T4SS-mediated CagA translocation, but the H. pylori-CEACAM interaction is of major importance.
Publications
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Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation. PLoS Pathogens, Oct 26;14(10):e1007359 (2018)
Zhao Q, Busch B, Jiménez-Soto L-F, Ishikawa-Ankerhold H, Massberg S, Terradot L, Fischer W, Haas, R
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The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs for host cell adherence and translocation of its oncoprotein CagA. EMBO J. 37(13):e98664. (2018)
Bonsor DA, Schmidinger B, Zhao Q, Deredge D, Wang J, Beadenkopf R, Dow B, Fischer W, Beckett D, Wintrode PL, Haas R, Sundberg EJ
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Different roles of integrin-β1 and integrin-αv for type IV secretion of CagA versus cell elongation phenotype and cell lifting by Helicobacter pylori. PLoS Pathogens PMID: 32497129 (2020)
Fischer W, Haas R
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The HopQ-CEACAM interaction controls CagA translocation, phosphorylation and phagocytosis of Helicobacter pylori in neutrophils. mBio. 11(1). pii: e03256-19 (2020)
Behrens I-K, Busch B, Ishikawa-Ankerhold H, Palamides P, Shively JE, Stanners C, Chan C, Leung N, Gray-Owen S, Haas R
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Kinetics of CagA type IV secretion by Helicobacter pylori and the requirement of substrate unfolding. Mol Microbiol. 116(3):794-807 (2021)
Lettl C., Haas R., Fischer W.
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Helicobacter pylori binds human Annexins via Lipopolysaccharide to interfere with Toll-like Receptor 4 signaling. PLoS Pathog. 2022 Feb 17;18(2):e1010326
Schmidinger B., Petri K., Lettl C., Li H., Namineni S., Ishikawa-Ankerhold H., Jimenez-Soto L.J., Haas R.
