Cysteine Residues in Helicobacter pylori Adhesin HopQ Are Required for CEACAM–HopQ Interaction and Subsequent CagA Translocation

Abstract

Attachment to the host gastric mucosa is a key step in Helicobacter pylori infection. Recently, a novel adhesin, HopQ, was shown to bind distinct host CEACAM proteins—an interaction that was found to be essential for the translocation of CagA, a key virulence factor of H. pylori. The HopQ–CEACAM1 co-crystal structure revealed a binding mode dependent on loops in HopQ that are clasped by disulfide bonds. In this study, we investigated the importance of these cysteine residues for CEACAM1 engagement by H. pylori. We observed a loss of CEACAM1 binding and CagA translocation upon disruption of the disulfide bond in loop CL1 (connecting C103 to C132 in HopQ). Deletion of the Dsb-like oxidoreductase HP0231 did not affect cell surface expression of HopQ or alter the interaction of H. pylori with target cells. Although HP0231 deletion was previously described to impede CagA translocation, our results indicate that this occurs through a HopQ-independent mechanism. Together, our results open up new avenues to therapeutically target the HopQ–CEACAM1 interaction and reduce the burden of pathogenic H. pylori.

Inhibition of Type IV Secretion Activity and Growth of Helicobacter pylori by Cisplatin and Other Platinum Complexes

“…That cysteine residues and disulfide bond formation are likely involved in the type IV secretion process, is indicated by the deficiency of an H. pylori deletion mutant in the disulfide bond oxidoreductase gene hp0231 for CagA translocation (Zhong et al, 2016). Furthermore, the outer membrane protein HopQ, which plays an important role in the translocation process, requires disulfide bonds for its interaction with CEACAM receptors (Hamway et al, 2020). However, a hopQ mutant of strain P12 is still able to translocate reduced levels of CagA into AGS cells (Zhao et al, 2018), and even this reduced translocation could be inhibited by adding cisplatin (data not shown), arguing against a major role of HopQ in the type IV secretion inhibition observed here.…”

Helicobacter pylori Virulence Factors—Mechanisms of Bacterial Pathogenicity in the Gastric Microenvironment

“…The synergy between HopQ and CagA co-expression is recognized as a facilitated activity of several pro-inflammatory pathways, including NF-κB or those associated with mitogen-activated protein kinases (MAP kinases) via T4SS since HopQ significantly facilitates T4SS activity [ 273 ]. The interaction with T4SS and CagA translocation and/or phosphorylation induced by HopQ is stimulated by HopQ binding to host the carcinoembryonic antigen-related cell adhesion molecules (CEACAM) proteins (primarily CEACAM1, CEACAM3, CEACAM5, and CEACAM6) expressed on gastric epithelial cells promoting NF-κB activation [ 274 , 275 , 276 , 277 , 278 , 279 , 280 ]. This, as a consequence, might induce the development of gastric ulcers and facilitate gastric carcinogenesis.…”

Engagement of CEACAM1 by Helicobacter pylori HopQ Is Important for the Activation of Non-Canonical NF-κB in Gastric Epithelial Cells

“…Binding of H. pylori to CEACAM receptors is mediated by the adhesin HopQ. This interaction is not only essential for binding of H. pylori to the gastric epithelium but also for cag pathogenicity island (cagPAI)-dependent CagA translocation and induction of IL8 secretion by gastric epithelial cells, thereby modulating the host immune response [ 19 , 37 , 39 ]. In this immune response, the activation of the non-canonical NF-κB pathway plays a crucial role, as we previously observed that this signalling pathway is important for the recruitment of immune cells to the stomach upon H. pylori infection [ 32 ].…”

“…H. pylori was grown on WC Dent agar plates for 48 h, the optical density was measured and adjusted to 2 × 10 8 bacteria in 1 mL media, and the bacteria was stained for 30 min with 10 µM carboxyfluorescein succinimidyl ester (CFSE, eBioscience, San Diego, CA, USA) [ 39 ]. The labeled bacteria were washed twice and added at an MOI of 10 to 1 × 10 5 cells, which were previously seeded in a 96-well V bottom plate.…”

“…Considering that HopQ-CEACAM binding is essential for T4SS functionality [ 33 , 37 , 38 , 39 ], we sought to investigate whether HopQ-CEACAM interaction influences the activation of non-canonical NF-κB signalling in gastric epithelial cells. Our results indicate that HopQ-CEACAM interaction not only effects CagA translocation or IL8 secretion, but also influences the activation of the non-canonical NF-ĸB pathway.…”