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Title: The interaction of Helicobacter pylori O-antigen with the immunomodulatory lectins DC-SIGN and galectin-3
Author: Flood, Warren
ISNI:       0000 0004 7655 1810
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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Helicobacter pylori are unique in their ability to colonise the human gastric mucosa. They persist lifelong in untreated individuals despite the presence of a continuous and specific immune response being mounted against it. H. pylori O-antigen is thought to be involved in immune-evasion and subversion by the bacteria and expression has been shown to facilitate colonisation and exacerbate pathology in murine models. This study investigates immuno-relevant roles of H. pylori O-antigen as a pathogen-associated molecular pattern (PAMP) and its interaction with two pattern recognition receptors (PRRs); galectin-3 and DC-SIGN. These PRRs possess distinct carbohydrate recognition domain (CRD) structures and binding affinities. Despite this, we have demonstrated that they compete for adhesion to both Lewis antigen glycoconjugates and whole cell H. pylori 26695 in solid phase binding assays. Galectin-3 significantly reduces DC-SIGN adhesion at a 2:1 stoichiometric ratio in both Lex glycoconjugate and whole cell H. pylori 26695 assays, and abrogates carbohydrate-specific binding in Lex glycoconjugate assays at a 22:1 ratio. These results suggest that galectin-3 may play a role in inhibiting or modulating the interaction between H. pylori O-antigen and DC-SIGN in vivo. Supporting this, we have shown that galectin-3 secreted by AGS cells during competitive infection with H. pylori 26695 is sequestered by H. pylori O-antigen. We have demonstrated that competitive infection of the O-antigen deficient mutant H. pylori 26695 galE in DC-SIGN expressing THP-1 cells reveals a significant reduction in intracellular survival at 8 hours compared to H. pylori 26695 Wt. Co-incubation of H. pylori 26695 Wt with 10 µg ml-1 galectin-3 reduced intracellular survival to the levels of H. pylori 26695 galE at 8 hours. Furthermore, H. pylori 26695 galE displayed rapid association of the endocytic markers Rab5 and Rab7 at 15 minutes compared to H. pylori 26695 Wt. Monoclonal antibody-mediated blocking of DC-SIGN in H. pylori 26695 Wt-THP-1 infections resulted in rapid association of the endocytic markers Rab5 and Rab7, corresponding to that of H. pylori 26695 galE, indicating that DC-SIGN-O-antigen interactions alters intracellular processing of the bacteria and reduces the rate at which these markers are recruited. Together these results elucidate novel mechanisms of H. pylori O-antigen and its interaction with galectin-3 and DC-SIGN that warrant further investigation in vivo. The identification of two PRRs competing for the same PAMP is unconventional and inspires a re-evaluation of PRRs in innate immune recognition.
Supervisor: Else, Kathryn Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Lectins ; Innate immunity ; Dendritic cells ; Helicobacter pylori ; DC-SIGN ; Galectin-3