Glycosphingolipid interactions of Helicobacter pylori and F1C-fimbriated Escherichia coli

Abstract

The aim of this thesis was to investigate glycosphingolipid interactions of the human pathogens Helicobacter pylori and F1C-fimbriated uropathogenic Escherichia coli. Of particular interest were interactions of H. pylori to complex gangliosides, the role played by H. pylori neutrophil activating protein (HP-NAP) and the SabA adhesin in the binding of H. pylori to complex gangliosides, and the factors influencing binding of H. pylori to such structures. Additionally, the interaction between F1C-fimbriated E. coli and glycosphingolipids from its target tissue was investigated. Glycosphingolipids were isolated from different sources and characterised using mass spectrometry, proton NMR, enzyme degradation and by the binding of relevant antibodies and lectins. Isolated glycosphingolipids were separated by TLC and binding by 35S-labelled bacteria was investigated by means of the chromatogram-binding assay. Binding by H. pylori to the complex gangliosides sialyl-neolactohexaosylceramide and sialyl-neolactooctaosylceramide isolated from human gastric adenocarcinoma and sialyl-dimeric-Lex from human gall bladder adenocarcinoma was obtained. To investigate the factors influencing the binding of H. pylori to complex gangliosides and the involvement of HP-NAP and the SabA adhesin a ganglioside bank was generated, against which the binding of H. pylori was tested. Binding to complex gangliosides was eliminated in a SabA knockout mutant but not in a HP-NAP knockout mutant. Binding by H. pylori to serial dilutions of linear, fucosylated and branched structures revealed that chain length, fucosylation and branching influence binding. Binding by F1C-fimbriated uropathogenic E. coli to the monoglycosylceramide region of rat, canine and human kidney with additional binding in the tri-tetraglycosylceramide regions of canine and human kidney was obtained. Structural analysis revealed the mono- and triglycosylceramide binding-active components to be Galb1Cer and Gala4Galb4Glcb1Cer with phytosphingosine and hydroxy fatty acids. The results obtained of this study show that H. pylori interacts with complex gangliosides, that this interaction is mediated solely by the SabA adhesin but not HP-NAP. Factors influencing binding were identified as increasing chain length, fucosylation and the degree of sialylation of branches. Two binding-active non-acid glycosphingolipids from human and canine kidney were characterised as Galb1Cer and Gala4Galb4Glcb1Cer with binding of E. coli requiring phytosphingosine and hydroxy fatty acid.