N-linked glycosylation in Campylobacter jejuni and Campylobacter fetus and N-linked glycans as targets for antibody-based detection

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Abstract

Campylobacter spp., especially C. jejuni and C. coli, are the leading cause of bacterial gastroenteritis in Europe. There is a recognised need to develop detection tools which can be performed on farms to facilitate reducing the presence of Campylobacter in poultry. A similar application could be beneficial for detection of C. fetus, a veterinary pathogen which causes significant economic loss in the cattle industry. Campylobacter species perform protein N-linked glycosylation and in C. jejuni at least 150 proteins, many of which are surface-exposed, may be modified. Therefore, the first portion of this thesis investigated the feasibility of using N-linked glycans as targets for antibody-based detection of Campylobacter species. To do this, a His-tagged N-glycoprotein was expressed and purified from C. fetus and used as immunogen to raise an antiserum termed CfNgp. The Campylobacter N-glycan reactivity of this antiserum was characterised and it was shown to react with N-glycoproteins and cells of C. fetus and other emerging Campylobacter species such as C. concisus. Immunoblotting techniques and flow cytometry were used to characterise an antiserum (CjNgp) raised against a C. jejuni N-linked glycoprotein and demonstrated that it can specifically detect cells of C. jejuni, C. coli and other emerging Campylobacter species found in poulty. This thesis also describes the investigation of the relatively uncharacterised C. fetus N-linked glycosylation system. Functional analysis of C. fetus predicted glycosyltransferases was acheived by developing glycocompetent E. coli containing a hybrid C. jejuni/C. fetus pgl system. The N-glycan structures biosynthesised were analysed using mass spectrometry and this novel approach discovered the activity of two C. fetus glycosyltransferase enzymes. Finally, this work used a bioinformatics pipeline to produce a C. fetus predicted N-linked glycoproteome and experimentally verified a newly identified N-linked glycoprotein. This pipeline was also applied to investigate the putative conservation of N-linked glycoproteins throughout the Campylobacter genus and highlighted ‘core’ N-linked glycoproteins which are key targets for experimental investigation. Overall, this work demonstrates that Campylobacter N-linked glycans are attractive targets for antibody-based detection, expands our knowledge of C. fetus N-linked glycosylation and contributes to the broader understanding of this intriguing aspect of Campylobacter biology.

Keyword(s)

Antibody-based detection; Bacterial glycosyltransferases; Campylobacter fetus; Campylobacter jejuni; N-linked glycoproteome; N-linked glycosylation

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