The pathogen C. jejuni is now recognised as the leading cause of bacterial foodborne enteritis in the industrial world. The yearly estimate for Campylobacter infections in the United States alone is 2.4 million people or 1% of the population. Illness caused by C. jejuni is self-limiting, however, some individuals develop complications resulting in autoimmune responses. Despite being a major health burden, the pathogenic process is not fully understood. One aspect of importance is the ability of C. jejuni to adhere to glycosaminoglycans (GAGs), such as heparin. GAGs, sulphated carbohydrates expressed on or in host cells, can serve as receptors for bacterial proteins. In the first study, five heparin-binding proteins of C. jejuni NCTC 11168H were identified. For PEB3 (Cj0289c), this work showed that native wild-type PEB3 and purified recombinant PEB3 produced in E. coli bind heparin. The location of two PEB3 heparin-binding clusters: 62KAKKD65 and 122NKKVRI127, was investigated via site-directed mutagenesis, resulting in impaired heparin-binding. These data suggest GAG-protein-binding may play a role in the pathogenesis of C. jejuni. As well as GAG-binding PEB3 binds 3-PG. Though its exact in vivo role remains unclear, it may act to deliver 3-PG. Scrutiny of the C. jejuni NCTC 11168H genome revealed an uncharacterised gene next to peb3 encoding glpT, or a putative 3-PG transporter. The location of glpT adjacent to peb3 may suggest a related function for the corresponding proteins with PEB3 as the periplasmic binding partner for the transport of 3-PG via GlpT. In this thesis, the roles of peb3 and glpT for two independent phenotypes, 3-PG dependent growth and fosfomycin sensitivity was studied in vitro. The findings indicate glpT has an effect on both, but not peb3. Furthermore, the NCTC 11168H glpT pseudogene, despite containing two frameshift mutations, has the capacity to encode a functional protein. Lastly, the NCTC 11168H peb3/glpT locus was compared with other C. jejuni strains and closely related species C. coli, C. lari and C. upsaliensis genome sequences. The majority of strains peb3/glpT locus followed the gene arrangement lpxB, peb3, glpT, surE. However, the findings indicate the gene loci between lpxB/surE in remaining strains to be hypervariable. Further analysis shows peb3 to be relatively conserved, whereas, the majority of glpT genes display genetic diversity due to interruptions such as indels and deletion. Lastly, I display the organisation of the peb3/glpT locus and glpT structure in their evolutionary context through MLST. In summary, the findings provide for further characterisation of the PEB3 protein and explores the importance of the uncharacterised GlpT of C. jejuni.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:694303 |
| Date | January 2016 |
| Creators | Cantu, Deborah |
| Contributors | Cavet, Jennifer ; Linton, Dennis |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-the-campylobacter-jejuni-peb3-and-glpt(75754190-96b3-46e7-9239-fe5bb9557dd8).html |
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