Delivery of bio-molecules by Helicobacter pylori

Fayth Good

Research output: ThesisMaster's Thesis

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Abstract

Live, recombinant delivery systems are produced by constructing attenuated, recombinant micro-organisms, such as bacteria or virus, which present foreign vaccine antigens or bioactive molecules to the host to elicit protective immunity or relieve pre-existing symptoms in the host without causing pathology. These delivery systems have several advantages; firstly viruses or bacteria presenting the antigen can behave as an adjuvant to facilitate the recognition of foreign antigen by the immune system; secondly it is possible to create a multivalent vaccine which can deliver both bioactive proteins and vaccine antigens, thereby reducing the need for several doses; thirdly these systems are needle-free and orally administered thus do not require trained personnel and finally they are stable, do not require refrigeration and are cheap to produce in large quantities.

To date, bacterial species such as lactic acid bacteria have effectively been used in animals and humans as an oral cytokine delivery system. Specifically, Lactococcus lactis expressing recombinant interleukin 10 was successfully used as a daily treatment for the relief of symptoms in inflammatory bowel disease.

The aim of this thesis was to construct a live, recombinant delivery system for small molecules such as cytokines (IL-2, IL-4, IL-10 and IFN) and a gut hormone (leptin) using Helicobacter pylori, a non-invasive bacterium which colonizes the gastric mucosa. Various delivery pathways were investigated including delivery of bio-molecules via fusion to a protein for extracellular secretion or surface display (HopE, Lpp20), fusion to auto-transporters (YadA, VacA) and periplasmic secretion signals (sec pathway). DNA coding recombinant proteins were inserted at various loci, such as the ureAB, mdaB and vacA loci, which were tested to ensure no interference with bacterial survival. Genetic characterization of the recombinant strains was done by colony PCR and Western Blot analysis. To ensure insertion at the ureAB locus did not disrupt urease activity, a urease-activity assay was performed for strains which contained insertions at this locus. As a final step, some strains were assessed for colonization in a H. pylori mouse model of infection.

Results showed that it was possible to engineer H. pylori to express cytokines or leptin using the various delivery pathways investigated. However, not all cytokines could be engineered by fusion to HopE or the periplasmic sec signal and the urease activity of strains with insertions at the ureAB locus showed approximately a 50% decrease compared to wild-type H. pylori. Recombinant strains using the VacA fusion were able to colonise the gastric mucosa of mice, although there was a noticeable impairment in colonization of the leptin- and a cytokine-expressing strains compared to wild-type H. pylori.

Results obtained from this study are promising but require further investigation to fully evaluate the use of H. pylori as a biological delivery system. This study has demonstrated that H. pylori can be genetically modified to deliver small proteins via multiple pathways, using secretion signals and autotransporter domains. Further experiments to determine localization (FACS analysis) and in vivo activity (mouse model) are essential to fully characterize whether cytokine and hormone-expressing H. pylori strains are a viable bio-molecular delivery system.

Taken together a live bacterial delivery system based on H. pylori provides a new and novel opportunity for the continuous delivery of bioactive molecule for the treatment of chronic diseases such as inflammatory bowel disease.
Original languageEnglish
QualificationMasters
Publication statusUnpublished - 2010

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