It is all about flagellin: Towards development of a Campylobacter jejuni flagellin-based vaccine

Abstract

Campylobacter jejuni is the most common cause of foodborne bacterial diarrhea in humans worldwide. C. jejuni is highly prevalent in livestock, poultry in particular. The most common source of human infection is C. jejuni-contaminated poultry meat products. The main objective of the work described in this thesis was to provide a scientific basis for the development and production of a C. jejuni vaccine for use in chickens. Effective vaccination of chickens would decrease C. jejuni entry into the food chain and in turn the incidence of infections in humans. We designed a subunit vaccine based on the flagellin. The flagellin protein is required for flagellar motility, which is an imperative to C. jejuni virulence. The recombinant flagellin was produced in Escherichia coli and administered to chickens at the embryonic stage of development (in ovo). In ovo vaccination with subunit vaccines is not common practice. Our successful immunization indicates that this approach is feasible. The protein was immunogenic and generated a humoral immune response but protection after challenge with C. jejuni was not achieved. One possible reason is the lack of the reactivity of the raised antibodies with intact C. jejuni cells. Since the native C. jejuni flagellin is heavily glycosylated, we hypothesized that glycans may contribute to the development of the protective immune response. This requires the production of a glycosylated variant of the vaccine. Since E. coli lacks the flagellin glycosylation machinery, we explored C. jejuni as a natural microbial factory to produce glycosylated flagellins. The establishment of a C. jejuni production strain that shows efficient biosynthesis, glycosylation and secretion of recombinant flagellins requires detailed knowledge of the biogenesis of flagella in this species. The experimental work described in this thesis identifies several flagellin-binding partners (FliW, FliS) that play a role in flagellin processing. We discovered that FliW protein, together with the post-transcriptional regulator CsrA, acts as a sensor of intracellular flagellin levels. The flagellin chaperone FliS was identified as crucial for C. jejuni filament assembly and bacterial motility. FliS was found to preferentially bind to glycosylated flagellin what favors the export of the glycoform of the protein, ensuring the proper filament assembly. We identified the potential flagellin glycosyltransferase that adds the sugars to the protein. This is a first step towards understanding of the post-translational processing of flagellins in C. jejuni. Detailed investigation revealed separate flagellin subdomains as binding sites for FliW, FliS and the glycotransferase. Overall, the thesis presents valuable insights into the production and processing of flagellins in C. jejuni. We propose a theoretical model describing the events occurring after the flagellin biosynthesis. In this model, FliW, FliS and the glycotransferase interact with flagellin sequentially to ensure the correct post-translational processing of the flagellin and its delivery to flagellar export machinery. The gained knowledge can be explored for the design of an optimal C. jejuni strain producing glycosylated flagellins for vaccination purposes.