Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Creuzenet, Carole

Abstract

With the majority of Canadian Campylobacter jejuni infections resulting from contaminated poultry, I investigated how the strain NCTC 11168 capsular heptose modulates host macrophage activation, and bacterial clearance. Activation was assessed by ELISAs, Griess assays, qRT-PCR, and cytokine multiplex, while looking at adhesion to, uptake by, and survival within host macrophages. The heptose was immunosuppressive in chicken, but not human, macrophages and did not significantly impact clearance by tested host macrophages. It is also likely that heptose biosynthesis enzymes engage in substrate channelling, as previous data indicates that these enzymes have a means to limit degradation of unstable intermediates within the bacterial cytosol. Interactions between heptose biosynthesis enzymes were assessed by surface plasmon resonance, which indicates that enzymes in the heptose modification pathways can bind. This work determines how the capsular heptose alters the activity of host macrophages, while investigating if enzymatic interactions can be exploited to modulate heptose production.

Summary for Lay Audience

Campylobacter jejuni is a common cause of diarrheal disease in humans, with most transmission in Canada resulting from ingestion of contaminated broiler chicken meat. The bacterium does not cause disease in the gastrointestinal tract of this avian host, with disparities behaviour across hosts potentially resulting from differences in innate immune activation. A key mediator of interactions with host cells may be 6-O-Me-L-gluco-heptose, a sugar residue which branches off the backbone of the C. jejuni NCTC 11186 capsule polysaccharide (CPS). To study the role this heptose plays in moderating the host immune response, I have co-cultured C. jejuni mutants that differentially express their capsular heptose with human or chicken macrophages. This work has permitted the study of immune activation using assays that assess the production of molecules that help kill bacteria. Bacterial clearance has been assessed using adhesion, uptake, and intracellular survival assays. I hypothesized that the C. jejuni NCTC 11186 capsular heptose residue acts to dampen activation in both hosts, leading to diminished clearance by host macrophages. Data indicate that the capsular heptose reduces immune activation when interfacing with chicken, but not human, macrophages. The CPS confers lower adhesion to and survival within chicken macrophages, while conferring diminished uptake by human macrophages, as well as diminished survival early after internalization.

I have also investigated how the enzymes that produce the heptose interact, as previous data indicates that these enzymes have a means to limit degradation of intermediates within the cytosol. I hypothesize this is accomplished through substrate channeling, a process by which enzymes transfer pathway intermediates through physical interactions. Interactions between enzymes are being studied primarily using surface plasmon resonance, which indicate that enzymes within the heptose synthesis pathways can bind.

These two subprojects merge synergistically with the goal of increasing clearance of C. jejuni from broiler chickens being raised for consumption, limiting transmission to humans. I have simultaneously investigated how the capsular heptose alters activation of, and clearance by, host macrophages while determining if physical interactions between enzymes can be exploited to modulate heptose production.

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