Monash University
Browse

Restricted Access

Reason: Access restricted by the author. A copy can be requested for private research and study by contacting your institution's library service. This copy cannot be republished

Interaction of Helicobacter pylori with host autophagic processes

thesis
posted on 2017-02-26, 22:34 authored by Deen, Nadia Sultana
Helicobacter pylori is a Gram-negative bacterium that causes gastritis, gastric ulcers and stomach cancers. The bacterium can modulate host autophagic processes in both phagocytic and non-phagocytic cells. Autophagy is a eukaryotic process that can degrade intracellular bacteria. However, some bacteria can modulate autophagic pathways for promoting their intracellular survival. A non-canonical form of autophagy, termed as LC3 (microtubule associated protein 1 light chain 3)-associated phagocytosis (LAP), has also been implicated in the intracellular life of several bacteria in conjunction with or independent of canonical autophagy. Despite sharing some characteristics of both canonical autophagy and classical phagocytosis, LAP has distinct functions in the immune system. Before this study, no report addressed whether the autophagic processes induced during H. pylori infection of macrophages represent canonical autophagy and/or LAP. Moreover, no reports were available on the effect of two major H. pylori virulence factors, namely cytotoxin-associated genes (cag) pathogenicity island (cagPAI) and vacuolating toxin A (VacA), on these pathways. Using a correlative microscopic approach involving both immunofluorescence microscopy and transmission electron microscopy, this study shows that in macrophages, H. pylori induces the formation of a low level of canonical autophagosomes, in which a small percentage of intracellular bacteria (0.5-7.5%) were found. The majority of intracellular bacteria (85-95%) were located in LC3-negative phagosomes, while only 4-14% were found ‘free’ in cytosol. These results suggest that H. pylori does not induce LAP and a small percentage of intracellular bacteria might escape from phagosomes into the cytosol from where they can be sequestered into autophagosomes. No statistically significant difference in the relative distribution of H. pylori in the various aforementioned intracellular compartments was observed between wild-type and ∆cagPAI mutant bacteria, indicating that the cagPAI of H. pylori has little, or no influence on these processes. Interestingly, although no statistically significant difference was observed in the overall extent of cellular autophagy or phagocytosis between wild-type and ∆vacA mutant bacteria, a difference in the percentage of intracellular wild-type and ∆vacA mutant bacteria were observed into autophagosomes, suggesting that VacA influences the autophagic sequestration of H. pylori. Additionally, a difference in the intracellular survival of wild-type and ∆vacA mutant bacteria was observed, demonstrating that VacA affects the intracellular survival of H. pylori in macrophages. However, future studies are required to confirm whether autophagy contributes to this VacA-dependent effect. Using a qRT-PCR array, the expression profiles of 84 autophagy-related genes suggest that a number of genes involved in autophagy machinery and regulation are differentially expressed during H. pylori infection of macrophages. Additionally, the mRNA levels of several genes were found to be H. pylori strain-, virulence factor- and macrophage cell-line-specific. Taken altogether, the results of this study indicate that the autophagic responses induced in H. pylori-infected macrophages are complex, involving the interplay of various bacterial and host factors.

History

Principal supervisor

Terry Kwok-Schuelein

Additional supervisor 1

Rodney Devenish

Additional supervisor 2

Lan Gong

Year of Award

2016

Department, School or Centre

Biomedical Sciences (Monash Biomedicine Discovery Institute)

Additional Institution or Organisation

Biochemistry and Molecular Biology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences

Usage metrics

    Faculty of Medicine, Nursing and Health Sciences Theses

    Categories

    No categories selected

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC