Research Interests

The Team




General overview:

We aim at identifying the role played by protein glycosylation in the virulence of two genetically related gastro-intestinal human pathogens that cause very different and specific pathologies: Campylobacter jejuni and Helicobacter pylori. We have made great progress in the elucidation of the N- and/or O-linked protein glycosylation pathways in these bacteria, and in the determination of their role in pathogenesis. We are now investigating the role of glycosylation on the function of select glycoproteins.
Other significant research topics in the laboratory include investigating the biosynthesis and role of C. jejuni capsular components, the role of amoeba in survival of C. jejuni in the environment and host transmission, and the function of novel secreted proteins in the virulence of H. pylori.


Figure 1: C. jejuni.

1/ Background:

Campylobacter jejuni is the leading cause of endemic and traveler's bacterial gastroenteritis. It has also recently been linked to the development of intestinal cancers. The endemic character of C. jejuni infections in developing countries, together with the high incidence of C. jejuni associated traveler's disease cases, and the emergence of antibiotic resistant clinical isolates of C. jejuni emphasize the need for the discovery of new potential therapeutic targets. However, this will require a better understanding of the virulence factors of this organism and of their specific roles during host infection and colonization.
Helicobacter pylori was only discovered in the early eighties by Warren and Marshall, who received the 2005 Physiology and Medicine Nobel Prize. Since then, H. pylori has been shown to be responsible for gastric ulcers and cancers. It is present chronically in 70-90% of the population in developing countries but its mode of transmission and its potential initial reservoir are not known. This organism is very well adapted to the hostile environment of the human stomach in which it thrives. Although numerous virulence factors have been identified, the pathogenesis of H. pylori is still far from fully understood. Additional factors that control the high stringency of host specificity and that could be critical for host colonization have yet to be identified and characterized.

2/ Research focus:

2.1/ Protein glycosylation in Campylobacter jejuni and Helicobacter pylori:
A common hallmark of these bacteria is the production of glycoproteins that contribute significantly to their virulence. Using a combination of bacterial genetics, microbiology and enzymology, we have made great progress in the elucidation of the biosynthetic pathways that generate the sugars that are necessary for protein glycosylation in both bacteria. We have characterized several key enzymes of these pathways at the biochemical and/or structural level (Creuzenet et al 2000; Creuzenet 2004; Obhi et al 2005; Vijayakumar et al 2006; Ishiyama et al 2006; Demendi and Creuzenet 2009) and produced evidence to suggest the existence of multiple glycoproteins in H. pylori (Hopf et al 2011). We have also produced knockout mutants of these enzymes in both bacteria, and analyzed how the mutations affected the production and function of virulence factors such as flagella, lipopolysaccharide and urease (Merkx-Jacques et al 2004; Vijayakumar et al 2006; Hopf et al 2011). Some of the mutants were also tested in animal models. Using similar techniques, we are now focusing on determining the role of protein glycosylation on the function of select glycoproteins. All our data indicate that the enzymes targeted are important for the production of multiple virulence factors and represent good candidates for the development of inhibitors with potential therapeutic value.

2.2/ Biosynthesis of modified heptoses in Campylobacter jejuni and Yersinia pseudotuberculosis:

            C. jejuni produces a capsule that is also essential for its virulence. It comprises uniquely modified heptoses that do not exist in Mammals. Consequently, the enzymes involved in their synthesis are potential new targets for therapeutic intervention against Campylobacter species. At onset of this research, the biosynthesis of these modified heptoses was not understood. Using a combination of bacterial genetics, cellular biology and microscopy techniques, we have identified 3 genes that are responsible for the synthesis of these modified heptoses, and shown that each of them contributes to the full virulence potential of C. jejuni strain NCTC 11168, both in vitro and in an animal model (Wong et al 2015). We are in the process of characterizing the encoded enzymes at the biochemical level, to allow subsequent development of inhibitors. We have characterized the enzymes involved in heptose modification in strain NCTC 11168 (McCallum et al 2013) and also fully characterized the heptose modification pathway of a highly virulent strain of C. jejuni that produces a slightly different heptose derivative than strain NCTC 11168 and revealed unexpected enzymatic activities which can be exploited for the development of inhibitors (McCallum 2011; McCallum 2012). A comparative structural and functional study of the enzymes involved in both strains is currently under way to determine common features that could be targeted for inhibitor development.

            Similar modified heptoses are also found in the lipopolysaccharide of another gastro-intestinal pathogen, Yersinia pseudotuberculosis but their contribution to the role of capsule or LPS as virulence factors and their biosynthetic pathways were unknown. Using a combination of molecular genetics and mass spectrometry analyses, we have identified the 2 genes responsible for 6-deoxyheptose biosynthesis in Y. pseudotuberculosis and shown that the 6-deoxyheptoses are important for resistance to components of the host’s innate immune defenses both in vitro and in vivo. (Ho et al 2008; Kondakova et al 2008). The enzymes involved were fully characterized at the biochemical level (Butty et al 2009).


Figure 2:
H. pylori

2.3/ Role of novel secreted proteins in the virulence of Helicobacter pylori:
We have identified novel secreted proteins of unknown function in H. pylori. We have demonstrated that these proteins are produced and secreted in laboratory strains. With the help of our collaborators Prof. C. Burucoa and Dr. H. Atanassov (Universite de Poitiers, France), we were able to show that our proteins of interest are produced by clinical isolates of H. pylori and that patients with H. pylori infection produce antibodies against these proteins. We are currently investigating the function of these proteins using tissue culture and in vivo models. We are also investigating the mechanism of folding and secretion of these proteins as this mechanism could be targeted by therapeutic inhibitors to prevent the secretion of our proteins of interest. We have identified a novel chaperone involved in their folding prior to secretion and performed an extensive biochemical and fucntional charactreization of this protein (Lester et al 2015).

2.4/ Role of amoeba in survival of Campylobacter jejuni in the environment:

Transmission of C. jejuni to humans from contaminated poultry is recognized as an important socio-economic and health problem. The mode of transmission of C. jejuni from the environment to poultry is not well understood. We recently determined that amoeba could play a role in the process. Contrary to the accepted dogma, we showed that intra-amoeba survival of C. jejuni is very limited. Instead, we showed that C. jejuni benefits from amoeba-mediated depletion of dissolved oxygen from the environment to thrive extracellularly in aerobic conditions (Bui et al 2012a). This may play an important role in the chain of contamination of new poultry flocks in broiler houses. We also investigated whether environmental exposure to stress affected the subsequent interactions of C. jejuni with amoeba, which in turn could affect dissemination (Bui et al 2012b). We are currently investigating if protein glycosylation or capsular heptose modification have any effect on the interactions of C. jejuni with amoeba.


3/ What you would get to do if you joined us as a Graduate student?

You would be in charge of a full project, covering genetic and functional aspects. This would provide you with have ample opportunity to acquire training in multiple disciplines, ranging from basic Microbiology to sugar and protein Biochemistry, encompassing Molecular Biology and Cell Biology.
You would get exposed to state-of-the-art techniques such as confocal microscopy and capillary electrophoresis as well as to traditional but ever so useful techniques such as HPLC/FPLC purifications of enzymes and sugars, cloning, PCR, electrophoresis, Western blotting, tissue culture etc…

To inquire about available positions, please email your CV and transcripts to Dr Creuzenet at ccreuzen@uwo.ca.


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Last updated: March 24, 2015