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General overview:
We aim at identifying the role played by surface
carbohydrates (glycolipids and glycoproteins) in the virulence of two
genetically related gastro-intestinal human pathogens that cause very
different and specific pathologies: Campylobacter
jejuni and Helicobacter pylori.
One hallmark of these two bacteria is to produce glycosylated
proteins. 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 also include
investigating the biosynthesis and role of C.
jejuni capsular components, and investigating the use of lactobacilli as probiotics to treat/prevent H. pylori
infections.
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Figure 1: C. jejuni.
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1/ Background:
Campylobacter jejuni is the leading cause of endemic and traveler's
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). 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). 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 we believe are responsible for
the synthesis of these modified heptoses, and shown that each of them
contributes to the full virulence potential of C. jejuni, both in
vitro and in an animal model. We are in the process of characterizing the
encoded enzymes at the biochemical level, to allow subsequent development of
inhibitors.
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. A
manuscript describing this work has been accepted for publication in
Molecular Microbiology (Ho et al 2008) and another one has been submitted to
Carbohydrate Research (Kondakova et al 2008).
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Figure 2:
H. pylori
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2.3/ Probiotic role of lactobacilli
against Helicobacter pylori.
We have been investigating whether lactobacilli, so-called "good
bacteria" often used for food production and present in the
gastro-intestinal tract as commensals, could offer a novel therapeutic or
prophylactic avenue against H. pylori infections. We are in the
process of investigating the mechanism involved, using a combination of
bacterial genetics and functional assays. This project is being performed in
collaboration with Dr J. Jass (UWO/LHRI).
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, Southern 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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