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71 INVESTIGATING THE GLYCOSYLATION OF GroEL AND ITS ROLE IN THE VIRULENCE OF CAMPYLOBACTER JEJUNI A Merkx-Jacques, I Gryski, C Creuzenet Campylobacter jejuni is a food-borne human pathogen and a major cause of gastroenteritis, reactive arthritis and neurological disorders. Due to emerging drug resistant strains, new treatments and/or vaccines need to be developed.
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario
Protein glycosylation, or the post-translational modification of proteins by sugar molecules, plays an important role in this bacterium's virulence including affecting cell-host interactions, immunogenicity and pathogenicity. C jejuni harbours numerous proteins glycosylated by a heptasaccharide containing diacetamidobacillosamine. Our laboratory investigates the enzymes required to make this sugar as well as the glycoproteins on which this glycan is found.
Analysis of C jejuni proteins by Western blot suggests that the molecular chaperone GroEL is post-translationally modified. A stress-induced protein as well as a major immunogenic antigen in C jejuni infections, GroEL prevents misfolding and aggregation of partially denatured proteins through an ATP-dependent process and may play an essential role in intestinal tract colonization and bacterial survival at high temperatures. GroEL and other chaperones have been shown to be glycosylated in other bacteria including another Campylobacter species. It has also been proposed that C jejuni GroEL is glycosylated based on its reactivity with a lectin. The focus of this work is to determine if GroEL is really glycosylated, identify the glycan present on GroEL and determine the function of the glycosylation modification on GroEL.
We are using ATP affinity followed by anion exchange chromatography to purify GroEL and will determine its exact molecular weight and the glycan attachment site by mass spectrometry (MS). Initial enzymatic deglycosylation using five different enzymes suggests that the glycan present on the protein is not recognized by the enzymes available. The glycan present will be identified by ion-exchange chromatography, MS and NMR. The effect of the sugar on the activity of GroEL will be ascertained. We will also determine the role of the glycosylated form of GroEL and its glycan in the virulence of C jejuni by looking at the ability of mutants with defects in either the expression of the glycoprotein or its glycosylation to adhere and invade intestinal epithelial cells.
This information will permit us to infer what enzymes are required to make these sugars, allowing the enzymes to later be targeted to inhibit protein glycosylation should glycosylation be shown to be important for virulence.
INVESTIGATING THE GLYCOSYLATION OF GroEL AND ITS ROLE IN THE VIRULENCE OF CAMPYLOBACTER JEJUNI
A Merkx-Jacques, I Gryski, C Creuzenet
Campylobacter jejuni is a food-borne human pathogen and a major cause of gastroenteritis, reactive arthritis and neurological disorders. Due to emerging drug resistant strains, new treatments and/or vaccines need to be developed.