Grant: Discovery Grants Program - Individual

The glycome represents an extra layer of information above the genome, transcriptome and proteome and deciphering its role is a fundamental question. This is very difficult for bacterial carbohydrates that are highly variable. Elucidating their biosynthesis will allow assessing their function and harnessing the potential of novel enzymes for biotechnology applications. We have focused on modified heptoses from Campylobacters or Yersiniae and characterized 9 heptose modification enzymes that affect the function of the capsule and lipopolysaccharide in which the modified heptoses reside, and that promote chicken colonization by <em>C. jejuni</em>. <strong>Our research addresses 3 hypotheses that arose from this work:</strong><br /> <br /> <strong>1/ The substrate specificity of heptose modification enzymes can lead to biotechnological applications.</strong> We will perform structure / function studies combining protein structure modeling, crystallography and site-directed mutagenesis to investigate the mechanism of action of these enzymes, explain their substrate specificity, and delineate features necessary for specific inhibition. The data will be used to select libraries to be screened <em>in vitro</em> for inhibitors, and the effect of identified inhibitors on survival of <em>C. jejuni </em>and capsule function will be assessed. The data will also be used to alter the enzymes' specificity to produce substrates for transferases that assemble complex carbohydrates, enabling their application to biotechnology, including vaccine epitope synthesis.<br /> <br /> <strong>2/ Modified heptoses are important for bacterial survival in amoeba and for amoeba-mediated chicken colonization.</strong> This hypothesis is based on our demonstration of a role of amoeba in survival of <em>C. jejuni</em> in water, of capsule in protection against intracellular killing and of heptose in chick colonization. We will test this using our heptose modification mutants and our established amoeba exposure and chick colonization assays. Inhibitors arising from structure/function studies above will be tested for their ability to prevent transmission of C. jejuni from water to chicken.<br /> <br /> <strong>3/ </strong><strong>Two novel genes may affect heptose modification and capsule function.</strong> We will use established biochemical and mutagenesis assays to elucidate their activity and their effect on heptose modification and capsule function. This may provide novel methods to control the transmission of <em>C. jejuni </em>to chickens.<br /> <br /> <strong>Our long-term goals </strong>are to understand how specific glycans affect the function of polysaccharides, and to decipher the biosynthesis of novel sugar derivatives for biotechnological applications. Our complementary fundamental research avenues merge towards the practical goal of reducing amoeba-mediated transmission of <em>C. jejuni </em>to chickens via inhibitor design or vaccine epitope synthesis, which will benefit the chicken industry and Canadian economy. This application therefore fulfills the mandate of the NSERC RG program.<br />