Investigating the Structure and Function of Two Rhamnosyltransferases Which Determine the Capping State of Lipopolysaccharide in P. aeruginosa

dc.contributor.advisorLam, Dr. Joseph
dc.contributor.authorPrevedel, Daniel
dc.date.accessioned2015-01-07T16:19:40Z
dc.date.available2015-01-07T16:19:40Z
dc.date.copyright2014
dc.date.created2014-12-19
dc.date.issued2015-01-07
dc.degree.departmentDepartment of Molecular and Cellular Biologyen_US
dc.degree.grantorUniversity of Guelphen_US
dc.degree.nameMaster of Scienceen_US
dc.degree.programmeMolecular and Cellular Biologyen_US
dc.description.abstractPseudomonas aeruginosa is an important opportunistic pathogen known to cause nosocomial infections in compromised individuals including those suffering from the genetic disorder cystic fibrosis. A major virulence factor associated with this organism is lipopolysaccharide (LPS), which is comprised of three domains: lipid A, core oligosaccharide (OS), and the distal O-antigen (O-Ag). The core OS can be produced as two structurally distinct glycoforms termed “capped” and “uncapped”. The capped glycoform contains L-rhamnose (L-Rha) that is α-1,3-linked to the core OS and acts as a receptor for the attachment of O-Ag; whereas the uncapped glycoform has an L-Rha residue α-1,6-linked to the core OS and this is devoid of O-Ag. The mucus-inducible gene (migA) encodes a putative α-1,6 rhamnosyltransferase responsible for the generation of the uncapped core. Conversely, another rhamnosyltransferase gene called wapR, found within the core synthesis gene cluster, encodes the enzyme involved in the production of capped core. To date, biochemical and structural data on MigA and WapR are not available. In this study, expression and purification of MigA and WapR were attained and sparse-matrix crystal screens were carried out on both rhamnosyltransferases, yielding preliminary crystal formation; however, as of yet, diffractable crystals have not been attained. Hence, structural models were generated using I-TASSER software, which predicted a high amount of structural similarity between both enzymes, most notably in the N-terminal region. Progress was also made towards the development of a MigA/WapR rhamnosyltransferase assay. Donor substrate, dTDP-L-Rha, was synthesized enzymatically in vitro and the acceptor core OS lacking L-Rha was purified using a phenol-based extraction method. Separation of dTDP-L-Rha from dTDP was achieved using capillary electrophoresis and will be used to monitor the progress of the assay.en_US
dc.identifier.urihttp://hdl.handle.net/10214/8671
dc.language.isoenen_US
dc.publisherUniversity of Guelphen_US
dc.rights.licenseAll items in the Atrium are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectdTDP-L-rhamnoseen_US
dc.subjectGlycoformen_US
dc.subjectGlycosyltransferaseen_US
dc.subjectLipopolysaccharideen_US
dc.subjectPseudomonas aeruginosaen_US
dc.subjectRhamnosyltransferaseen_US
dc.titleInvestigating the Structure and Function of Two Rhamnosyltransferases Which Determine the Capping State of Lipopolysaccharide in P. aeruginosaen_US
dc.typeThesisen_US

Files

Original bundle
Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Prevedel_Daniel_201501_Msc.pdf
Size:
2.33 MB
Format:
Adobe Portable Document Format
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.71 KB
Format:
Item-specific license agreed upon to submission
Description: