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