Enterobacterial Common Antigen Biosynthesis in Shigella flexneri

Abstract

The complex outer membrane (OM) polysaccharides of bacteria play crucial roles in cellular homeostasis, fitness and provide resistances to extracellular pressures. Enterobacteriaceae possess two major OM polysaccharides; lipopolysaccharide (LPS), which is characteristic of Gram negative bacteria and Enterobacterial Common Antigen (ECA), which is ubiquitously expressed on the OM of all Enterobacteriaceae. Both of these polysaccharides are in-part biosynthesised by separate homologs of the Wzy-dependent pathway, the most common bacterial polysaccharide biosynthetic pathway. Due to the importance of LPS in virulence, the majority of the studies directed towards the key proteins of the Wzy-dependent pathway have been directed towards the LPS homologs, with little research being directed towards the ECA specific homologs. The objective of this thesis was to investigate ECA biosynthesis in Shigella flexneri, with particular focus on WzyE, the Wzy protein from ECA biosynthesis. For the first time, a WzyE protein was directly investigated and the data showed that WzyE is uncharacteristic of other Wzy proteins; showing a high amount of sequence conservation amongst Enterobacteriales. Furthermore, through experimental topology mapping and site-directed mutagenesis, the data showed a plausible central cavity which may be involved in the polymerisation mechanism of WzyE. Through the necessity of the project, for the first time, two different wzyE mutants were generated through lambda Red mutagenesis which showed alternative sensitivities to Colicin E2 and deoxycholate. I ultimately determined that this was due to the regulatory disruption of the adjacent gene, wecG, in one of the mutants and highlighted WecG’s importance in the biosynthetic pathway. Subsequently I investigated WecG which, like WzyE, had not been directly investigated. The data revolutionised the understanding of WecG revealing that WecG is a protein which is peripherally associated with the inner membrane (IM) via its three, C-terminal helices. Further, critical residues along the second helix were shown to be important for both WecG’s membrane association as well as its function and, demonstrated that WecG is likely maintained to the IM via interactions with ECA lipid-I. Ultimately this allowed me to place WecG as the second protein in the novel glycosyltransferase fold family, GT-E. Through investigating WzyE, I noticed an interdependence between ECA and LPS O antigen (Oag) biosynthesis. I subsequently demonstrated that the two OM polysaccharide pathways are fundamentally linked due to their reliance on undecaprenyl phosphate (Und-P) where, wzy mutations in one of the pathways caused a reduction in the OM polysaccharide of the un-mutated pathway. Overall, the work presented here provides new insights and revolutionises our understanding of the ECA biosynthetic pathway. I demonstrated indirect cross-talk between the two OM polysaccharide pathways of S. flexneri for the first time and provide a platform for future studies to investigate Wzy proteins and other key proteins from the ECA biosynthetic pathway.