Investigation of the Wzy 0 antigen polym.erase and the Wzz co-polymerase interface

Abstract

Complex polysaccharide chains such as the O antigen (Oag) component of lipopolysaccharide (LPS) and the enterobacterial common antigen (ECA) are located on the bacterial cell surface. Oag and ECA are synthesised by the most common polysaccharide synthesis pathway in bacteria known as the Wzy-dependent pathway. The Oag and ECA are polymerized into chains via the inner membrane proteins WzyB and WzyE respectively, while the co polymerases WzzB and WzzE respectively, modulate the number of chains or “the modal length” of the polysaccharide. The objective of this thesis is to understand how the Wzy and Wzz proteins interact to control Oag and ECA modal length, using Shigella flexneri as a model. Particular focus was placed on WzyB and its binding partners; throughout this thesis each of the co-polymerases, WzzB, WzzE and WzzpHS2, were all shown to physically interact with WzyB. For the first time “cross-talk” between Oag and ECA synthesis was shown and identified novel physical protein protein interactions between proteins in these systems. These findings further the understanding of how these systems function to control polysaccharide chain length. The data suggests that the TM 2 region of the co-polymerases is likely the binding site to WzyB. Additionally, the data suggests that the aa 352-354 region of WzyB appears to be the binding site of WzzpHS2 but is also likely an active site involved with Oag modal length control. Using co-purification experiments, novel WzyB binding partners with no previous link were also discovered via mass spectrometry. The physical interaction between the WzyB and Wzz members of the Wzy-dependent pathway appear to be the only members of the pathway that interact. This interaction is likely required for function and as such furthering the understanding of this interaction is of critical importance, which has great implications for novel biotechnologies and/or the combat of bacterial diseases. Finally, whole genome sequencing of previously identified colicin E2 sensitive mutants reveals novel genes involved with unknown aspects of LPS synthesis, transport or length regulation. Future mutagenic investigation may shed light on these genes which may be critical to LPS