Regulation of O-antigen modification in Salmonella

The extracellular surface of the pathogen Salmonella contains the lipopolysaccharide (LPS) molecule, which provides structural support and defence against predators. A polysaccharide component of the LPS, the O-antigen has a highly variable composition and is a primary target for the antibody mediated immune response. The O-antigen displays vast structural diversity across the Salmonella genus, and this variation is further enhanced through the action of genes such as glycosyltransferase (gtr) operons, which add carbohydrates to the O-antigen and affect phage resistance and antibody recognition. Importantly, certain gtr operons are also capable of phase variation, meaning their expression can be switched ON or OFF in a heritable, but reversible manner. A switched ON, gtr expressing cell, can produce daughter cells with switched OFF gtr expression and vice versa. gtr phase variation produces modified and unmodified O-antigen molecules, thereby creating population heterogeneity. Previous work identified the mechanism of gtr phase variation: methylation of GATC sequences by Dam and binding of the oxidative stress response regulator OxyR. GATC sequences overlap with OxyR half sites in the gtr regulatory region. Depending on which GATC sequences are methylated and which half-sites OxyR occupies, gtr expression switches between the ON and OFF phase. This research aimed to identify influences on gtr phase variation and expression. Naturally occurring sequence variations of Salmonella serovars were found to alter the phase variation switch frequency, or even abrogate the process. Stationary phase growth caused reduced gtr expression. The role of OxyR in gtr regulation was also further defined. The OxyR regulator can switch between an oxidised and a reduced state. Although the reduced state was previously found to be sufficient for phase variation to occur, the role of oxidised OxyR in this process was not understood. In this work, a possible role for oxidised OxyR was found in regulating expression of a short RNA (STnc1870), which was divergently transcribed from the gtr regulatory region. Finally, a potential target for STnc1870 was found: the ssaO mRNA which encodes a component of the Salmonella Pathogenicity Island 2 secretion needle. Artificial overexpression of STnc1870 reduced expression of ssaO. This connection indicates a possible further role for the gtr regulatory region beyond controlling O-antigen modification.