Proteomic analysis of the oxidative stress response of Salmonella enterica serovar Typhimurium

This thesis describes an analysis of the protein components involved in the oxidative stress responses of the facultatively intracellular pathogen Salmonella enterica serovar Typhimurium (strain SL1344), as well as a study of their control and integration with other key adaptive processes. Following dose response studies with adapted and non-adapted cells, benchmark lethal and sub-lethal dose values for H2O2 were derived for proteomic studies. Comparison of the protein expression profiles of cells of S. Typhimurium that has exposed to H2O2 or left untreated showed that at least 15 proteins were differentially expressed. These included phase 1 and phase 2 flagellin, D-galactose binding protein and glycerophosphoryl diester phosphodiesterase. A putative peroxidase corresponding to the STM0402 gene, whose predicted sequence shares 42% identity with ahpC of S. Typhimurium, was strongly down regulated under the same conditions. The AhpC and STM0402 proteins of Salmonella are paralogues but show opposite responses to oxidative stress. Furthermore, STM0402 does not appear to be regulated by the OxyR or SoxRS global regulatory systems, in contrast to the AhpC protein. The thesis also describes the construction and functional characterisation of a null mutant of oxyS, which codes for an RNA molecule that integrates responses to peroxide stress with other key adaptive processes. The putative oxyS gene product of S. Typhimurium is 110 nt in length and, like its E. coli counterpart, is predicted to contain three stem loops. In support of the structure of the OxyS RNA of Salmonella, base changes in a region predicted to be double stranded were accompanied by complementary base changes on the other strand. The oxyS mutant had an adaptive response to H2O2 that mirrored that of the wild-type but, surprisingly, it was much more resistant to H2O2.