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Salmonella enterica is an important zoonotic pathogen of clinical and veterinary significance. The species is divided into seven subspecies; subspecies I, enterica, is further divided in to Over 1530 serovars based on different epitopes of two surface antigens. Clinical and veterinary isolalions of Salmonella are frequently typed to the serovar level of classirication. This epidemiological data shows that some serovars are isolated from distinct subsets of species. In this study I have focused on two serovars, S. Derby and S. Mbandaka, which are frequently isolated from distinct subsets of livestock species in the UK. The majority of S. Derby isolations are from pigs and turkeys, whereas the majority of S. Mbandaka isolations are from cattle and chickens. To begin to identify potential mechanisms of host adaptation, I sequenced two strains of each serovar and compared their nucleotide sequences and functional annotations. This lead to the discovery of a new Salmonella pathogenicity is land, SPI-23, in the chromosome sequence of S. Derby, that I go on to show is regulated in a ti ssue specific manner in a porcine Ivac model. Mutagenesis of the most highly upregulated gene with in SPI-23 , po/R, generated unique phenotypes that have enabled me to pos it a ro le for SPI-23 in tropism to porcine jejunum. To interrogate the role of metabolite utilisation in constraining co loni sation of certain niches, I performed high-th roughput phenotyping using Biolog phenotypic microarray technology, at ambient and body temperature!;, under aerobic and anaerobic conditions. This, along with other phenotypic studies, lead me to propose a partitioned niche model 3 Matthew R. Hayward: Thesis between host adaptation in the case of S. Derby and adaptation to persistencc in the environment on soybean based feed in the case of S. Mbandaka. To identify the contribution of an environmcnt composed of a complex set of mctabolites 10 host adaptation, I produced genome-scale metabol ic reconstructions for both serovars. The models were confronted with metabolites found in porcine colon and jejunum; these I identified through metabolomics of gut sections using NMR. These models were used to observe which transport and secondary metabolic reactions contribute most to the incorporation of biomass by S. Derby and S. Mbandaka when in a porcine host. Finally I relate the lindings of these studies to a representat ive population of isolates, for which' have produced a phylogenetic recon struction. I discovered two di stinct lineages of S. Derby each with a distinct set of genotypes and phenotypes. I postulate that one lineage is adapted La turkeys and environmental persistence, and the other adapted to pathogenicity in figs. 1 also show that S. Mbandaka is clonal in thc UK, and is adapted to growth in soybean based feeds. at ambient temperatures and is adapted to environmental persistence. 4

Despite successful sequencing and subsequent comparison of Salmonella Typhimurium (S. Typhimurium) and Salmonella Typhi (S. Typhi) genomes, mechanisms driving S. Typhi host specificity and pathogenicity remain unclear. The characterisation of the surface proteomes of the two serotypes may reveal proteins that play a role in host pathogen interaction, thereby permitting S. Typhi to establish persistent infections. Surface proteins represent a small fraction of the total cell and are difficult to extract preferentially. This study evaluated a range of methods for the extraction of outer membrane proteins (OMPs) in S. Typhimurium, including previously published protocols based on differential solubilisation and a novel approach based on the LPI™ FlowCell. Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified these proteins, and their association with the outer membrane confirmed against a database containing in silica predictions of the sub-cellular location of all ORFs in both genomes. This comprehensive approach identified 73 OMPs, representing 63% of all predicted S. Typhimurium aMPs. When applied to S. Typhi, several OMPs undetected in S. Typhimurium extracts were identified including proteins involved in capsule synthesis. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) detected OMPs potentially upregulated in S. Typhi, resulting in the identification of 20 upregulated spots containing 15 OMPs, five previously undetected. OMPs with various functions were identified, including porins, receptor proteins, transporters and enzymes. Several OMPs with potential roles in virulence, such as the two regulators SlyB and YfgL modulati ng the expression of pathogenicity islands-encoded proteins, were detected. Additionally, the TolC protein previously implicated in antibiotic resistance and invasion as part of the AcrAB-ToIC efflux system was present in both serotypes. Other examples include the FepA and IroN, involved in iron uptake, potentially upregulated in S. Typhi. The expression of many hypothetical aMPs with poorly defined functions was also confirmed, providing useful targets for future studies.