title: Exploring the fitness landscape of gastroenteritis-associated and bloodstream infection-associated Salmonella Enteritidis
Creators: name: Fong, Wai Yee
Publisher: name: University of Liverpool Repository
Year: 2021

description: Non-typhoidal Salmonella (NTS) typically causes gastroenteritis in humans. However, in recent years NTS have emerged as a major causative agent of bloodstream infections in sub Saharan Africa, with Salmonella Enteritidis ranked as the second most common serovar associated with this invasive disease. Genomic and phylogenetic characterisation of S. Enteritidis isolates from human bloodstream has identified the Central/Eastern African clade (CEAC) and Western African clade that are significantly different to the gastroenteritis-associated Global Epidemic clade (GEC). The African clades have distinct genetic features such as genomic degradation, novel prophage repertoires and multi-drug resistance. The molecular basis for the enhanced propensity of African S. Enteritidis to cause bloodstream infection remains poorly understood. This thesis explores the molecular basis for African S. Enteritidis virulence, by investigating the genetic determinants of the GEC-representative strain P125109 and the CEAC-representative strain D7795 for growth in vitro in LB, NonSPI2 and InSPI2 conditions, and replication in RAW 264.7 murine macrophages. Two functional genomic approaches, RNA sequencing (RNA-seq) and transposon insertion sequencing (TIS), were considered. Previously, a SDS-phenol-ethanol-based differential lysis approach had proved successful for isolating Salmonella RNA from infected eukaryotic cells for RNA-seq analyses. Unfortunately, RNA yields were often low or highly contaminated with eukaryotic RNA. I performed experiments to optimise the separation and recovery of intra-macrophage Salmonella and found that needle-and-syringe-mediated lysate homogenisation showed most promise in improving intra-macrophage Salmonella recovery, although the associated increase in intra-macrophage Salmonella RNA yield proved to be unreproducible. Separately, a comparative analysis of four commonly-used RNA extraction methods showed that the RNA extracted with TRIzol was superior in terms of RNA quality and concentration. In light of the unresolved technical challenges with intracellular Salmonella RNA extraction, TIS was used to investigate the genetic requirements of P125109 and D7795 for in vitro growth and macrophage infection. I identified 207 in vitro-required genes common to both strains that were also required by other Salmonella serovars and Escherichia coli, and 63 genes that showed a strain-specific requirement. Distinct patterns for gene requirements for optimal growth in LB, NonSPI2 and InSPI2 were observed between P125109 and D7795, suggesting that the two strains have differing metabolic capabilities. Screening the transposon libraries during macrophage infection identified about 200 genes belonging to each strain that contribute to bacterial survival and replication in mammalian cells. The majority of the genes have characterised roles in Salmonella virulence. I identified candidate strain-specific virulence genes, including genes with no known orthologues in the other Salmonella strains referenced in this study, representing potential novel Salmonella virulence factors. I anticipate that my TIS findings will serve as a useful community resource for investigation of S. Enteritidis gene functions. Future integrative analyses of my TIS-based fitness data with other “-omics” datasets should yield new insights into African S. Enteritidis infection biology.