Early interactions of non-typhoidal Salmonella with human epithelium

Non-typhoidal Salmonella is an important bacterial pathogen causing worldwide morbidity and mortality. Early interactions between Salmonella Typhimurium and intestinal epithelium have been demonstrated in animal models, but little is known in humans. The aims of this thesis were to a) establish in vitro and ex vivo models to study such interactions, b) seek evidence of Salmonella invasion in human tissues in vivo, and c) identify virulence genes responsible for Salmonella adherence, invasiveness, and intracellular growth using a high throughput screening model based on transposon mediated differential hybridisation (TMDH). HEp-2 cell cultures were validated as a reproducible in vitro model at different early stages and for investigating host cell cytokine responses and signalling pathways. The model discriminated between different wild-type and characterised mutant strains; it also demonstrated the ability of probiotic lactobacilli to inhibit IL-8 expression in IL- 1β-pretreated and Salmonella-co-treated cells. This was not via IκBα degradation, phosphorylation of NF-κB p65 or its nuclear translocation. Lactobacilli did not postinfectiously affect intracellular Salmonella proliferation or IL-8 production. Polarised Caco-2 cells were employed to investigate host responses to various bacterial pathogen associated molecular patterns (PAMPs). Exposure to Salmonella Typhimurium DNA triggered IL-8/hBD-2 mRNA expression and infection with its FliC-deficient mutant ΔfliM stimulated IL-8 mRNA expression. Thus, both flagellin and bacterial DNA could elicit pro-inflammatory responses. Histology and immunohistochemistry of in vivo and ex vivo human tissues showed bacterial/Salmonella invasion in epithelial cells, macrophages, and neutrophils. After HEp-2 cell infection and extraction of genomic DNA using 1,440 transposon mutants of Salmonella Typhimurium, Transposon Directed Insertion-site Sequencing (TraDIS) identified 47 genes responsible for bacterial colonisation, among which 5 genes (intergenic sucD-cydA, glyA, yqiC, wzxE, and rfaI) account for bacteria-cell association. Furthermore, we discovered that speG is responsible for intracellular replication. Selected mutants will be re-characterised for pathophysiology investigations and prospective adaptation to prevention/treatment in non-typhoidal salmonellosis