In vitro, ex vivo and clinical characterization of novel Salmonella virulence genes

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. After HEp-2 cell infection and extraction of genomic DNA using 1,440 transposon mutants of Salmonella infection and extraction of genomic DNA using 1,440 transposon mutants of Salmonella Typhimurium, Transposon Directed Insertion-site Sequencing (TraDIS) identified 6 novel Salmonella virulence genomic loci responsible for adhesion/invasion (intergenic sucD-cydA, glyA, yqiC, wzxE, and rfaI) and intracellular replication (speG). The aims of this project were to a) create the single-gene mutants by site-directed mutagenesis, b) characterize the discovered genes by testing the created mutants in the established in vitro human epithelial cells (e.g. HEp-2 cells, polarized Caco-2 cells, and LS174T cells) and polarized human intestinal ex vivo organ culture models to study Salmonella virulence (bacterial adhesion, invasion, and intracellular replication) and host responses (e.g. protein and mRNA expression of IL-8, defensins), c) seek clinical importance of these genes by detecting their expression levels in the isolated strains from patients with and without bacteremia/clinical toxic signs, and in those strains with and without antibiotic resistance to specific antibiotic (e.g. ampicillin). After comprehensive characterization of these unreported genes, their representative mutants can be promising candidates to develop novel strategies for prevention (e.g. oral vaccines) and treatment (e.g. targeted drug vectors) of non-typhoidal salmonellosis.