Identification and characterisation of Salmonella enterica serovar Typhimurium factors playing a role in the colonisation of the porcine gut

Salmonella is an important food borne pathogen. Over 100,000 cases of human Salmonella infection are reported in the European Union each year, resulting in an economic burden estimated to be around 3 billion Euros per year (EFSA, 2012). In a European Food Safety Authority (EFSA) survey between 2006 and 2007 S. Typhimurium was the most common serovar of Salmonella isolated from pig carcasses (EFSA, 2008a). Pigs can be asymptomatic carriers of S. Typhimurium (Berends et al., 1996) and contaminated pork contributes significantly to the number of human infections. It has been estimated that the porcine Salmonella reservoir contributes between 10-20% of human salmonellosis cases per year (VLA, 2010). In addition to improvements in biosecurity and husbandry practices, immune-prophylaxis is an important method to reduce the prevalence of food borne pathogens such as Salmonella in reservoir species. An understanding of the molecular basis of bacterial colonisation and persistence in the reservoir host is crucial to rational vaccine design and targeting relevant species. S. Typhimurium expresses multiple surface factors involved in adherence and colonisation of gut epithelium in several host species. The aim of this project was to identify factors involved in S. Typhimurium colonisation of the porcine gut. The work presented here specifically focuses on the role of flagella in the colonisation of porcine gut epithelium. Flagella are motility organelles possessed by many bacterial species. Flagella can also function as surface adhesins, shown in Escherichia coli O157:H7 (Mahajan et al., 2009), and Pseudomonas (De Bentzmann et al., 1996, Lillehoj et al., 2002). Flagellin is the major flagellar filament structural protein approximately 50kDa in size. Salmonella enterica has the ability to switch between two alternate, antigenic forms of its flagellin filament protein, expressing either FliC or FljB (Macnab, 1996). The biological relevance of these two types of flagella filament protein is still not understood. It has been postulated that the presence of a second phase type of flagella may offer an advantage to the bacteria by avoiding recognition by the immune system. However, studies have shown that both FliC and FljB flagella activate Toll-like receptor-5 (TLR-5) mediated by nuclear factor (NF)-κB signalling (Simon and Samuel, 2007b). One specific objective of this research was to compare the role of flagellar phase types in S. Typhimurium adherence and colonisation of porcine gut. To this end a porcine colonic primary epithelial cell culture and ex vivo tissue explants were developed as in vitro infection models. Primary colonic cell cultures were phenotypically characterised using specific markers for epithelial and M cells. In addition to primary epithelial cell culture, porcine intestinal epithelial cell line, IPEC-J2, was also used for specific flagellar interaction studies. The role of flagella in interaction of S. Typhimurium to porcine intestinal epithelium was tested using S. Typhimurium strain SL1344 and flagella mutant derivative strains. Flagella mutant strains exhibited reduced binding to porcine intestinal epithelial cells. Purified flagella proteins were also shown to bind porcine intestinal epithelial cells. Moreover, flagella specific anti-sera suppressed S. Typhimurium adherence to both porcine intestinal epithelial cells as well as porcine colonic explants. The immuno-protective role of flagella as a potential S. Typhimurium vaccine candidate was tested during vaccine efficacy studies in pigs. Parenteral immunisation of pigs with purified FliC and FljB flagella proteins induced production of both IgG and IgA antibodies. The vaccination of pigs with Salmonella flagella provided some protection against challenge as fewer ileum tissue samples from the pigs in the vaccinated group tested positive for Salmonella. The intestinal contents from the vaccinated pigs tested for Salmonella post mortem appeared to also have lower levels of Salmonella compared to un-vaccinated controls, though these were not significantly different between groups. This project has identified flagella as one potential subunit of a multivalent subunit vaccine to help control salmonellosis in the porcine reservoir

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively

Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor

The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following endoplasmic reticulum (ER) stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors dimethyloxalylglycine (DMOG) and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels. Hypoxic induction of ATF-4 was due to increased protein stability, but was independent of the ubiquitin ligase von Hippel-Lindau protein (pVHL). A novel oxygen-dependent degradation (ODD) domain was identified adjacent to the zipper II domain. Mutations of 5 prolyl residues within this ODD domain or siRNA-mediated down-regulation of PHD3, but not of PHD2, was sufficient to stabilize ATF-4 under normoxic conditions. These data demonstrate that PHD-dependent oxygen-sensing recruits both the hypoxia-inducible factor (HIF) and ATF-4 systems, and hence not only confers adaptive responses but also cell fate decisions

Comprehensive and Integrated Genomic Characterization of Adult Soft Tissue Sarcomas

Sarcomas are a broad family of mesenchymal malignancies exhibiting remarkable histologic diversity. We describe the multi-platform molecular landscape of 206 adult soft tissue sarcomas representing 6 major types. Along with novel insights into the biology of individual sarcoma types, we report three overarching findings: (1) unlike most epithelial malignancies, these sarcomas (excepting synovial sarcoma) are characterized predominantly by copy-number changes, with low mutational loads and only a few genes (, , ) highly recurrently mutated across sarcoma types; (2) within sarcoma types, genomic and regulomic diversity of driver pathways defines molecular subtypes associated with patient outcome; and (3) the immune microenvironment, inferred from DNA methylation and mRNA profiles, associates with outcome and may inform clinical trials of immune checkpoint inhibitors. Overall, this large-scale analysis reveals previously unappreciated sarcoma-type-specific changes in copy number, methylation, RNA, and protein, providing insights into refining sarcoma therapy and relationships to other cancer types