Project description:Defense against attaching and effacing (A/E) bacteria requires the sequential generation of IL-23 and IL-22 to induce protective mucosal responses. While the critical source of IL-22 has been identified as CD4+ and Nkp46+ innate lymphoid cells (ILCs), the precise source of IL-23 is unclear. Here, we use genetic techniques to deplete specific classical dendritic cell (cDC) subsets and analyze immunity to the A/E pathogen Citrobacter rodentium. We find that Zbtb46+ cDCs, and specifically Notch2-dependent intestinal CD11b+ cDCs, but not Batf3-dependent CD103+ cDCs, are required for IL-23 production and immunity against C. rodentium. Notch2 controls cDC differentiation at a terminal step mediated by lymphotoxin signaling. Importantly, these results provide the first demonstration of a non-redundant function of CD11b+ cDCs in vivo. Analysis of genes differentially expressed between WT, Batf3 KO and Notch2 KO colons following C. rodentium infection. Mice were infected with 2 x 10^9 C. rodentium and colons harvested at either Day 4 or Day 9.
Project description:Profiling of a total of 34,790 genes revealed a wide range of expression changes during the course of C. rodentium infection in murine colon. The majority of changes were observed during weeks 1 and 2, while relatively fewer changes were seen at week 3. Interestingly, chemokines made up 20% of the top twenty upregulated genes. Overall design: Mouse colon from pooled control or C. rodentium-infected mice.
Project description:Whole genome trancription study of Citrobacter rodentium grown in rich media. Publication Title: Citrobacter rodentium is an Unstable Pathogen Showing Evidence of Significant Genomic Flux Publication Author List: Nicola K. Petty, Theresa Feltwell, Derek Pickard, Simon Clare, Ana L. Toribio, Maria Fookes, Kevin Roberts, Rita Monson, Satheesh Nair, Robert A. Kingsley, Richard Bulgin, Siouxsie Wiles, David Goulding, Craig Corton, Nicola Lennard, David Harris, David Willey, Richard Rance, Lu Yu, Jyoti S. Choudhary, Carol Churcher, Michael A. Quail, Julian Parkhill, Gad Frankel, Gordon Dougan, George P.C. Salmond, Nicholas R. Thomson ArrayExpress Release Date: 2011-02-12 Person Roles: investigator Person Last Name: Thomson Person First Name: Nicholas Person Mid Initials: Person Email: firstname.lastname@example.org Person Phone: Person Address: Wellcome Trust Genome Campus, Hinxton, Cambridge, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: submitter Person Last Name: Service Person First Name: Submission Person Mid Initials: Person Email: email@example.com Person Phone: Person Address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom Person Affiliation: Wellcome Trust Sanger Institute Overall design: Experimental Design: individual_genetic_characteristics_design Experimental Design: in_vitro_design Experimental Design: co-expression_design Experimental Factor Name: GENOTYPE Experimental Factor Type: genotype
Project description:It is crucial to decipher the host-microbiota interactions as they are involved in intestinal homeostasis and diseases. Caspase Recruitment Domain 9 (Card9) is an inflammatory bowel disease (IBD) susceptibility gene coding for an adapter protein for innate immunity toward many microorganisms. Card9-/- mice are more susceptible to colitis induced by Citrobacter rodentium as a result of impaired of the IL-22 pathway. C. rodentium is a natural mouse pathogen widely used to model human infections with Enteropathogenic Escherichia coli and Enterohaemorrhagic E. coli. To explore the role of the gut microbiota in the susceptibility of Card9-/- mice to C. rodentium infection, we colonized WT germ-free (GF) mice with the microbiota of WT (WT-->GF) or Card9-/- (Card9-/- -->GF) mice and challenged them with C. rodentium. Card9-/- -->GF mice were more susceptible than WT-->GF mice to C. rodentium. To examine the mechanisms responsible for this defect, we compared the cecum transcriptomes of WT -->GF and Card9-/- -->GF mice before and during C. rodentium-induced colitis. The number of down-regulated and up-regulated genes on day 12 after C. rodentium infection was lower in Card9-/- -->GF mice than WT-->GF mice. Card9-/- -->GF mice showed a significant down-regulation of gut morphogenesis and wound healing pathways suggesting that recovery is impaired in Card9-/- -->GF mice after C. rodentium infection. Immune response and cell division pathways were up-regulated in WT-->GF mice but not in Card9-/- -->GF confirming the defect of global response to infection when only the Card9-/- microbiota was transferred. The most induced and differentially expressed genes between Card9-/- -->GF and WT-->GF mice on day 4 after C. rodentium infection were Reg3g (encoding REGIIIγ) and Reg3b (encoding REGIIIβ). Overall design: Germ-free (GF) C57BL/6 wild-type (WT) mice were inoculated by oral gavage with fresh stools from conventional WT (WT-->GF) or Card9-/- (Card9-/- -->GF) mice. Three weeks after the colonization, WT-->GF and Card9-/- -->GF mice were infected by oral gavage with 1x10^9 CFU of C. rodentium strain DBS100. 5 mice of each groups (WT-->GF and Card9-/- -->GF) were sacrified before infection. 6 mice of each group were sacrified 4 days after infection, 5 mice of each group were sacrified at day 12 and 3 WT-->GF mice and 5 Card9-/- -->GF mice were sacrified at day 22.
Project description:The mechanisms by which the mouse pathogen Citrobacter rodentium triggers effacement of the brush border microvilli, colitis, hyperplasia and dysbiosis remain poorly understood. We investigated the impact of C. rodentium infection on the proteomic and metabolic landscapes of intestinal epithelial cells (IECs) in vivo using isobaric labeling proteomics and targeted metabolomics. We found that infection depletes proteins involved in butyrate uptake, glycolysis, TCA cycle, lipid metabolism and oxidative phosphorylation with aparallel, increased production of creatine/phosphocreatine and cholesterol. The evolving ecological niche within the infected gut was concomitant with a reduction in butyrate-producing commensal bacteria and expansion of Proteobacteria that can metabolize cholesterol. These changes coincide with the modulation of IEC transcription factors by C. rodentium, specifically phosphorylation of Kdm5a, demethylation of histone H3 (Lys-4) and cleavage of Srebp2. Taken together our results show that whilst engaging with the host in a race to control innate immune responses, C. rodentium and the changing microbiota shape the metabolism flow in IECs.
Project description:Identification of the targets of RegA with and without bicarbonate stimulation by comparing RegA knockout to multicopy RegA transgenics. RegA is an AraC like transcription factor identified in a mutational screen for virulence genes in Citrobacter rodentium, an attaching and effacing pathogen that causes transmissible colonic hyperplasia in mice. This experiment compares the RegA null strain with a multicopy plasmid rescue of this null strain in the presence and absence of bicarbonate with the aim of identifying pathogenesis related genes related to the early and late stages of attachment and effacement. Keywords: genetic modification, transcription factor, induction Overall design: A strain of Citrobacter rodentium with a knockout of RegA was compared to the same strain rescued with a multicopy plasmid containing the wildtype RegA gene. These strains were analyzed with and without bicarbonate in an unconnected two factor design with dye balanced biological replicates.