The gut bacterium and pathobiont Bacteroides vulgatus activates NF-κB in a human gut epithelial cell line in a strain and growth phase dependent manner

The gut microbiota is increasingly implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) although the identity of the bacteria that underpin these diseases has remained elusive. The pathobiont Bacteroides vulgatus has been associated with both diseases although relatively little is known about how its growth and functional activity might drive the host inflammatory response. We identified an ATP Binding Cassette (ABC) export system and lipoprotein in B. vulgatus ATCC 8482 and B. vulgatus PC510 that displayed significant sequence similarity to an NF-κB immunomodulatory regulon previously identified on a CD-derived metagenomic fosmid clone. Interestingly, the ABC export system was specifically enriched in CD subjects suggesting that it may be important for colonization and persistence in the CD gut environment. Both B. vulgatus ATCC 8482 and PC510 activated NF-κB in a strain and growth phase specific manner in a HT-29/kb-seap-25 enterocyte like cell line. B. vulgatus ATCC 8482 also activated NF-κB in a Caco-2-NF-κBluc enterocyte like and an LS174T-NF-κBluc goblet cell like cell lines, and induced NF-κB-p65 subunit nuclear translocation and IL-6, IL-8, CXCL-10 and MCP-1 gene expression. Despite this, NF-κB activation was not coincident with maximal expression of the ABC exporter or lipoprotein in B. vulgatus PC510 suggesting that the regulon may be necessary but not sufficient for the immunomodulatory effects

The microbiome and rheumatoid arthritis

Rheumatoid Arthritis (RA) is a severe, chronic autoimmune disease that affects 1% of the world's population. Familial risk contributes 50% of the risk of seropositive RA, with strongest risks seen in first-degree relatives. Smoking increases the risk of developing anti-citrullinated peptide antibody (ACPA) RA, particularly in individuals with high-risk RA-susceptibility alleles. Other contributory environmental risks including particulate exposure, periodontal disease, bronchiectasis, diet, obesity and the oral contraceptive impact respiratory, oral, intestinal and genital tract mucosal sites. Furthermore, the first signs of autoimmunity may appear at mucosal sites e.g. sputum ACPA-IgA and IgG. While oral and faecal dysbiosis are well described, there is no consistent single bacterial species that appears to drive RA. Animal and human data suggest a model in which multiple environmental influences impact mucosal immune function through the host genetics through enhanced mucosal permeability and the traffic of pro-inflammatory PAMPs and the amplification of autoimmune responses. In some cases, autoimmunity may be driven by cross-reactivity, or mimicry, to pathogen-specific antigens, particularly where the host immune system fails to support their rapid control and elimination

Enterococcus faecalis AHG0090 is a genetically tractable bacterium and produces a secreted peptidic bioactive that suppresses nuclear factor Kappa B activation in human gut epithelial cells

is an early coloniser of the human infant gut and contributes to the development of intestinal immunity. To better understand the functional capacity of , we constructed a broad host range RP4 mobilizable vector, pEHR513112, that confers chloramphenicol resistance and used a metaparental mating approach to isolate AHG0090 from a fecal sample collected from a healthy human infant. We demonstrated that AHG0090 is genetically tractable and could be manipulated using traditional molecular microbiology approaches. AHG0090 was comparable to the gold-standard anti-inflammatory bacterium A2-165 in its ability to suppress cytokine-mediated nuclear factor kappa B (NF-κB) activation in human gut-derived LS174T goblet cell like and Caco-2 enterocyte-like cell lines. AHG0090 and A2-165 produced secreted low molecular weight NF-κB suppressive peptidic bioactives. Both bioactives were sensitive to heat and proteinase K treatments although the AHG0090 bioactive was more resilient to both forms of treatment. As expected, AHG0090 suppressed IL-1β-induced NF-κB-p65 subunit nuclear translocation and expression of the NF-κB regulated genes IL-6, IL-8 and CXCL-10. Finally, we determined that AHG0090 is distantly related to other commensal strains and likely encodes niche factors that support effective colonization of the infant gut

A nucleotide analog prevents colitis associated cancer via Β-catenin independently of inflammation and autophagy

Chronic bowel inflammation increases the risk of colon cancer: colitis associated cancer (CAC). Thiopurine use is associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/β-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/β-catenin signalling. Autophagy is implicated in colonic tumours, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms.Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harbouring intestinal epithelial cell (IEC)-specific deletion of autophagy related 7 gene (Atg7). TG or vehicle were administered intrarectally, and the effect on tumour burden and β-catenin activity assessed. The mechanisms of action of TG were investigated in vitro and in vivo.TG ameliorated DSS colitis in wild type (WT) but not Atg7 mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both WT and Atg7 mice. This was associated with decreased β-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines and the dependency on Rac1 GTPase demonstrated by siRNA knock-down and overexpression of constitutively active Rac1.Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.Originally included in thesis in manuscript form. </p

MUC13 promotes the development of colitis-associated colorectal tumors via β-catenin activity

Many adenocarcinomas, including colorectal cancer (CRC), overexpress the MUC13 cell surface mucin, but the functional significance and mechanisms are unknown. Here, we report the roles of MUC13 in colonic tumorigenesis and tumor progression. High-MUC13 expression is associated with poor survival in two independent patient cohorts. In a comprehensive series of in vivo experiments, we identified a critical role for MUC13 in the development of this malignancy, by promoting survival and proliferation of tumor-initiating cells and driving an immunosuppressive environment that protects tumors from checkpoint inhibitor immunotherapy. In Muc13-deficient mice, fewer tumors are generated after exposure to carcinogens and inflammation, they have markedly reduced β-catenin signaling, have more tumor-infiltrating CD103 dendritic cells and CD8 T lymphocytes, fewer myeloid-derived suppressor cells, and are rendered sensitive to checkpoint inhibitor immunotherapy (anti-PD-L1). Mechanistically, we show that MUC13 protects β-catenin from degradation, by interacting with GSK-3β, which increases β-catenin nuclear translocation and promotes its signaling, thereby driving cancer initiation, progression, invasion, and immune suppression. Therefore, MUC13 is a potential marker of poor prognosis in colorectal cancer, and inhibiting MUC13 may be useful in the treatment of colitis-associated cancer and sensitizing tumors to immunotherapy