The intestinal flora may promote colon tumor formation. Here we explore immunologic mechanisms of colonic carcinogenesis by a human colonic bacterium, enterotoxigenic Bacteroides fragilis (ETBF). ETBF that secretes B. fragilis toxin (BFT) causes human inflammatory diarrhea but also asymptomatically colonizes a proportion of the human population. Our results indicate that whereas both ETBF and nontoxigenic B. fragilis (NTBF) chronically colonize mice, only ETBF triggers colitis and strongly induces colonic tumors in multiple intestinal neoplasia (Min) mice. ETBF induces robust, selective colonic signal transducer and activator of transcription-3 (Stat3) activation with colitis characterized by a selective T helper type 17 (TH17) response distributed between CD4+ T cell receptor-αβ (TCRαβ)+ and CD4−8−TCRγδ+ T cells. Antibody-mediated blockade of interleukin-17 (IL-17) as well as the receptor for IL-23, a key cytokine amplifying TH17 responses, inhibits ETBF-induced colitis, colonic hyperplasia and tumor formation. These results show a Stat3- and TH17-dependent pathway for inflammation-induced cancer by a common human commensal bacterium, providing new mechanistic insight into human colon carcinogenesis.
Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of Escherichia coli and Bacteroides fragilis. Genes for colibactin (clbB) and Bacteroides fragilis toxin (bft), encoding secreted oncotoxins, were highly enriched in FAP patients’ colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with E. coli (expressing colibactin), and enterotoxigenic B. fragilis showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria.
Significance We demonstrate, to our knowledge for the first time, that bacterial biofilms are associated with colorectal cancers, one of the leading malignancies in the United States and abroad. Colon biofilms, dense communities of bacteria encased in a likely complex matrix that contact the colon epithelial cells, are nearly universal on right colon tumors. Most remarkably, biofilm presence correlates with bacterial tissue invasion and changes in tissue biology with enhanced cellular proliferation, a basic feature of oncogenic transformation occurring even in colons without evidence of cancer. Microbiome profiling revealed that biofilm communities on paired normal mucosa cluster with tumor microbiomes but lack distinct taxa differences. This work introduces a previously unidentified concept whereby microbial community structural organization exhibits the potential to contribute to disease progression.
It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, although the direct molecular events linking inflammation and carcinogenesis are poorly defined. In the context of gastrointestinal disease, the bacterium enterotoxigenic Bacteroides fragilis (ETBF) is a significant source of chronic inflammation and has been implicated as a risk factor for colorectal cancer. Spermine oxidase (SMO) is a polyamine catabolic enzyme that is highly inducible by inflammatory stimuli resulting in increased reactive oxygen species (ROS) and DNA damage. We now demonstrate that purified B. fragilis toxin (BFT) upregulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent generation of ROS and induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in C57BL/6 mice is associated with increased SMO expression and treatment of mice with an inhibitor of polyamine catabolism, N 1 ,N 4 -bis(2,3-butandienyl)-1,4-butanediamine (MDL 72527), significantly reduces ETBF-induced chronic inflammation and proliferation. Most importantly, in the multiple intestinal neoplasia (Min) mouse model, treatment with MDL 72527 reduces ETBF-induced colon tumorigenesis by 69% (P < 0.001). The results of these studies indicate that SMO is a source of bacteria-induced ROS directly associated with tumorigenesis and could serve as a unique target for chemoprevention.inflammatory bowel diseases | adenomatous polyposis coli I t is estimated that chronic inflammation associated with microbial infection directly contributes to the etiology of about 20% of epithelial cancers. The chronic inflammatory microenvironment is characterized by immune dysregulation and elevated levels of reactive oxygen species [ROS; including superoxide, hydrogen peroxide (H 2 O 2 ), and singlet oxygen]. These features result in activation of stress response pathways and oncogenes, down-regulation of tumor suppressor genes, cell and tissue damage, and contribute to tumor initiation, promotion, and progression. However, the precise molecular links between inflammation and carcinogenesis remain to be clarified (1, 2).In the colon, alterations in the physiological balance between the diverse and abundant microbiota (w10 12 organisms per gram feces) and the host are a common source of inflammation. Bacteroides spp comprise a significant proportion of colonic commensal bacteria and members of this group include symbionts and the leading human anaerobic pathogen, Bacteroides fragilis. Enterotoxigenic B. fragilis (ETBF) represent a molecular subtype characterized by a single unique virulence determinant, the production and secretion of a 20-kDa metalloprotease enterotoxin (B. fragilis toxin; BFT). ETBF have been epidemiologically associated with acute diarrheal diseases in humans and livestock, inflammatory bowel diseases (IBD), and colorectal cancer (reviewed in ref.3). Exposure of intestinal epithelial cell lines to BFT results in cleavage of the cell adhesion and tumor suppressor protein E-cadherin, ...
Strains of Bacteroides fragilis associated with diarrheal disease (enterotoxigenic B. fragilis) produce a 20-kDa zinc-dependent metalloprotease toxin (B. fragilis enterotoxin; BFT) that reversibly stimulates chloride secretion and alters tight junctional function in polarized intestinal epithelial cells. BFT alters cellular morphology and physiology most potently and rapidly when placed on the basolateral membrane of epithelial cells, suggesting that the cellular substrate for BFT may be present on this membrane. Herein, we demonstrate that BFT specifically cleaves within 1 min the extracellular domain of the zonula adherens protein, Ecadherin. Cleavage of E-cadherin by BFT is ATP-independent and essential to the morphologic and physiologic activity of BFT. However, the morphologic changes occurring in response to BFT are dependent on target-cell ATP. E-cadherin is shown here to be a cellular substrate for a bacterial toxin and represents the identification of a mechanism of action, cell-surface proteolytic activity, for a bacterial toxin.
Enterotoxigenic Bacteroides fragilis (ETBF) causes diarrhea and is implicated in inflammatory bowel diseases and colorectal cancer. The only known ETBF virulence factor is the Bacteroides fragilis toxin (BFT), which induces E-cadherin cleavage, interleukin-8 secretion, and epithelial cell proliferation. A murine model for ETBF has not been characterized. Specific pathogen-free (SPF) C57BL/6J or germfree 129S6/SvEv mice were orally inoculated with wild-type ETBF (WT-ETBF) strains, a nontoxigenic WT strain of B. fragilis (WT-NTBF), WT-NTBF overexpressing bft (rETBF), or WT-NTBF overexpressing a biologically inactive mutated bft (rNTBF). In SPF and germfree mice, ETBF caused colitis but was lethal only in germfree mice. Colonic histopathology demonstrated mucosal thickening with inflammatory cell infiltration, crypt abscesses, and epithelial cell exfoliation, erosion, and ulceration. SPF mice colonized with rETBF mimicked WT-ETBF, whereas rNTBF caused no histopathology. Intestinal epithelial E-cadherin was rapidly cleaved in vivo in WT-ETBF-colonized mice and in vitro in intestinal tissues cultured with purified BFT. ETBF mice colonized for 16 months exhibited persistent colitis. BFT did not directly induce lymphocyte proliferation, dendritic cell stimulation, or Toll-like receptor activation. In conclusion, WT-ETBF induced acute then persistent colitis in SPF mice and rapidly lethal colitis in WT germfree mice. Our data support the hypothesis that chronic colonization with the human commensal ETBF can induce persistent, subclinical colitis in humans.
Summary Pro-carcinogenic bacteria have the potential to initiate and/or promote colon cancer, in part via immune mechanisms that are incompletely understood. Using ApcMin mice colonized with the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) as a model of microbial-induced colon tumorigenesis, we show that the Bacteroides fragilis toxin (BFT) triggers a pro-carcinogenic multi-step inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CECs). Although necessary, Stat3 activation in CECs is not sufficient to trigger ETBF colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1 that mediates the recruitment of CXCR2-expressing polymorphonuclear immature myeloid cells with parallel onset of ETBF-mediated distal colon tumorigenesis. Thus, BFT induces a procarcinogenic signaling relay from the CEC to a mucosal Th17 response that results in NFκB activation selectively in distal colon CECs, that collectively triggers myeloid cell-dependent distal colon tumorigenesis.
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