Shaoguang Wu

A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses.

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.

Microbiota organization is a distinct feature of proximal colorectal cancers

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. Environmental factors clearly affect colorectal cancer (CRC) incidence, but the mechanisms through which these factors function are unknown. One prime candidate is an altered colonic microbiota. Here we show that the mucosal microbiota organization is a critical factor associated with a subset of CRC. We identified invasive polymicrobial bacterial biofilms (bacterial aggregates), structures previously associated with nonmalignant intestinal pathology, nearly universally (89%) on right-sided tumors (13 of 15 CRCs, 4 of 4 adenomas) but on only 12% of left-sided tumors (2 of 15 CRCs, 0 of 2 adenomas). Surprisingly, patients with biofilm-positive tumors, whether cancers or adenomas, all had biofilms on their tumor-free mucosa far distant from their tumors. Bacterial biofilms were associated with diminished colonic epithelial cell E-cadherin and enhanced epithelial cell IL-6 and Stat3 activation, as well as increased crypt epithelial cell proliferation in normal colon mucosa. High-throughput sequencing revealed no consistent bacterial genus associated with tumors, regardless of biofilm status. However, principal coordinates analysis revealed that biofilm communities on paired normal mucosa, distant from the tumor itself, cluster with tumor microbiomes as opposed to biofilm-negative normal mucosa bacterial communities also from the tumor host. Colon mucosal biofilm detection may predict increased risk for development of sporadic CRC.

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis.

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) up-regulates 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, N1,N4-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.

Induction of Persistent Colitis by a Human Commensal, Enterotoxigenic Bacteroides fragilis, in Wild-Type C57BL/6 Mice

ABSTRACT 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.

Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and γ-secretase-dependent E-cadherin cleavage

Enterotoxigenic Bacteroides fragilis – organisms that live in the colon – secrete a metalloprotease toxin, B. fragilis toxin. This toxin binds to a specific intestinal epithelial cell receptor and stimulates cell proliferation, which is dependent, in part, on E-cadherin degradation and β-catenin–T-cell-factor nuclear signaling. γ-Secretase (or presenilin-1) is an intramembrane cleaving protease and is a positive regulator of E-cadherin cleavage and a negative regulator of β-catenin signaling. Here we examine the mechanistic details of toxin-initiated E-cadherin cleavage. B. fragilis toxin stimulated shedding of cell membrane proteins, including the 80 kDa E-cadherin ectodomain. Shedding of this domain required biologically active toxin and was not mediated by MMP-7, ADAM10 or ADAM17. Inhibition of γ-secretase blocked toxin-induced proteolysis of the 33 kDa intracellular E-cadherin domain causing cell membrane retention of a distinct β-catenin pool without diminishing toxin-induced cell proliferation. Unexpectedly, γ-secretase positively regulated basal cell proliferation dependent on the β-catenin–T-cell-factor complex. We conclude that toxin induces step-wise cleavage of E-cadherin, which is dependent on toxin metalloprotease and γ-secretase. Our results suggest that differentially regulated β-catenin pools associate with the E-cadherin–γ-secretase adherens junction complex; one pool regulated by γ-secretase is important to intestinal epithelial cell homeostasis.

Enterotoxigenic bacteroides fragilis: a potential instigator of colitis.

Background: Inflammatory bowel disease (IBD) is proposed to result from a dysregulated mucosal immune response to the colonic flora in genetically susceptible individuals. Enterotoxigenic Bacteroides fragilis (ETBF), a molecular subclass of the common human commensal, B. fragilis, has been associated with IBD. This study investigated whether ETBF colonization of mice initiated colitis or modified the clinical course of a colitis agonist, dextran sodium sulfate (DSS). Methods: Four‐ and 6‐week‐old C57BL/6 mice were inoculated with buffer, nontoxigenic B. fragilis (NTBF) strain 9343(pFD340), or ETBF strain 86‐5443‐2‐2 via orogastric tube. A subset of mice received 2% DSS several days pre‐ or post‐inoculation of bacteria. Clinical status was assessed throughout the experiment and severity of colonic inflammation was scored after sacrifice. Results: All mice, including those receiving DSS, were clinically well prior to bacterial inoculation. NTBF and ETBF colonization was similar. Regardless of mouse age or timing of DSS administration, mice who received ETBF+DSS experienced worse colitis reflected by less weight gain, enhanced gross disease, and greater inflammation in their colons (P < 0.05), especially in the cecum. In particular, younger mice had more extensive disease. Mice inoculated only with ETBF also exhibited colitis with more severe inflammation when compared to all other groups (P < 0.05) except the ETBF+DSS group. Conclusions: ETBF, a colonic commensal, alone stimulates colitis and significantly enhances colonic inflammation in DSS‐treated mice. This study suggests that acquisition of ETBF colonization may be a potential factor in initiation and/or exacerbation of colitis. (Inflamm Bowel Dis 2007)

Bacteroides fragilis enterotoxin induces intestinal epithelial cell secretion of interleukin-8 through mitogen-activated protein kinases and a tyrosine kinase-regulated nuclear factor-κB pathway

ABSTRACT Enterotoxigenic Bacteroides fragilis (ETBF) secretes a 20-kDa metalloprotease toxin termed B. fragilis toxin (BFT). ETBF disease in animals is associated with an acute inflammatory response in the intestinal mucosa, and lethal hemorrhagic colitis may occur in rabbits. In this study, we confirmed recent reports (J. M. Kim, Y. K. Oh, Y. J. Kim, H. B. Oh, and Y. J. Cho, Clin. Exp. Immunol. 123:421-427, 2001; L. Sanfilippo, C. K. Li, R. Seth, T. J. Balwin, M. J. Menozzi, and Y. R. Mahida, Clin. Exp. Immunol. 119:456-463, 2000) that purified BFT stimulates interleukin-8 (IL-8) secretion by human intestinal epithelial cells (HT29/C1 cells) and demonstrate that stimulation of IL-8 production is dependent on biologically active BFT and independent of serum. Induction of IL-8 mRNA expression occurs rapidly and ceases by 6 h after BFT treatment, whereas IL-8 secretion continues to increase for at least 18 h. Our data suggest that BFT-stimulated IL-8 secretion involves tyrosine kinase-dependent activation of nuclear factor-κB (NF-κB) as well as activation of the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-related kinase. Simultaneous activation of NF-κB and MAPKs appears necessary for secretion of IL-8 by HT29/C1 cells treated with BFT.

The Bacteroides fragilis Toxin Binds to a Specific Intestinal Epithelial Cell Receptor

ABSTRACT The Bacteroides fragilis toxin (BFT) is the only known virulence factor of enterotoxigenic B. fragilis. BFT has previously been shown to act, at least in part, through cleavage of the intercellular adhesion protein E-cadherin. A specific cellular receptor for BFT has not been identified. The goal of this study was to determine if the initial interaction of BFT with intestinal epithelial cells was consistent with binding to a specific cellular receptor. Purified BFT was labeled with a fluorophore or iodide to assess specific cellular binding and the properties of BFT cellular binding. BFT binds specifically to intestinal epithelial cell lines in vitro in a polarized manner. However, specific binding occurs only at 37°C and requires BFT metalloprotease activity. The BFT receptor is predicted to be a membrane protein other than E-cadherin or a known protease-activated receptor (PAR1 to PAR4). BFT binding is resistant to acid washing, suggesting an irreversible interaction. Sugar or lipid residues do not appear to be involved in the mechanism of BFT cellular binding, but binding is sensitive to membrane cholesterol depletion. We conclude that intestinal epithelial cells in vitro possess a specific membrane BFT receptor that is distinct from E-cadherin. The data favor a model in which the metalloprotease domain of BFT processes its receptor protein, initiating cellular signal transduction that mediates the biological activity of BFT. However, activation of recognized protease-activated receptors does not mimic or block BFT biological activity or binding, suggesting that additional protease-activated receptors on intestinal epithelial cells remain to be identified.

Regulatory T-cell Response to Enterotoxigenic Bacteroides fragilis Colonization Triggers IL17-Dependent Colon Carcinogenesis

UNLABELLED Many epithelial cancers are associated with chronic inflammation. However, the features of inflammation that are procarcinogenic are not fully understood. Regulatory T cells (Treg) typically restrain overt inflammatory responses and maintain intestinal immune homeostasis. Their immune-suppressive activity can inhibit inflammation-associated cancers. Paradoxically, we show that colonic Tregs initiate IL17-mediated carcinogenesis in multiple intestinal neoplasia mice colonized with the human symbiote enterotoxigenic Bacteroides fragilis (ETBF). Depletion of Tregs in ETBF-colonized C57BL/6 FOXP3(DTR) mice enhanced colitis but diminished tumorigenesis associated with shifting of mucosal cytokine profile from IL17 to IFNγ; inhibition of ETBF-induced colon tumorigenesis was dependent on reduced IL17 inflammation and was independent of IFNγ. Treg enhancement of IL17 production is cell-extrinsic. IL2 blockade restored Th17 responses and tumor formation in Treg-depleted animals. Our findings demonstrate that Tregs limit the availability of IL2 in the local microenvironment, allowing the Th17 development necessary to promote ETBF-triggered neoplasia, and thus unveil a new mechanism whereby Treg responses to intestinal bacterial infection can promote tumorigenesis. SIGNIFICANCE Tregs promote an oncogenic immune response to a common human symbiote associated with inflammatory bowel disease and colorectal cancer. Our data define mechanisms by which mucosal Tregs, despite suppressing excessive inflammation, promote the earliest stages of immune procarcinogenesis via enhancement of IL17 production at the expense of IFNγ production.

Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria

Biofilms provide refuge for cancerous bacteria Familial adenomatous polyposis (FAP) causes benign polyps along the colon. If left untreated, FAP leads to a high incidence of colon cancer. To understand how polyps influence tumor formation, Dejea et al. examined the colonic mucosa of FAP patients. They discovered biofilms containing the carcinogenic versions of the bacterial species Escherichia coli and Bacteroides fragilis. Colon tissue from FAP patients exhibited greater expression of two bacterial genes that produce secreted oncotoxins. Studies in mice showed that specific bacteria could work together to induce colon inflammation and tumor formation. Science, this issue p. 592 Bacterial biofilms are linked to colon cancer. 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.