Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis

Abstract

Enterotoxigenic Bacteroides fragilis (ETBF) is associated with noninvasive diarrheal diseases (1, 2), inflammatory bowel diseases (1), and colorectal cancers (3-5). B. fragilis enterotoxin (BFT), a virulence factor of ETBF, is responsible for these diseases (1). BFT interacts with a single layer of intestinal epithelial cells and can provoke signals that induce mucosal inflammation (1, 6-9).In mammalian cells, two genetically distinct isozymes of heme oxygenase (HO) have been clearly identified. HO-1 is inducible, whereas HO-2 is constitutively expressed. HO-1 catalyzes the degradation of free heme into carbon monoxide, biliverdin, and free iron (10, 11). Within mammalian cells, biliverdin reductase converts biliverdin to bilirubin. Pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), lipoteichoic acid, and peptidoglycan, as well as several proinflammatory cytokines, can induce HO-1 expression (12). Upregulated HO-1 expression can lead to adaptive immune responses that protect cells from immunopathogenesis or stress damage (12, 13). In addition, HO-1 expression is involved in clearance of pathogenic bacteria and downregulation of inflammatory responses. For example, HO-1 deficiency not only results in inadequate pathogen clearance (14) but also promotes the development of necrotizing enterocolitis-like intestinal injury in mice (15). HO-1 and HO-1-induced carbon monoxide can ameliorate intestinal inflammation through promotion of bacterial clearance (16). The HO-1/carbon monoxide pathway also suppresses Toll-like receptor 4 (TLR4) signaling, leading to downregulation of proinflammatory signaling induced by stimulation with LPS (17). Based on these findings, we hypothesized that the induction of HO-1 may regulate inflammatory responses induced by BFT. However, there are no reports regarding BFT-induced HO-1 expression.Signals from transcription factors, including nuclear factor-B (NF-B), activator protein-1 (AP-1), and NF-E2-related factor 2 (Nrf2, or nuclear factor [erythroid-derived 2]-like 2 [NFE2L2]), regulate the expression of HO-1 (11). Stimulation of intestinal epithelial cells with BFT can activate NF-B and AP-1 signaling (6)(7)(8)(9)(18)(19)(20). We have previously demonstrated that exposure of intestinal epithelial cells to BFT results in delayed apoptosis, suggesting that protection of cells after BFT stimulation is related to the generation of signals that activate or suppress mucosal inflammation (21). These observations raise the possibility that signaling molecules that regulate HO-1 expression may be activated in BFTexposed cells. However, there is no evidence that BFT-induced signaling results in HO-1 induction in intestinal epithelial cells. We therefore investigated HO-1 induction in response to stimulation of intestinal epithelial cells with BFT. We found that a signaling pathway involving p38 mitogen-activated protein kinases (MAPKs)-IB kinase (IKK)-NF-B in intestinal epithelial cells is required for HO-1 induction following exposure to BFT.

Epigenetic changes induced by Bacteroides fragilis toxin (BFT)

“…Areas of increased chromatin accessibility after BFT2 treatment are enriched in binding motifs for many transcription factors belonging to the AP-1/ATF family of transcription factors, including FOSL1, JUN and JDP. These transcription factors function downstream of the JNK pathway known to be activated by BFT2 (18, 20, 21) . After BFT2 treatment, we expected to see increased expression of genes which are regulated by these particular transcription factors, but chromatin opening alone appeared insufficient to drive changes in gene expression of these JNK pathway genes.…”

Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway

“…Purification of BFT and cell culture conditions. BFT was purified from culture supernatants of a toxigenic ETBF strain (ATCC 43858) as described previously (42)(43)(44). The purity of BFT preparations was confirmed by SDS-PAGE.…”

“…Cells were harvested and nuclear extracts were prepared as described previously (42,44). Concentrations of protein in the extracts were determined using the Bradford assay (Bio-Rad, Hercules, CA, USA).…”

“…Lentiviral systems containing mammalian expression vectors were used to block CHOP, AP-1, and MAPK activation, as described previously (44,45). Specifically, lentiviral systems containing mammalian expression vectors carrying a hemagglutinin epitope-tagged mutant c-jun gene (TAM67) with deletions of amino acids at positions 3 to 122 were used to block AP-1.…”

“…In brief, 5 g of nuclear extract was incubated for 30 min at room temperature with a ␥-32 P-labeled oligonucleotide probe (5=-CGC TTG ATG ACT CAG CCG GAA-3= for the AP-1 binding site). After incubation, both bound DNA and free DNA were resolved on 5% polyacrylamide gels, as described previously (42,44).…”