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

Abstract

Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogenactivated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy.KEYWORDS autophagy, Bacteroides fragilis enterotoxin, endothelial cells E nterotoxigenic Bacteroides fragilis (ETBF) is known to be associated with diarrheal diseases, inflammatory bowel diseases, and colorectal cancers. B. fragilis enterotoxin (BFT), a virulence factor of ETBF, is responsible for these diseases (1). Exposure to BFT results in the infiltration of inflammatory cells through endothelial cells (ECs) (2-4). Although aspects of pathogenesis regarding ETBF infection have been investigated, a more detailed understanding of the interaction between BFT and the host, especially BFT-induced changes in host cells, could reveal indispensable characteristics of ETBF infection.

Intestinal Epithelial Cells Exposed to Bacteroides fragilis Enterotoxin Regulate NF-κB Activation and Inflammatory Responses through β-Catenin Expression

“…N-(6-Methyl-2-benzothiazolyl)-2-[ (3,4,6,7- Purification of BFT and cell culture conditions. BFT was purified from culture supernatants of a toxigenic strain of ETBF (ATCC 43858) as described previously (18,26,34). The purity of the BFT preparations was verified, as they appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels.…”

Bacteroides fragilis Enterotoxin Induces Sulfiredoxin-1 Expression in Intestinal Epithelial Cell Lines Through a Mitogen-Activated Protein Kinases- and Nrf2-Dependent Pathway, Leading to the Suppression of Apoptosis

“…Delayed apoptosis has been observed in BFT-exposed IECs [ 27 , 37 ]. These findings raise the possibility that the generation of signals for preventing apoptosis is evoked in BFT-treated IECs.…”

“…In a series of experiments to demonstrate this hypothesis, we already reported that the enhanced expression of cellular inhibitor of apoptosis protein-2 attenuates the apoptosis in IECs [ 27 ]. In addition, BFT-induced heme oxygenase-1 (HO-1) upregulation is also closely connected with the suppression of apoptosis [ 37 ]. However, overexpression of HO-1 did not completely prevent apoptosis.…”

Molecular interplay of autophagy and endocytosis in human health and diseases

“…The final steps of autophagy are the fusion of an autophagosome and lysosome to form the autolysosome, and then the degradation of its inner membrane and internal cargos by lysosomal acid hydrolases. The core mechanisms of autophagosome trafficking and lysosomal fusion, and their regulation, are still unclear (Ko et al , ). The movement of lysosomes and endosomes has a decisive role in the fusion process to form autolysosomes, and is supported by microtubular motor proteins like myosin, kinesin, and dynein.…”

“…In addition, levels of p62, LC3‐II, ATG5 and ATG12, proteins which are essential for autophagosome formation, increased when cells were treated with the Bacteroides fragilis enterotoxin (BFT). Like rapamycin, BFT promotes autophagosome formation via the fusion process, although rapamycin shows a greater influence on the fusion process (Ko et al , ). Heavy metals like cadmium are known to induce oxidative stress and autophagy, and accumulation of both cadmium and calcium ions block the autophagosome–lysosome fusion process (Liu et al , ).…”