Table 3.
In vitro studies (n = 49) included in review.
| Reference | Study design | Method of analysis | Major findings |
|---|---|---|---|
| Van Tassell et al. 1992 [10] | Colon carcinoma HT-29 cells were treated with BFT | Cytotoxicity assay | BFT induced cytotoxic response (cell rounding) |
| Weikel et al. 1992 [117] | Human intestinal epithelial cells T84, Caco-2, HT-29 cell lines were co-cultured with ETBF and NTBF cultures | Cell morphology (bright-field light microscopy) | Exposure to ETBF induced morphological changes |
| Pantosti et al. 1994 [120] | 146 B fragilis strains and 64 Bacteroides isolates were tested for ability to produce BFT | Anti-serum testing, Cytotoxicity assay | 16 strains of ETBF were identified (11% of B. fragilis strains examined)Clinical isolates were associated with tissue destruction |
| Moncrief et al. 1995 [115] | HT29 cells were treated with BFT | Cytotoxicity, SSP-PCT, protein assays, ELISA, PAGE, Western blot | BFT exhibited cytotoxic activity that was inhibited by pretreatment with a metal chelator |
| Donelli et al. 1996 [79] | HT-29 cells were treated with BFT | Fluorescence and electron microscopy | BFT induces morphological cell changes by reversibly modifying the actin cytoskeleton |
| Koshy et al. 1996 [80] | Cloned human colonic epithelial cells (HT29/C1) were treated with BFT | Fluorescent phallicidin staining. Cell volume | BFT exposure resulted in distribution of F-actin with loss of stress fibers and cellular membrane blebbing |
| Saidi and Sears 1996 [81] | HT29/C1 cells were treated with BFT | Cytotoxicity assay | BFT rapidly and irreversibly intoxicates HT29/C1 cells in a concentration- and temperature-dependent manner |
| Wells et al. 1996 [82] | HT-29 enterocytes were treated with BFT and then co-cultured with enteric bacteria | Viability, transepithelial electrical resistance (TEER), Light and electron microscopy, bacterial internalization | BFT treatment decreased transepithelial electrical resistance, decreased Listeria monocytogenes internalization, increased internalization of other enteric species |
| Obiso Jr. et al. 1997 [83] | HT-29, rat lung type II, and canine kidney epithelium cells were treated with BFT | Mannitol flux assay, Tight junction resistance recovery assay, epifluorescence microscopy | BFT increased permeability of the paracellular barrier of epithelial cells |
| Chambers et al. 1997 [84] | T84 cells were treated with BFT | Light and electron microscopy, Cell viability, F-actin staining, Ussing chambers | BFT treated induced morphological changes, loss of cellular microvilli, and complete dissolution of some tight junctions |
| Saidi et al. 1997 [85] | HT29/C1 cells were treated with BFT | Spectrofluorimetry, Confocal microscopy, Western blot | BFT alters the F and G-actin cytoskeletal architecture of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content |
| Sanfilippo et al. 1998 [86] | Human primary colon cells were treated with BFT | Cytotoxicity, Electron microscopy | BFT treatment induced morphological changes (cell rounding, separation from adjacent cells, detachment from basement membrane) and cell cytotoxicity |
| Wu et al. 1998 [87] | HT29/C1 cells were treated with BFT | Western blot, Immunofluorescent, confocal microscopy, Northern blot, Reverse transcription PCR | BFT cleaves the extracellular domain of E-cadherin |
| Chung et al. 1999 [116] | 89 B. fragilis strains were tested for BFT productionHT29/C1 cells were co-cultured with NTBF and ETBF | Colony blot hybridization, PCR, Western blot | 38% of B. fragilis strains examined were ETBF, BFT cleaved E-cadherin |
| Riegler et al. 1999 [88] | Treated colonic mucosa with BFT | Ussing chambers, confocal microscopy | BFT treatment increased cell permeability and damaged crypt and surface colonocytes |
| Sanfilippo et al. 2000 [89] | Intestinal epithelial cell lines HT29, T84, Caco-2, and IEC-6 were treated with BFT | Transmission electron microscopy, reverse transcription PCR, sandwich ELISA | BFT exposure increased expression of IL-8 and secretion of TGF-β (T84), induced morphology changes (HT29), loss of tight junctions (T84), and detachment (T84) |
| Kim et al. 2001 [90] | HT29 and Caco-2 cells were treated with BFT | Quantitative real-time (qRT)-PCR, ELISA | BFT exposure increased expression of neutrophil chemoattractant and activators (ENA-78, GRO-α, IL-8) |
| Kim et al. 2002 [91] | HT29, T84, and primary human colon epithelial cells were treated with BFT | Supershift EMSA, Western blot, qRT-PCR, ELISA | BFT induced NF-κB activation and IκB degradation |
| Franco et al. 2002 [6] | HT29/C1 cells were treated with BFT | Reverse transcription PCR | The B. fragilis pathogenicity island and its flanking regions modulate bft expression |
| Wu et al. 2003 [92] | HT29/C1 cells were treated with BFT | Western blot, Immunofluorescent confocal microscopy, Reverse transcription PCR | BFT activates T-cell factor-dependent transcriptional activation and promotes cell proliferation |
| Wu et al. 2004 [93] | HT29/C1 cell were treated with BFT | Western blot, ELISA, reverse transcription PCR | BFT stimulates IL-8 secretion |
| Kim et al. 2005 [38] | HT29 cells were treated with BFT | qRT-PCR, ELISA, EMSA, Western blot | BFT activated three major MAPK cascades (p38, JNK, ERK1/2) and AP-1 signals composed of c-Jun/c-Fos heterodimers |
| Kim et al. 2006 [50] | HT29 cells were treated with BFT | qRT-PCR, Western blot, Luciferase assay | BFT exposure increased expression of COX-2 and prostaglandin E2 |
| Sears et al. 2006 [94] | HT29/C1 cells were treated with BFT | Western blot, reverse transcription PCR | The deletion of 2 amino acids in the C terminus of BFT reduced biological activity |
| Wu et al. 2006 [95] | HT29/C1 cells were treated with BFT | Confocal microscopy, flow cytometry, acid wash | BFT binds irreversibly to intestinal epithelial cells in a polarized, metalloprotease-dependent manner |
| Wu et al. 2007 [96] | HT29/C1 cells were treated with BFT | Western blot, RNA interference, immunostaining | BFT mediated shedding of cell membrane proteins. Cleavage of E-cadherin was dependent on toxin metalloprotease and γ-secretase. |
| Kim et al. 2008 [97] | HT29 cells were treated with BFT | Cell Death detection ELISA, flow cytometry, qRT-PCR, Western blot, luciferase assay | BFT induced apoptosis and activated the phosphorylation of ERK1/2, p38, and JNK |
| Kim et al. 2009 [98] | HT29 cells were treated with BFT | Quantitative reverse transcription PCR, RT-PCR, ELISA, Western blot | BFT-induced phosphorylation of both IκBα and IκB kinase (IKK) signals was prevented in eupatilin-pretreated HT29 cells |
| Yoon et al. 2010 [99] | HT-29 and Caco-2 cells were treated with BFT | qRT-PCR, ELISA, EMSA, Western blot | BFT induced human ß-defensin 2 in a dose- and time-dependent manner that could be regulated by a MAPK, IKK-, and NF-kB-dependent signaling pathway. BFT also activated ERK1/2, p38, and JNK |
| Goodwin et al. 2011 [65] | HT29/C1 and T84 cells were treated with BFT | qRT-PCR, Western blot, enzyme activity assays, | BFT upregulates spermine oxidase (SMO), resulting in SMO-dependent generation of ROS and induction of a DNA damage marker (γ-H2A.x) |
| Roh et al. 2011 [100] | HUVECs and rat aortic endothelial cells were treated with BFT | qRT-PCR, flow cytometry, immunofluorescence assay, EMSA, ELISA | BFT induced ICAM-1 expression. Upregulation of ICAM-1 was dependent on the activation of IkB and NF-kB signaling pathways. |
| Hwang et al.2013 [101] | HT29/C1 wells were treated with BFT | ELSA and Western blot | BFT induced E-cadherin degradation and IL-8 secretion |
| Yoo et al. 2013 [102] | HT29 cells were treated with BFT | Quantitative reverse transcriptase PCR, ELISA, EMSA, luciferase assay, Western blot | BFT induced upregulation of lipocalin 2 in an AP-1 signaling dependent manner that was regulated by MAPKs (ERK, p38) |
| Remacle et al. 2014 [103] | Human colorectal carcinoma cell lines (HTC116, HT29, HT29/C1) were treated with BFT | Immunofluorescence microscopy, immunoprecipitation of E-cadherin | BFT cleaved E-cadherin, |
| Shiryaev et al. 2014 [7] | HT29 cells were treated with BFT | Immunoprecipitation of E-cadherin, cell aggregation assay | BFT repressed cell aggregation |
| Kharlampieva et al. 2015 [104] | HT29 cells were treated with BFT | Site-directed mutagenesis, recombination, Western blot | BFT induced endogenous E-cadherin cleavage. Cleavage activity required the native structure of zinc-binding motif |
| Ko et al. 2016 [105] | Murine intestinal epithelial cells were treated with BFT | Quantitative reverse transcriptase PCR, EMSA, transfection assay, Western blot, ELISA immunofluorescence, apoptosis assay | BFT upregulated expression of heme oxygenase-1 (HO-1) in a p38 and IKK-NF-xB dependent manner |
| Ko et al. 2017 [106] | HUVECs were treated with BFT | Western blot, ELISA, immunofluorescence assay, EMSAs, transfection assay | BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. BFT increased indices of autophagosomal fusion with lysosomes, activated ATP-1, and upregulated expression of C/EBP |
| Jeon et al. 2019 [107] | Human colon epithelial cells (HCT 116) were treated with BFT | Quantitative reverse transcriptase PCR, ELISA, Western blot | BFT reduced expression of β-catenin. Suppression of β-catenin resulted in increased NF-kB activity and IL-8 expression. |
| Metz et al. 2019 [109] | Ht29/C1 cells were treated with BFT | Morphological assay, thermal shift assay | Chenodeoxycholic acid inhibits BFT |
| Allen et al. 2019 [110] | HT29/C1 cells were treated with BFT | Quantitative PCR, RNA-seq assay | BFT induced differential expression of genes related to bacterial interactions with colon epithelial cells. Ceacam1 was increased and Muc2 was decreased |
| Jeon et al. 2020 [108] | HCT 116 cells were treated with BFT | Western blot, ELISA, EMSA, Cell death detection ELISA | BFT increased expression of sulfiredoxin 1 (Srx-1) in a time-dependent manner. BFT also activated transcriptional signals (Nrf2, AP-1, and NF-kB). Srx-1 induction was dependent on the activation of Nrf2 signals. Overexpression of Srx-1 attenuated apoptosis |
| Ko et al. 2020 [111] | Murine dendritic cells were exposed to BFT | Quantitative reverse transcriptase-PCR, EMSA, transfection assays, Western blot, ELISA, ROS assay | BFT upregulated HO-1expression and activated transcription factors (NF-kB, AP-1, Nrf2). Upregulation of HO-1 was dependent on Nrf2 activation and regulated by ERK and p38. BFT also increased production of ROS. |
| Liu et al. 2020 [58] | Murine colonoids were co-cultured with ETBF | qRT- PCR, sphere forming assay, Western blot, immunohistochemistry | ETBF increased cell stemness and enhanced expression of core stemness transcription factors (NANOG, SOX2). ETBF also activated the Toll-Like 4 pathway |
| Patterson et al. 2020 [59] | Colon organoids and HT29/C1 cells were treated with BFT | Lipidomic analysis, confocal microscopy, q RT-PCR, Western blot, flow cytometry | BFT increased glucosylceramide levels and decreased colonoid permeability and bursting. |
| Becker et al. 2021 [112] | Caco-2 cells were exposed to bft-positive and -negative strains of B. fragilis | TEER, Real-time qPCR, whole genome sequencing, NMR spectroscopy. | BFT increased intestinal barrier function |
| Cao et al. 2021 [113] | Human CRC cell lines (HCT116, SW480) were co-cultured with NTBF and ETBF | microRNA sequencing, semiquantitative reverse-transcription PCR, RT-PCR | ETBF promoted CRC cell proliferation by down-regulating miR-149-3p |
| Xie et al. 2021 [114] | Human CRC cell line SW620 and normal colon cell line NCM460 were cultured/ treated with Recombinant BFT (rBFT) | ELISA, Western blot, cell proliferation assays, | rBFT promoted CRC cell proliferation and accelerated tumor growth. This was associated with upregulation of CCL3, CCR5, NF-kB, and TRAF-6 |
| Boleij et al. 2021 [60] | HT29/C1 cells were treated with BFT | CRISPRcas GPR35-knockout, Western blot, ELISA, immunofluorescence, confocal imaging, RT-PCR | GPR35 identified as a signaling molecule for BFT |