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1.
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 degrees 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. 相似文献
2.
Human intestinal epithelial cells swell and demonstrate actin rearrangement in response to the metalloprotease toxin of Bacteroides fragilis. 总被引:6,自引:3,他引:3
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Enterotoxigenic Bacteroides fragilis (ETBF) cells produce a 20-kDa heat-labile metalloprotease toxin which is potentially important in the pathogenesis of diarrhea associated with this infection. Previous studies indicate that subconfluent HT29/C1 cells treated with the B. fragilis toxin (BFT) develop morphologic changes with dissolution of tight clusters and apparent swelling. Such alterations suggest toxin-stimulated reorganization of the cellular cytoskeleton. The purpose of the current study was to evaluate the effect of BFT on actin microfilaments (F-actin) and cell volume. As assessed by fluorescent phallicidin staining which detects F-actin, BFT treatment of HT29/C1 cells resulted in redistribution of F-actin with loss of stress fibers, a floccular staining pattern, and cellular membrane blebbing without quantitative changes in F-actin fluorescence intensity. The F-actin redistribution was time and concentration dependent. In contrast to the cell shrinkage observed in response to the F-actin-depolymerizing agents cytochalasin D and Clostridium difficile toxin A, BFT stimulated an increase in HT29/C1 cell volume of 10 to 25% (compared with control cells) over a 24-h time course. Only 10 to 30 ng of BFT per ml was necessary to stimulate a maximal increase in HT29/C1 cell volume. The effect of BFT on cell volume was persistent and dependent on the proteolytic activity of BFT. In agreement with cell viability assays indicating that BFT did not injure HT29/C1 cells, intoxicated cells exhibited regulatory volume decrease, suggesting that toxin-treated cells remain physiologically dynamic. We conclude that BFT acts on the intestinal epithelial cell cytoskeleton to alter F-actin structure and to stimulate an increase in HT29/C1 cell volume. Although these two activities of BFT appear to be linked, the precise sequence of cellular events following intoxication of HT29/C1 cells with BFT remains unclear. We hypothesize that these F-actin and cell volume changes may lead to an alteration in tight junction function in the polarized intestinal epithelium, contributing to the pathogenesis of diarrhea in ETBF infections. 相似文献
3.
Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro. 总被引:5,自引:0,他引:5
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F G Chambers S S Koshy R F Saidi D P Clark R D Moore C L Sears 《Infection and immunity》1997,65(9):3561-3570
Strains of Bacteroides fragilis associated with diarrhea in children (termed enterotoxigenic B. fragilis, or ETBF) produce a heat-labile ca. 20-kDa protein toxin (BFT). The purpose of this study was to examine the activity of BFT on polarized monolayers of human intestinal epithelial cells (T84 cells). In Ussing chambers, BFT had two effects. First, BFT applied to either the apical or basolateral surfaces of T84 monolayers diminished monolayer resistance. However, the time course, magnitude, and concentration dependency differed when BFT was applied to the apical versus basolateral membranes. Second, only basolateral BFT stimulated a concentration-dependent and short-lived increase in short circuit current (Isc; indicative of C1- secretion). Time course experiments indicated that Isc returned to baseline as resistance continued to decrease, indicating that these two electrophysiologic responses to BFT are distinct. Light microscopic studies of BFT-treated monolayers revealed only localized cellular changes after apical BFT, whereas basolateral BFT rapidly altered the morphology of nearly every cell in the monolayer. Transmission and scanning electron microscopy after basolateral BFT confirmed a striking loss of cellular microvilli and complete dissolution of some tight junctions (zonula occludens) and zonula adherens without loss of desmosomes. The F-actin structure of BFT-treated monolayers (stained with rhodamine-phalloidin) revealed diminished and flocculated staining at the apical tight junctional ring and thickening of F-actin microfilaments in focal contacts at the basolateral monolayer surface compared to those in similarly stained control monolayers. BFT did not injure T84 monolayers, as assessed by lactic dehydrogenase release and protein synthesis assays. These studies indicate that BFT is a nonlethal toxin which acts in a polar manner on T84 monolayers to stimulate C1- secretion and to diminish monolayer resistance by altering the apical F-actin structure of these cells. BFT may contribute to diarrheal disease associated with ETBF infection by altering epithelial barrier function and stimulating C1- secretion. 相似文献
4.
Bacteroides fragilis toxin rapidly intoxicates human intestinal epithelial cells (HT29/C1) in vitro. 总被引:5,自引:3,他引:2
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Enterotoxigenic Bacteroides fragilis strains associated with childhood diarrhea produce a 20-kDa protein toxin (BFT). Purified BFT causes striking morphologic changes in subconfluent human colonic epithelial cells (HT29/C1). In a 3-h HT29/C1 cell assay, the estimated half-maximal effective concentration of BFT was 12.5 pM, and morphologic effects were detectable as early as 30 min and nearly complete by 1.5 h. Concentrations as low as 0.5 pM could also cause intoxication, but morphologic changes were detectable only when the assay was extended to 18 h. The onset of this intoxication was concentration dependent and rapid, occurring within minutes (<7 min at 0.25 nM, <2 min at 2.5 nM). Notably, the onset of intoxication at 37 degrees C became irreversible to washing within 2 min after exposure to BFT. Morphologic changes were completely inhibited by treatment of HT29/C1 cells with BFT at 4 degrees C but could be demonstrated by subsequent warming to temperatures of 15 degrees C or higher after washing. The time required for the association of BFT with HT29/C1 cells at 4 degrees C was inversely correlated with concentration. Inhibitors of endosomal and Golgi trafficking (NH4Cl and brefeldin A) prevented the intoxication of HT29/C1 cells by Clostridium difficile toxin A and cholera toxin, respectively, but not by BFT. Agents altering microtubule structure did not affect the cellular activity of BFT. These data indicate that a purified toxin from B. fragilis strains associated with diarrhea rapidly and irreversibly intoxicates human intestinal epithelial cells (HT29/C1) in a concentration- and temperature-dependent manner and that the process of intoxication may not involve internalization mechanisms utilizing microtubules or sensitive to pH or brefeldin A. 相似文献
5.
Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene. 总被引:4,自引:0,他引:4
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A A Franco L M Mundy M Trucksis S Wu J B Kaper C L Sears 《Infection and immunity》1997,65(3):1007-1013
Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86-5433-2-2). 相似文献
6.
Prevalence of fragilysin gene in Bacteroides fragilis isolates from blood and other extraintestinal samples 总被引:1,自引:0,他引:1
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Foulon I Piérard D Muyldermans G Vandoorslaer K Soetens O Rosseel P Lauwers S 《Journal of clinical microbiology》2003,41(9):4428-4430
Of 166 Bacteroides fragilis isolates, 26.2% of 103 isolates from blood and 20.6% of 63 extraintestinal isolates harbored the fragilysin gene (difference not statistically significant). Clinical characteristics and evolution were comparable in patients with B. fragilis bacteremia with or without this enterotoxin. Fragilysin seems not to be an important virulence factor in B. fragilis disease. 相似文献
7.
Diversity of the metalloprotease toxin produced by enterotoxigenic Bacteroides fragilis 总被引:1,自引:0,他引:1
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Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a 20-kDa zinc metalloprotease toxin (BFT) associated with diarrheal disease of animals, young children, and adults. BFT stimulates secretion in intestinal loops in vivo and modifies epithelial cell morphology in vitro. The B. fragilis toxin (bft) gene from ETBF strain 86-5443-2-2 (piglet; bft-2) revealed significant nucleotide and predicted amino acid differences when compared to the bft gene from ETBF strain VPI 13784 (lamb; bft-1). This study compares BFT-1 and BFT-2, respectively, produced by ETBF strains VPI 13784 and 86-5443-2-2 purified using the Van Tassell method (38) and a modified purification scheme described herein. Multiple differences in the protein toxins produced by these ETBF strains were identified. First, purified BFT-1 eluted from a high-resolution anion-exchange column (Mono Q) at 0.22 +/- 0.005 M NaC1 versus 0.18 +/- 0.001 M NaC1 for BFT-2 (P < 0.001). Second, BFT-1 and BFT-2 exhibited different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase fast protein liquid chromatography. Third, each BFT reacted with greater specificity to homologous rather than heterologous antisera. Fourth, BFT-2 had modest, but consistently, greater biological activity than BFT-1 when tested on HT29/C1 cells (P < or = 0.01). Together, these data indicate that these ETBF strains produce two distinct isotypes of BFT, termed BFT-1 (VPI 13784 BFT) and BFT-2 (86-5443-2-2 BFT) to recognize the order in which the proteins were purified and genetic sequences identified. The modified purification scheme described in this report yields about two to three times more purified BFT protein than previous protocols and is less time consuming. 相似文献
8.
A fatal case of pyogenic meningitis due to Bacteroides fragilis in a 6-year-old boy is reported. The need for processing cerebrospinal fluid of patients with underlying conditions such as chronic otitis media for recovery of both aerobes and anaerobes is discussed. 相似文献
9.
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11.
Enterotoxigenic Bacteroides fragilis produces an approximately 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in generating mucosal inflammation. Although it is well known that proinflammatory signals are expressed in BFT-stimulated intestinal epithelial cells, cell death processes have not been elucidated. BFT induced apoptosis in HT-29 cells, but the apoptosis was first apparent 36 h after stimulation. During the early period of BFT stimulation, expression of cellular inhibitor of apoptosis protein-2 (c-IAP2) increased, and inhibition of c-IAP2 augmented the apoptotic cell death. Inhibition of BFT-induced COX-2 expression decreased prostaglandin E(2) (PGE(2)) production, which led not only to a decrease of c-IAP2 activity but also to an enhancement of DNA fragmentation in the early period of BFT stimulation. Furthermore, apoptosis inhibition through PGE(2) and c-IAP2 was mainly regulated by a p38 mitogen-activated protein kinase (MAPK). These results suggest that the inhibition of apoptosis may be mediated by a sequential pathway, including MAPK, COX-2, PGE(2) and c-IAP2, in the early period of stimulation. The delay in the onset of epithelial cell apoptosis after enterotoxigenic B. fragilis infection may be important to the host since it can provides sufficient time for epithelial cells to generate signals for the activation of mucosal inflammation and it may increase the chances of bacterial colonization. 相似文献
12.
A micro broth-dilution method was developed for the antibiotic susceptibility testing of Bacteroides frgilis. Eighty strains of B. fragilis were tested against six antibiotics using the agar dilution test as the reference method. The microdilution test yielded results in 24 hours, and agreement with the reference test was satisfactory. Certain subspecies-specific patterns of antibiotic susceptibility were observed. However, larger numbers of subspecies should be evaluated before subspeciation of B. fragilis can be used as a reliable predictor of antibiotic susceptibility. 相似文献
13.
Rosenthal R Günzel D Finger C Krug SM Richter JF Schulzke JD Fromm M Amasheh S 《Biomaterials》2012,33(9):2791-2800
Chitosan is employed as an absorption enhancer for drug delivery strategies. Aim of this study was to investigate the rapid effects on barrier properties of the intestinal epithelial cell model HT-29/B6. Chitosan (0.005%) induced a fast decrease in transepithelial resistance (Rt) which was completely reversible after wash-out. Two-path impedance spectroscopy revealed that chitosan affects both, the paracellular (Rpara) and the transcellular (Rtrans) resistance. pH-dependence and inhibition of both effects by negatively charged heparin indicated a chitosan action only in the protonated form. The decrease in Rtrans was mediated by activation of a chloride-bicarbonate exchanger involved in intracellular pH regulation. This activation was coupled to the decrease in Rpara which was associated with an increase in ion permeability and permeability for paracellular flux markers up to 10 kDa. No effects on expression and subcellular distribution of tight junction (TJ) proteins or the actin cytoskeleton were observed. Accordingly, inhibition of actin-myosin interaction, Ca2+-dependent intracellular signaling, PKC, PI3K/Akt, MAP kinase p38, and endocytosis pathways did not impair the chitosan effect. These results suggest that the rapid and reversible absorption-enhancing chitosan effect is due to changes in intracellular pH caused by the activation of a chloride-bicarbonate exchanger resulting in the opening of the TJ. 相似文献
14.
Radioimmunoassay methods were evaluated for immunoglobulin G and immunoglobulin M antibodies against Bacteroides fragilis antigen. Of 12 serum samples from patients with B fragilis infections, 9 had higher concentrations of immunoglobulin G antibodies than any from 11 control subjects. Of 9 serum samples from infected patients, 6 had higher concentrations of immunoglobulin M than any from control subjects. Six serum samples from patients with Escherichia coli bacteremia did not contain elevated concentrations of immunoglobulin G or immunoglobulin M antibodies against B. fragilis antigen. 相似文献
15.
Superoxide dismutase in Bacteroides fragilis and related Bacteroides species. 总被引:5,自引:0,他引:5
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Superoxide dismutase (SOD) activity was demonstrated in cell-free extracts of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides ovatus, and Bacteroides thetaiotaomicron. The strains were grown under anaerobic conditions in Trypticase soy broth, and the specific activity of SOD in the extracts was, in most strains, higher than in cell-free extracts of Escherichia coli B grown under anaerobic conditions. Isoelectric focusing of the extracts in polyacrylamide gel demonstrated distinct forms of SOD in the different species. 相似文献
16.
The ability of piliated and capsulated Bacteroides fragilis and Bacteroides ovatus to adhere to intestinal cells and mucus was investigated. The adherence of piliated and capsulated strains was at least five times greater than the adherence of their nonpiliated and noncapsulated or capsulated only counterparts. These data illustrate the importance of pili as promoters of adherence of B. fragilis group species to the gastrointestinal mucosa. 相似文献
17.
A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture. 总被引:3,自引:3,他引:3
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Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO. 相似文献
18.
Fluorescent-antibody studies on selected strains of Bacteroides fragilis subspecies fragilis.
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Antisera against seven strains of Bacteroides fragilis subspecies fragilis were produced from dense suspensions of whole cells. These sera exhibited high agglutination titers with homologous antigens. Reciprocal cross-reactions in agglutination tests with each immunizing strain yielded lower titers. Both the indirect and direct fluorescent-antibody techniques were used to evaluate these reagents in the serological identification of 24 defined strains of B. fragilis subspecies fragilis. Subspecies and even strain specificities were noted with particular antisera. A pooled antiserum and conjugate were prepared and studied. Study results showed that specific and high-titered antisera against strains within this subspecies can be produced by the methods described herein and that possibly more than one serotype exists within the seven strains studied. The development of more antibody pools will be necessary to encompass a wider antigenic coverage before the fluorescent-antibody technique can be relied upon altogether for serologically identifying isolates of B. fragilis subspecies fragilis. Test data showed that the indirect method of fluorescent-antibody staining with whole antiserum is an excellent means of identifying strains of this organism. 相似文献
19.
Lectinlike adhesins were identified in the Bacteroides fragilis group by using sugars immobilized on agarose beads either with whole bacteria by direct microscopic examination or with soluble extracts by immunoaffinoelectrophoresis. These two methods allowed the identification of two sugars reacting with whole bacteria and with the corresponding extracts: alpha-D-glucosamine and D-galactosamine. Among eight strains tested representing seven species, the two strains of B. fragilis were equally adhesive and showed the greatest adhesions. The lectinlike adhesin was purified by affinity chromatography on glucosamine-agarose or galactosamine-agarose and showed one band at 70,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This lectinlike adhesin may help to elucidate the roles of the B. fragilis group in the colonization of intestinal surfaces and in the predominance of B. fragilis in infections alone and in synergy with other bacteria. 相似文献
20.
Detection of Bacteroides fragilis and Bacteroides melaninogenicus by direct immunofluorescence. 总被引:1,自引:1,他引:1
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M Labbé N Delamare F Pepersack F Crokaert E Yourassowsky 《Journal of clinical pathology》1980,33(12):1189-1192
A new diagnostic kit, which contains a polyvalent antiserum for either Bacteroides fragilis or Bacteroides melaninogenicus, was tested for reliability and specificity on 146 clinical samples of different origin. A correlation between the culture and immunofluorescence was observed for B. fragilis in 87.39% of cases and for B. melaninogenicus in 81.48% of cases. When pure cultures were tested, aerobically as well as anaerobically, false-positive reactions were observed with staphylococci and Clostridium ramosum spores. The well-defined morphology of these bacteria and spores allows for the elimination of any diagnostic error. The method is rapid, and the margin for error is limited. The test gives a semiquantitative idea of the number of bacteroides organisms present in the clinical specimens even in the presence of a mixed flora. 相似文献