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Tracy D. Wilkins - One of the best experts on this subject based on the ideXlab platform.
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Fragilysin, the enterotoxin from Bacteroides fragilis, enhances the serum antibody response to antigen co-administered by the intranasal route.
Vaccine, 2000Co-Authors: Richard R. Vines, J. Scott Moncrief, Rhonda L. Wright, Lisa Barroso, Samuel S Perdue, Danielle R Sentz, Tracy D. WilkinsAbstract:Fragilysin, an extracellular zinc metalloprotease produced by enterotoxigenic strains of the anaerobic bacterium Bacteroides fragilis, disrupts the paracellular barrier by cleavage of the intercellular proteins between epithelial cells resulting in fluid secretion. Intranasal immunization of mice with Fragilysin and co-administered ovalbumin (Ova) resulted in an Ova-specific serum IgG response that was over 18 000-fold higher than Ova alone, as well as detectable levels of serum IgA. Serum IgG titers were comparable with those seen when whole cholera toxin was used as the adjuvant, although the responses obtained with Fragilysin showed more variability between mice. Metalloproteases to which Fragilysin is structurally related were ineffective as mucosal adjuvants. Our results and similar studies with enterotoxins that affect the paracellular barrier suggest that alteration of mucosal permeability may play an important role in the mechanisms of adjuvanticity.
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Molecular Characterization of the Fragilysin Pathogenicity Islet of Enterotoxigenic Bacteroides fragilis
Infection and immunity, 1998Co-Authors: J. Scott Moncrief, A. Jane Duncan, Rhonda L. Wright, Lisa Barroso, Tracy D. WilkinsAbstract:Enterotoxigenic strains of Bacteroides fragilis produce an extracellular metalloprotease toxin (termed Fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the Fragilysin gene is contained within a small genetic element termed the Fragilysin pathogenicity islet. The pathogenicity islet of B. fragilis VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the B. fragilis chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the Fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of Fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to Fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.
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The Bacteroides fragilis toxin Fragilysin disrupts the paracellular barrier of epithelial cells.
Infection and immunity, 1997Co-Authors: Richard J. Obiso, Ali Azghani, Tracy D. WilkinsAbstract:Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the most commonly isolated anaerobe from human clinical specimens. Some strains produce a toxin (Fragilysin, a zinc-metalloproteinase) implicated as a cause of diarrheal disease in farm animals and humans. Studies in our laboratory confirm that the proteolytic activity of this toxin is responsible for the fluid secretion and tissue damage observed in vivo. In this study, we investigated the effects of Fragilysin on the paracellular barrier of epithelial cells. Researchers suggest that, since the toxin rapidly intoxicates HT-29 cells, it may be internalized. However, we could not prevent cell rounding by using inhibitors of receptor-mediated endocytosis, which indicates that the toxin may act outside the cell. Based on these observations, we studied the effects of the highly purified B. fragilis Fragilysin on the barrier function of cultured epithelial cells. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). We tested a human colon cell line and cell lines from the lung and the kidney; the human colon cell line was most sensitive, but all three were affected in the same manner. Our studies show that B. fragilis Fragilysin alters the barrier function of the epithelial lining, possibly by degrading the tight junction proteins, such as ZO-1. The proteolytic activity is required to cause this effect. The toxin's action has been assumed to be limited to the intestine; however, our studies show that Fragilysin could also contribute to the pathogenesis of B. fragilis in extraintestinal infections.
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The Bacteroides fragilis Toxin Fragilysin Disrupts the
1997Co-Authors: Paracellular Barrier, Ali Azghani, Richard J. Obiso, Epithelial Cells, Tracy D. WilkinsAbstract:Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the most commonly isolated anaerobe from human clinical specimens. Some strains produce a toxin (Fragilysin, a zinc-metallo-proteinase) implicated as a cause of diarrheal disease in farm animals and humans. Studies in our laboratory confirm that the proteolytic activity of this toxin is responsible for the fluid secretion and tissue damage observed in vivo. In this study, we investigated the effects of Fragilysin on the paracellular barrier of epithelial cells. Researchers suggest that, since the toxin rapidly intoxicates HT-29 cells, it may be internalized. However, we could not prevent cell rounding by using inhibitors of receptor-mediated endocytosis, which indicates that the toxin may act outside the cell. Based on these observations, we studied the effects of the highly purified B. fragilis Fragilysin on the barrier function of cultured epithelial cells. Fragilysin rapidly increased the perme-ability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). We tested a human colon cell line and cell lines from the lung and the kidney; the human colon cell line was most sensitive, but all three were affected in the same manner. Our studies show that B. fragilis Fragilysin alters the barrier function of the epithelial lining, possibly by degrading the tight junction proteins, such as ZO-1. The proteolytic activity is required to cause this effect. The toxin’s action has been assumed to be limited to the intestine; however, our studies sho
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Molecular Modeling and Analysis of Fragilysin, the Bacteroides fragilis Toxin
Clinical Infectious Diseases, 1997Co-Authors: Richard J. Obiso, David R. Bevan, Tracy D. WilkinsAbstract:Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the anaerobe most commonly isolated from human clinical specimens. It is associated with abscesses, soft-tissue infections, and bacteremias [1]. In the mid 1980s, Myers et al. [la] reported that some strains of B. fragilis produced a toxin that could be detected in ligated ileal loops of lambs. Since then, enterotoxigenic B. fragilis strains have been implicated as the cause of diarrhea in calves, piglets, foals, rabbits, and, more recently, humans [2]. The results from these studies together support the role of enterotoxigenic B. fragilis in gastrointestinal diseases in humans and animals. In 1992, Weikel et al. [3] showed that culture filtrates from the enterotoxigenic strains caused rapid morphological changes on human colon carcinoma cell lines, particularly HT-29, which are characterized by cell rounding and dissociation of F-actin. Furthermore, in 1996, Donelli et al. [4] showed that B. fragilis toxin-treated HT-29 cells demonstrated membrane blebbing and reorganization of their F-actin structure. We have purified the toxin (Fragilysin) from culture supematants of enterotoxigenic B. fragilis. This toxin is a small polypeptide (~20,600 molecular weight) that causes fluid secretion in intestinal loops at 10 /g/mL and rounding of HT-29 cells at -10 ng/mL [5, 6]. Recently, in collaboration with Wells et al. [7], we showed that Fragilysin increases bacterial intemalization and modulates the epithelial permeability of HT-29 enterocytes. Further studies at our laboratory have confirmed that Fragilysin affects the tight junctions of epithelial cells (authors' unpublished data). These data suggest that Fragilysin may disrupt the epithelial paracellular barrier through proteolytic degradation. The degradation of these extracellular proteins would then allow for cytoskeletal rearrangements because these proteins are directly linked through the cytoplasmic membrane [8]. We sequenced peptides obtained by proteolytic and chemical digestion of Fragilysin and obtained sequences that were then used to generate primers for single specific primer (SSP)-PCR [9]. This process generated a fragment that contained a match to a consensus motif with zinc-metalloproteinases, especially within the metzincin superfamily. Further studies show that Fragilysin has proteolytic activity. Fragilysin degrades a number of cellular proteins including fibrinogen, human complement C3, cytoskeletal proteins and some extracellular matrix proteins such as collagen IV [9]. We found that the purified toxin causes fluid secretion and exfoliation of the surface epithelial cells in vivo, and we have demonstrated that the proteolytic activity of the toxin is responsible for this effect [6]. Metalloproteinases have emerged as important virulence factors in a number of diverse pathogenic organisms including bacteria
F. Xavier Gomis-rüth - One of the best experts on this subject based on the ideXlab platform.
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A novel mechanism of latency in matrix metalloproteinases
The Journal of biological chemistry, 2015Co-Authors: Mar López-pelegrín, Joan L. Arolas, Miroslaw Ksiazek, Abdulkarim Y. Karim, Tibisay Guevara, Jan Potempa, F. Xavier Gomis-rüthAbstract:The matrix metalloproteinases (MMPs) are a family of secreted soluble or membrane-anchored multimodular peptidases regularly found in several paralogous copies in animals and plants, where they have multiple functions. The minimal consensus domain architecture comprises a signal peptide, a 60-90-residue globular prodomain with a conserved sequence motif including a cysteine engaged in "cysteine-switch" or "Velcro" mediated latency, and a catalytic domain. Karilysin, from the human periodontopathogen Tannerella forsythia, is the only bacterial MMP to have been characterized biochemically to date. It shares with eukaryotic forms the catalytic domain but none of the flanking domains. Instead of the consensus MMP prodomain, it features a 14-residue propeptide, the shortest reported for a metallopeptidase, which lacks cysteines. Here we determined the structure of a prokarilysin fragment encompassing the propeptide and the catalytic domain, and found that the former runs across the cleft in the opposite direction to a bound substrate and inhibits the latter through an "aspartate-switch" mechanism. This finding is reminiscent of latency maintenance in the otherwise unrelated astacin and Fragilysin metallopeptidase families. In addition, in vivo and biochemical assays showed that the propeptide contributes to protein folding and stability. Our analysis of prokarilysin reveals a novel mechanism of latency and activation in MMPs. Finally, our findings support the view that the karilysin catalytic domain was co-opted by competent bacteria through horizontal gene transfer from a eukaryotic source, and later evolved in a specific bacterial environment.
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Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Theodoros Goulas, Joan L. Arolas, F. Xavier Gomis-rüthAbstract:Enterotoxigenic Bacteroides fragilis is the most frequent disease-causing anaerobe in the intestinal tract of humans and livestock and its specific virulence factor is Fragilysin, also known as B. fragilis toxin. This is a 21-kDa zinc-dependent metallopeptidase existing in three closely related isoforms that hydrolyze E-cadherin and contribute to secretory diarrhea, and possibly to inflammatory bowel disease and colorectal cancer. Here we studied the function and zymogenic structure of Fragilysin-3 and found that its activity is repressed by a ∼170-residue prodomain, which is the largest hitherto structurally characterized for a metallopeptidase. This prodomain plays a role in both the latency and folding stability of the catalytic domain and it has no significant sequence similarity to any known protein. The prodomain adopts a novel fold and inhibits the protease domain via an aspartate-switch mechanism. The catalytic Fragilysin-3 moiety is active against several protein substrates and its structure reveals a new family prototype within the metzincin clan of metallopeptidases. It shows high structural similarity despite negligible sequence identity to adamalysins/ADAMs, which have only been described in eukaryotes. Because no similar protein has been found outside enterotoxigenic B. fragilis, our findings support that Fragilysins derived from a mammalian adamalysin/ADAM xenolog that was co-opted by B. fragilis through a rare case of horizontal gene transfer from a eukaryotic cell to a bacterial cell. Subsequently, this co-opted peptidase was provided with a unique chaperone and latency maintainer in the time course of evolution to render a robust and dedicated toxin to compromise the intestinal epithelium of mammalian hosts.
Richard J. Obiso - One of the best experts on this subject based on the ideXlab platform.
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Characterization and Molecular Analysis ofFragilysin: The Bacteroides fragilis toxin
1997Co-Authors: Richard J. ObisoAbstract:CHARACTERIZATION AND MOLECULAR ANALYSIS OF Fragilysin: THE BACTEROIDES FRAGILIS TOXIN by Richard Joseph Obiso, Jr. Dr. Tracy D. Wilkins, chairman Department of Biochemistry and Anaerobic Microbiology (ABSTRACT) Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed Fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified Fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When Fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250
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The Bacteroides fragilis toxin Fragilysin disrupts the paracellular barrier of epithelial cells.
Infection and immunity, 1997Co-Authors: Richard J. Obiso, Ali Azghani, Tracy D. WilkinsAbstract:Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the most commonly isolated anaerobe from human clinical specimens. Some strains produce a toxin (Fragilysin, a zinc-metalloproteinase) implicated as a cause of diarrheal disease in farm animals and humans. Studies in our laboratory confirm that the proteolytic activity of this toxin is responsible for the fluid secretion and tissue damage observed in vivo. In this study, we investigated the effects of Fragilysin on the paracellular barrier of epithelial cells. Researchers suggest that, since the toxin rapidly intoxicates HT-29 cells, it may be internalized. However, we could not prevent cell rounding by using inhibitors of receptor-mediated endocytosis, which indicates that the toxin may act outside the cell. Based on these observations, we studied the effects of the highly purified B. fragilis Fragilysin on the barrier function of cultured epithelial cells. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). We tested a human colon cell line and cell lines from the lung and the kidney; the human colon cell line was most sensitive, but all three were affected in the same manner. Our studies show that B. fragilis Fragilysin alters the barrier function of the epithelial lining, possibly by degrading the tight junction proteins, such as ZO-1. The proteolytic activity is required to cause this effect. The toxin's action has been assumed to be limited to the intestine; however, our studies show that Fragilysin could also contribute to the pathogenesis of B. fragilis in extraintestinal infections.
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The Bacteroides fragilis Toxin Fragilysin Disrupts the
1997Co-Authors: Paracellular Barrier, Ali Azghani, Richard J. Obiso, Epithelial Cells, Tracy D. WilkinsAbstract:Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the most commonly isolated anaerobe from human clinical specimens. Some strains produce a toxin (Fragilysin, a zinc-metallo-proteinase) implicated as a cause of diarrheal disease in farm animals and humans. Studies in our laboratory confirm that the proteolytic activity of this toxin is responsible for the fluid secretion and tissue damage observed in vivo. In this study, we investigated the effects of Fragilysin on the paracellular barrier of epithelial cells. Researchers suggest that, since the toxin rapidly intoxicates HT-29 cells, it may be internalized. However, we could not prevent cell rounding by using inhibitors of receptor-mediated endocytosis, which indicates that the toxin may act outside the cell. Based on these observations, we studied the effects of the highly purified B. fragilis Fragilysin on the barrier function of cultured epithelial cells. Fragilysin rapidly increased the perme-ability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). We tested a human colon cell line and cell lines from the lung and the kidney; the human colon cell line was most sensitive, but all three were affected in the same manner. Our studies show that B. fragilis Fragilysin alters the barrier function of the epithelial lining, possibly by degrading the tight junction proteins, such as ZO-1. The proteolytic activity is required to cause this effect. The toxin’s action has been assumed to be limited to the intestine; however, our studies sho
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Molecular Modeling and Analysis of Fragilysin, the Bacteroides fragilis Toxin
Clinical Infectious Diseases, 1997Co-Authors: Richard J. Obiso, David R. Bevan, Tracy D. WilkinsAbstract:Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the anaerobe most commonly isolated from human clinical specimens. It is associated with abscesses, soft-tissue infections, and bacteremias [1]. In the mid 1980s, Myers et al. [la] reported that some strains of B. fragilis produced a toxin that could be detected in ligated ileal loops of lambs. Since then, enterotoxigenic B. fragilis strains have been implicated as the cause of diarrhea in calves, piglets, foals, rabbits, and, more recently, humans [2]. The results from these studies together support the role of enterotoxigenic B. fragilis in gastrointestinal diseases in humans and animals. In 1992, Weikel et al. [3] showed that culture filtrates from the enterotoxigenic strains caused rapid morphological changes on human colon carcinoma cell lines, particularly HT-29, which are characterized by cell rounding and dissociation of F-actin. Furthermore, in 1996, Donelli et al. [4] showed that B. fragilis toxin-treated HT-29 cells demonstrated membrane blebbing and reorganization of their F-actin structure. We have purified the toxin (Fragilysin) from culture supematants of enterotoxigenic B. fragilis. This toxin is a small polypeptide (~20,600 molecular weight) that causes fluid secretion in intestinal loops at 10 /g/mL and rounding of HT-29 cells at -10 ng/mL [5, 6]. Recently, in collaboration with Wells et al. [7], we showed that Fragilysin increases bacterial intemalization and modulates the epithelial permeability of HT-29 enterocytes. Further studies at our laboratory have confirmed that Fragilysin affects the tight junctions of epithelial cells (authors' unpublished data). These data suggest that Fragilysin may disrupt the epithelial paracellular barrier through proteolytic degradation. The degradation of these extracellular proteins would then allow for cytoskeletal rearrangements because these proteins are directly linked through the cytoplasmic membrane [8]. We sequenced peptides obtained by proteolytic and chemical digestion of Fragilysin and obtained sequences that were then used to generate primers for single specific primer (SSP)-PCR [9]. This process generated a fragment that contained a match to a consensus motif with zinc-metalloproteinases, especially within the metzincin superfamily. Further studies show that Fragilysin has proteolytic activity. Fragilysin degrades a number of cellular proteins including fibrinogen, human complement C3, cytoskeletal proteins and some extracellular matrix proteins such as collagen IV [9]. We found that the purified toxin causes fluid secretion and exfoliation of the surface epithelial cells in vivo, and we have demonstrated that the proteolytic activity of the toxin is responsible for this effect [6]. Metalloproteinases have emerged as important virulence factors in a number of diverse pathogenic organisms including bacteria
Joan L. Arolas - One of the best experts on this subject based on the ideXlab platform.
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A novel mechanism of latency in matrix metalloproteinases
The Journal of biological chemistry, 2015Co-Authors: Mar López-pelegrín, Joan L. Arolas, Miroslaw Ksiazek, Abdulkarim Y. Karim, Tibisay Guevara, Jan Potempa, F. Xavier Gomis-rüthAbstract:The matrix metalloproteinases (MMPs) are a family of secreted soluble or membrane-anchored multimodular peptidases regularly found in several paralogous copies in animals and plants, where they have multiple functions. The minimal consensus domain architecture comprises a signal peptide, a 60-90-residue globular prodomain with a conserved sequence motif including a cysteine engaged in "cysteine-switch" or "Velcro" mediated latency, and a catalytic domain. Karilysin, from the human periodontopathogen Tannerella forsythia, is the only bacterial MMP to have been characterized biochemically to date. It shares with eukaryotic forms the catalytic domain but none of the flanking domains. Instead of the consensus MMP prodomain, it features a 14-residue propeptide, the shortest reported for a metallopeptidase, which lacks cysteines. Here we determined the structure of a prokarilysin fragment encompassing the propeptide and the catalytic domain, and found that the former runs across the cleft in the opposite direction to a bound substrate and inhibits the latter through an "aspartate-switch" mechanism. This finding is reminiscent of latency maintenance in the otherwise unrelated astacin and Fragilysin metallopeptidase families. In addition, in vivo and biochemical assays showed that the propeptide contributes to protein folding and stability. Our analysis of prokarilysin reveals a novel mechanism of latency and activation in MMPs. Finally, our findings support the view that the karilysin catalytic domain was co-opted by competent bacteria through horizontal gene transfer from a eukaryotic source, and later evolved in a specific bacterial environment.
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Protein expression and purification trials using the pCri System.
2014Co-Authors: Theodoros Goulas, Joan L. Arolas, Anna Cuppari, Raquel Garcia-castellanos, Scott Snipas, Rudi Glockshuber, Xavier F. Gomis-rüthAbstract:(A) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in E. coli BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. (B) The gene coding for Fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in E. coli Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. (C) Partially purified MBP-Fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of Fragilysin (white) and the MBP (black) after TEV proteinase cleavage. (D) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in E. coli cells, and extracellularly (lanes 5 and 6) in P. pastoris cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. (E) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in E. coli BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. (F) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). (G) MecR1 was expressed in E. coli BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “Materials and Methods”. A black arrow indicates the detected MecR1. (H) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. (I) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.
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Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Theodoros Goulas, Joan L. Arolas, F. Xavier Gomis-rüthAbstract:Enterotoxigenic Bacteroides fragilis is the most frequent disease-causing anaerobe in the intestinal tract of humans and livestock and its specific virulence factor is Fragilysin, also known as B. fragilis toxin. This is a 21-kDa zinc-dependent metallopeptidase existing in three closely related isoforms that hydrolyze E-cadherin and contribute to secretory diarrhea, and possibly to inflammatory bowel disease and colorectal cancer. Here we studied the function and zymogenic structure of Fragilysin-3 and found that its activity is repressed by a ∼170-residue prodomain, which is the largest hitherto structurally characterized for a metallopeptidase. This prodomain plays a role in both the latency and folding stability of the catalytic domain and it has no significant sequence similarity to any known protein. The prodomain adopts a novel fold and inhibits the protease domain via an aspartate-switch mechanism. The catalytic Fragilysin-3 moiety is active against several protein substrates and its structure reveals a new family prototype within the metzincin clan of metallopeptidases. It shows high structural similarity despite negligible sequence identity to adamalysins/ADAMs, which have only been described in eukaryotes. Because no similar protein has been found outside enterotoxigenic B. fragilis, our findings support that Fragilysins derived from a mammalian adamalysin/ADAM xenolog that was co-opted by B. fragilis through a rare case of horizontal gene transfer from a eukaryotic cell to a bacterial cell. Subsequently, this co-opted peptidase was provided with a unique chaperone and latency maintainer in the time course of evolution to render a robust and dedicated toxin to compromise the intestinal epithelium of mammalian hosts.
Theodoros Goulas - One of the best experts on this subject based on the ideXlab platform.
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Protein expression and purification trials using the pCri System.
2014Co-Authors: Theodoros Goulas, Joan L. Arolas, Anna Cuppari, Raquel Garcia-castellanos, Scott Snipas, Rudi Glockshuber, Xavier F. Gomis-rüthAbstract:(A) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in E. coli BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. (B) The gene coding for Fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in E. coli Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. (C) Partially purified MBP-Fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of Fragilysin (white) and the MBP (black) after TEV proteinase cleavage. (D) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in E. coli cells, and extracellularly (lanes 5 and 6) in P. pastoris cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. (E) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in E. coli BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. (F) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). (G) MecR1 was expressed in E. coli BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “Materials and Methods”. A black arrow indicates the detected MecR1. (H) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. (I) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.
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Chapter 186 – Fragilysin
Handbook of Proteolytic Enzymes, 2013Co-Authors: Theodoros GoulasAbstract:Capítulo en: Rawlings, Neil D.; Salvesen, Guy (eds.). Handbook of Proteolytic Enzymes. 3rd ed. London: Academic Press, 2013, vol. 1, chapter 186, p.887-891. ISBN 978-0-12-382219-2. ISBN (V1) 978-0-12-407744-7Peer Reviewe
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Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Theodoros Goulas, Joan L. Arolas, F. Xavier Gomis-rüthAbstract:Enterotoxigenic Bacteroides fragilis is the most frequent disease-causing anaerobe in the intestinal tract of humans and livestock and its specific virulence factor is Fragilysin, also known as B. fragilis toxin. This is a 21-kDa zinc-dependent metallopeptidase existing in three closely related isoforms that hydrolyze E-cadherin and contribute to secretory diarrhea, and possibly to inflammatory bowel disease and colorectal cancer. Here we studied the function and zymogenic structure of Fragilysin-3 and found that its activity is repressed by a ∼170-residue prodomain, which is the largest hitherto structurally characterized for a metallopeptidase. This prodomain plays a role in both the latency and folding stability of the catalytic domain and it has no significant sequence similarity to any known protein. The prodomain adopts a novel fold and inhibits the protease domain via an aspartate-switch mechanism. The catalytic Fragilysin-3 moiety is active against several protein substrates and its structure reveals a new family prototype within the metzincin clan of metallopeptidases. It shows high structural similarity despite negligible sequence identity to adamalysins/ADAMs, which have only been described in eukaryotes. Because no similar protein has been found outside enterotoxigenic B. fragilis, our findings support that Fragilysins derived from a mammalian adamalysin/ADAM xenolog that was co-opted by B. fragilis through a rare case of horizontal gene transfer from a eukaryotic cell to a bacterial cell. Subsequently, this co-opted peptidase was provided with a unique chaperone and latency maintainer in the time course of evolution to render a robust and dedicated toxin to compromise the intestinal epithelium of mammalian hosts.
