A tissue culture method for the detection of bacterial enterotoxins

Maneval, David R.; Colwell, Rita R.; Joseph, Sam W.; Grays, R.; Donta, Sam T.

doi:10.1007/bf01666023

Cited by 38 publications

(29 citation statements)

References 21 publications

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“…Proteolytic cleavage of CTA into the 22-kDa CTA 1 fragment was assessed by SDS-PAGE and Western blotting. Relative toxicity of variant holotoxins was determined by exposing mouse Y1 adrenal cells to various concentrations of toxin and assessing cell rounding after overnight incubation, as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B₅Interface: Mutational Analysis and Development of an In Vitro Assembly System

et al. 2003

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Cholera toxin (CT) and related Escherichia coli enterotoxins LTI and LTIIb have a conserved hydrophobic region at the AB 5 interface postulated to be important for toxin assembly. Hydrophobic residue F223 in the A subunit of CT (CTA) as well as residues 174, L77, and T78 in the B subunit of CT (CTB) were replaced individually with aspartic acid, and the resulting CTA and CTB variants were analyzed for their ability to assemble into holotoxin in vivo. CTA-F223D holotoxin exhibited decreased stability and toxicity and increased susceptibility to proteolysis by trypsin. CTB-L77D was unable to form functional pentamers. CTB-I74D and CTB-T78D formed pentamers that bound to GM 1 and D-galactose but failed to assemble with CTA to form holotoxin. In contrast, CTB-T78D and CTA-F223H interacted with each other to form a significant amount of holotoxin in vivo. Our findings support the importance of hydrophobic interactions between CTA and CTB in holotoxin assembly. We also developed an efficient method for assembly of CT in vitro, and we showed that CT assembled in vitro was comparable to wild-type CT in toxicity and antigenicity. CTB-I74D and CTB-T78D did not form pentamers or holotoxin in vitro, and CTA-F223D did not form holotoxin in vitro. The efficient system for in vitro assembly of CT described here should be useful for future studies on the development of drugs to inhibit CT assembly as well as the development of chimeric CT-like molecules as potential vaccine candidates.Cholera toxin (CT) is a heterohexameric AB 5 toxin that is produced by Vibrio cholerae and is responsible for the profuse and life-threatening diarrheal disease resulting from infection. The pentameric B subunit of CT (CTB) binds to ganglioside GM 1 receptors and triggers uptake of CT holotoxin into intestinal epithelial cells by endocytosis. Subsequent reduction of the proteolytically nicked A subunit (CTA) within the endoplasmic reticulum releases the active toxin component CTA 1 (18). The CTA 1 fragment is translocated to the cytoplasm, where it catalyzes the NAD-dependent ADP-ribosylation of G s ␣, leading to the constitutive activation of adenylate cyclase. The subsequent increase in cyclic AMP concentration induces the secretion of fluids and electrolytes into the lumen of the small intestine (4). The resulting diarrhea may exceed 6 liters per hour, and there are estimated to be over 185,000 cases of V. cholerae infection worldwide each year (2, 34).CT is a member of the V. cholerae-Escherichia coli family of AB 5 heat-labile enterotoxins, which includes the highly homologous E. coli heat-labile type I enterotoxin (LTI) and the structurally related but less homologous type II enterotoxins (LTIIa and LTIIb) (8, 9). The LTIIa and LTIIb variants share the AB 5 structure and enzymatic activity of CT and LTI, but they differ significantly in the amino acid sequences of their A 2 domains and B subunits (33). The A 2 peptides of these enterotoxins penetrate the central pore of the corresponding B pentamers. The interactions between the A 2 domain a...

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Section: Methodsmentioning

confidence: 99%

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B₅Interface: Mutational Analysis and Development of an In Vitro Assembly System

et al. 2003

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“…Binding of mouse monoclonal antibody (MAb) was detected with rabbit anti-mouse immunoglobulin G (heavy plus light chains), followed by 125 I-labeled goat anti-rabbit immunoglobulin G. GM 1 enzyme-linked immunosorbent assays (ELISAs) were performed in a similar manner, except wells of low-binding easy-wash modified flat-bottom ELISA plates (Corning Glassworks, Corning, N.Y.) were coated with 50 l of 0.15 M GM 1 , and bound rabbit antibody was reacted with 1/5,000 goat anti-rabbit-horseradish peroxidase conjugate (BioRad) and detected with OPD substrate (Sigma). Toxicity of culture supernatants was assayed using the mouse Y1 adrenal cell assay (30). One toxic unit is defined as the smallest amount of toxin or supernatant that caused rounding of 75 to 100% of the cells in a well after overnight incubation in RPMI 1640 medium with 10% fetal calf serum.…”

Section: Methodsmentioning

confidence: 99%

Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

Jobling

Holmes

2001

J Bacteriol

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Cholera toxin (CT) is the prototype for the Vibrio cholerae-Escherichia coli family of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68؉V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10-and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12-to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.The massive diarrhea characteristic of the disease cholera is in large part due to the action of cholera toxin (CT), produced by Vibrio cholerae of serogroup O1. A better understanding of the structure and function of CT will provide new insights into the pathogenesis of cholera and may aid in the design of safe and effective vaccines against cholera and related diarrheas.CT is a heterohexameric complex consisting of one A polypeptide and five identical B polypeptides (11). The B pentamer is required for binding to the cell surface receptor ganglioside GM 1 (11). The A subunit can be proteolytically cleaved within the single disulfide-linked loop between C187 and C199 to produce the enzymatically active A1 polypeptide (23) and the smaller A2 polypeptide that links fragment A1 to the B pentamer (32). Upon entry into enterocytes by endocytosis and following reduction and translocation, CT-A1 ADPribosylates a regulatory G-protein (Gs␣), which leads to constitutive activation of adenylate cyclase, increased intracellular concentration of cyclic AMP, and secretion of fluid and electrolytes into the lumen of the small intestine (12). ADP-ribosyl transferase activity of CT is stimulated in vitro by the presence of accessory proteins called ARFs (49), small GTP-binding proteins known to be involved in vesicle trafficking within the eukaryotic cell, but the role of ARFs in the activity of CT in vivo has not yet been determined.CT is the prototype for the V. cholerae-Escherichia coli family of heat-labile enteroto...

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“…CT expression was induced with 0.5% L-arabinose for 3 h at 30°C, and periplasmic extracts were made in Tris-buffered saline containing 1 mg͞ml polymyxin B sulfate. Holotoxin and free B pentamer were quantitated by GM 1 -ELISA (24) by using anti-CTA and anti-CTB specific primary antibodies and purified CT holotoxin as the standard, and toxicity was assayed in mouse Y1 adrenal cell bioassays (25). Sensitivity of variant holotoxins to limited treatment with trypsin was determined by treating them for 30 min with 2% (wt͞vol) bovine trypsin at 30°C.…”

Section: Generation and Characterization Of Clones Expressing Variantmentioning

confidence: 99%

Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system

Jobling

Holmes

2000

Proc. Natl. Acad. Sci. U.S.A.

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The latent ADP-ribosyltransferase activity of cholera toxin (CT) that is activated after proteolytic nicking and reduction is associated with the CT A1 subunit (CTA1) polypeptide. This activity is stimulated in vitro by interaction with eukaryotic proteins termed ADP-ribosylation factors (ARFs). We analyzed this interaction in a modified bacterial two-hybrid system in which the T18 and T25 fragments of the catalytic domain of Bordetella pertussis adenylate cyclase were fused to CTA1 and human ARF6 polypeptides, respectively. Direct interaction between the CTA1 and ARF6 domains in these hybrid proteins reconstituted the adenylate cyclase activity and permitted cAMP-dependent signal transduction in an Escherichia coli reporter system. We constructed improved vectors and reporter strains for this system, and we isolated variants of CTA1 that showed greatly decreased ability to interact with ARF6. Amino acid substitutions in these CTA1 variants were widely separated in the primary sequence but were contiguous in the three-dimensional structure of CT. These residues, which begin to define the ARF interaction motif of CTA1, are partially buried in the crystal structure of CT holotoxin, suggesting that a change in the conformation of CTA1 enables it to bind to ARF. Variant CTA polypeptides containing these substitutions assembled into holotoxin as well as wild-type CTA, but the variant holotoxins showed greatly reduced enterotoxicity. These findings suggest functional interaction between CTA1 and ARF is required for maximal toxicity of CT in vivo. C holera, caused by the Gram-negative intestinal pathogenVibrio cholerae, remains a significant cause of morbidity and mortality in the developing world. Cholera toxin (CT), the primary virulence factor produced by the bacterium, is responsible for the massive dehydrating diarrhea characteristic of the disease (1). CT is secreted by the bacterium into the external milieu as an oligomeric complex of a single enzymatic A subunit (CTA) noncovalently coupled to a pentamer (CTB) of identical B subunits, and it has latent ADP-ribosyltransferase activity. The A subunit has a single intramolecular disulfide bond between Cys-187 and Cys-199, forming a protease-sensitive loop (2). Nicking within this loop by bacterial or host proteases generates the CTA1 and CTA2 polypeptides. CTA1 contains the enzyme active site, and CTA2 tethers the A1 polypeptide to CTB. Both nicking and reduction of the disulfide bond are required for full enzymatic activity (3). CTB binds to its receptor, ganglioside GM 1 , on the surface of enterocytes. After internalization of CT and reduction of CTA, CTA1 gains access to the cell cytosol and catalyzes the NAD-dependent ADP ribosylation of the ␣ subunit of the regulatory heterotrimeric G protein, Gs, inhibiting its GTPase activity and locking it in an active form. The consequence is constitutive stimulation of adenylate cyclase activity and increased intracellular cAMP levels (4), leading to fluid and electrolyte secretion by the affected enterocytes and the mas...

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A tissue culture method for the detection of bacterial enterotoxins

Cited by 38 publications

References 21 publications

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B₅Interface: Mutational Analysis and Development of an In Vitro Assembly System

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B₅Interface: Mutational Analysis and Development of an In Vitro Assembly System

Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system

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A tissue culture method for the detection of bacterial enterotoxins

Cited by 38 publications

References 21 publications

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B5Interface: Mutational Analysis and Development of an In Vitro Assembly System

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B5Interface: Mutational Analysis and Development of an In Vitro Assembly System

Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system

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Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B₅Interface: Mutational Analysis and Development of an In Vitro Assembly System

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B₅Interface: Mutational Analysis and Development of an In Vitro Assembly System