In Vitro Assembly of Novel Cholera Toxin-like Complexes

Hatic, Safet O.; McCann, Jameson A.; Picking, William D.

doi:10.1006/abio.2001.5084

Cited by 10 publications

(13 citation statements)

References 20 publications

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“…In 2001 Hatic et al reported the successful production of a GFP-CT chimera by the purification of individual subunits and in vitro reassembly (12). We have also assembled GFP-CT chimeras, as well as a toxin-coregulated pilus A (TcpA)-CT chimera in vitro (data not shown).…”

Section: Discussionmentioning

confidence: 80%

“…These chimeric molecules were functional, immunoreactive, and able to bind to ganglioside GM 1 (17). Subsequently, our group and other investigators have continued to develop CT chimeras, as well as E. coli heat-labile toxin (LTI and LTIIb) chimeras as promising mucosal immunogens (9,10,12,19,20,22,27,31,35,39,43,46). Bacterial enterotoxins have recently generated interest for a number of other uses as well, including vectors to transport novel therapeutics to intracellular targets and agents to combat autoimmune disease (5,8,14,33).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies from our laboratory reported an immunogenic CT chimera containing the serine-rich Entamoeba histolytica protein (39) and a CT chimera containing the MrpH pilus tip antigen from Proteus mirabilis, which protected mice against P. mirabilis urinary tract infection following intranasal immunization (27). Additional chimeras derived from CT, LTI, or LTIIb were characterized by other investigators (9,12,30,35). Assembly of holotoxin-like chimeras in E. coli requires that both the CTA 2 fusion protein and the B subunit are transported to the periplasm, where CTA 2 of the fusion protein interacts with the nascent B pentamer to promote the assembly process (11,17).…”

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confidence: 99%

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Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

2005

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Cholera toxin (CT) is an AB 5 toxin responsible for the profuse secretory diarrhea resulting from Vibrio cholerae infection. CT consists of a pentameric, receptor-binding B subunit (CTB) and a monomeric A subunit (CTA) that has latent enzymatic activity. In addition to its enterotoxicity, CT has potent mucosal adjuvant activity and can also function as a carrier molecule with many potential applications in cell biology. In earlier studies, the toxic CTA 1 domain was replaced by several other antigenic protein domains to produce holotoxinlike chimeras for use as potential mucosal vaccines. In the present study we utilized the twin arginine translocation (tat) system to produce fluorescent CT chimeras, as well as fluorescent chimeras of Escherichia coli heat-labile toxins LTI and LTIIb. Fusion proteins containing either green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP) and the A 2 domain of CT, LTI, or LTIIb were transported to the periplasm of E. coli by the tat system, and the corresponding B polypeptides of CT, LTI, and LTIIb were transported to the periplasm by the sec system. The fluorescent fusion proteins were shown to assemble spontaneously and efficiently with the corresponding B polypeptides in the periplasm to form chimeric holotoxin-like molecules, and these chimeras bound to and entered cultured cells in a manner similar to native CT, LTI, or LTIIb. The GFP and mRFP derivatives of CT, LT, and LTIIb developed here are useful tools for studies on the cell biology of trafficking of the CT/LT family of bacterial enterotoxins. In addition, these constructs provide proof in principle for the development of novel chimeric CT-like or LT-like vaccine candidates containing CTA 2 fusion proteins that cannot be delivered to the periplasm of E. coli by use of the sec secretion pathway.The AB 5 -type enterotoxins produced by Vibrio cholerae and Escherichia coli have generated interest as potent mucosal adjuvants and immunomodulators, as well as molecular tools to study endocytosis and trafficking from the cell surface to the endoplasmic reticulum (ER) (14,25). Cholera toxin (CT) and E. coli type I heat-labile enterotoxin (LTI) are approximately 80% identical at the amino acid level and consist of an enzymatically active A subunit and a pentameric, receptor-binding B subunit. The B subunit of CT (CTB) binds to ganglioside GM 1 and triggers uptake of the toxin into epithelial cells by endocytosis. Subsequent reduction of the proteolytically nicked A subunit (CTA) releases the active CTA 1 fragment within the endoplasmic reticulum, and CTA 1 is then translocated to the host cytoplasm to catalyze ADP-ribosylation of Gs␣ (25). The E. coli type II enterotoxins, LTIIa and LTIIb, are structurally and functionally homologous to CT and LTI; however, they are more divergent at the amino acid sequence level, and their pentameric B subunits bind preferentially to different ganglioside receptors.Our laboratory and others have constructed stable holotoxin-like molecules with another protein of interest r...

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Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

2005

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“…This method has consistently produced a high efficiency of reassembly of wild-type toxin. In 2001, Hatic et al reported the assembly of recombinant green fluorescent protein (GFP)-CTA 2 polypeptides with CTB in vitro to form GFP-CT chimeras with approximately 20% efficiency based on the concentration of resulting protein (6). From our GM 1 ELISA analysis of in vitro-assembled CT compared to a CT holotoxin standard produced in vivo (Fig.…”

Section: Discussionmentioning

confidence: 91%

“…To determine if denaturation of these CTB variants under less extreme acidic conditions would facilitate their subsequent reassembly, we generated monomeric CTB-T74D and CTB-T78D with citric acid at pH 2.3, as described by Hatic et al (6). Although wild-type CTB monomers produced under these milder conditions were assembly competent, the variant CTB-I74D and CTB-T78D polypeptides remained unable to reassemble into pentamers (data not shown).…”

Section: Sds-page Analysis Of the Ctb-i74d And Ctb-t78d Variants Is Smentioning

confidence: 96%

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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An in vitro and in vivo study of the brain-targeting effects of an epidermal growth factor-functionalized cholera toxin-like chimeric protein

Lin

Sun

et al. 2020

Journal of Controlled Release

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In Vitro Assembly of Novel Cholera Toxin-like Complexes

Cited by 10 publications

References 20 publications

Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

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

An in vitro and in vivo study of the brain-targeting effects of an epidermal growth factor-functionalized cholera toxin-like chimeric protein

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In Vitro Assembly of Novel Cholera Toxin-like Complexes

Cited by 10 publications

References 20 publications

Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

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

An in vitro and in vivo study of the brain-targeting effects of an epidermal growth factor-functionalized cholera toxin-like chimeric protein

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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