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

Abstract

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...

Ganglioside GD1a Is an Essential Coreceptor for Toll-like Receptor 2 Signaling in Response to the B subunit of Type IIb Enterotoxin

“…The so-called "lower end" of the B pentamer pore of LT-IIb interacts through hydrophilic interactions with the extracytoplasmic oligosaccharide moiety of GD1a, which is anchored to the cell membrane via an intramembrane ceramide lipid. The "upper end" of the B pentamer pore contains a large hydrophobic surface (549 Å 2 ) that engages in hydrophobic interactions with the A2 subcomponent of the A subunit; however, this hydrophobic surface area is solvent-accessible in the absence of the A subunit (42,43). Because of the absence of an A subunit, LT-IIb-B 5 might, therefore, be free to interact with TLR2 or other receptors that depend on hydrophobic interactions for ligand binding (44 -46).…”

“…LT-IIb-B 5 and other B pentamers of heat-labile enterotoxins can participate in both hydrophilic and hydrophobic interactions (42,43). The so-called "lower end" of the B pentamer pore of LT-IIb interacts through hydrophilic interactions with the extracytoplasmic oligosaccharide moiety of GD1a, which is anchored to the cell membrane via an intramembrane ceramide lipid.…”

Mucosal Immunization with a Staphylococcus aureus IsdA-Cholera Toxin A2/B Chimera Induces Antigen-Specific Th2-Type Responses in Mice

“…MRSA USA300 (pvl mutant) was also used in adhesion assays (14). E. coli TE1, a ⌬endA derivative of TX1, and BL21(DE3)/pLysS (Invitrogen, Carlsbad, CA) were used for protein expression (56). All strains were cultured using Luria-Bertani (LB) agar or broth at 37°C with chloramphenicol (35 g/ml), ampicillin (100 g/ml), and/or kanamycin (50 g/ml).…”

The A Subunit of Type IIb Enterotoxin (LT-IIb) Suppresses the Proinflammatory Potential of the B Subunit and Its Ability to Recruit and Interact with TLR2

“…The so-called "lower end" of the B pentamer pore of LT-IIb interacts with the oligosaccharide moiety of GD1a through hydrophilic interactions, whereas the "upper end" of the pore contains a large hydrophobic surface (549-Å 2 ), which interacts with the A2 segment of the A subunit. Because this hydrophobic surface area is solvent accessible in the absence of the A subunit (2,30), it is possible that the isolated B pentamer may interact hydrophobically with molecules such as TLRs that also depend on hydrophobic interactions for ligand binding (31)(32)(33). Presumably, the presence of the A subunit in the holotoxin could preclude or suppress TLR interactions.…”

“…Some insights, however, may be gleaned from the crystallographic structure of LT-IIb (2). LT-IIb-B 5 and other B pentamers of heat-labile enterotoxins interact via both hydrophobic and hydrophilic interactions with other molecules (2,30). The so-called "lower end" of the B pentamer pore of LT-IIb interacts with the oligosaccharide moiety of GD1a through hydrophilic interactions, whereas the "upper end" of the pore contains a large hydrophobic surface (549-Å 2 ), which interacts with the A2 segment of the A subunit.…”