A single native ganglioside GM1-binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway

Abstract

Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476-1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573-586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT. Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants of CT with zero to five functional receptor-binding sites (BS). One BS enabled CT to intoxicate cells, supporting the conclusion that CT can enter cells by coopting an endogenous lipid-sorting pathway. Although multivalent receptor binding is not essential, it does increase CT toxicity. These findings suggest that achieving higher receptor binding avidity or affecting membrane dynamics by lipid clustering and membrane remodeling may be driving forces for evolution of AB(5) subunit toxins that can bind multivalently to cell membrane lipid receptors.

FIG 1

Plasmid constructs and purification of variant holotoxins from strain AMBT. (A) Schematic representation of plasmid constructs showing the following: the promoters (arrowheads online) pBAD, arabinose inducible, and pLac, IPTG inducible; replication origins (shaded boxes); antibiotic resistance determinants (solid arrows); and other genes (toxin subunits, shaded arrows, and AraC regulatory protein, open arrow). (B) Cation exchange (HS20) chromatographic separation of holotoxin and CTB pentamers from Talon eluates of extracts from strain AMBT. Holotoxins elute in the flowthrough, whereas CTB pentamers bind and are eluted from the column with a salt gradient (dashed line, 0 to 1 M NaCl). Inside ticks on the x axis mark fractions collected. (C) Anion exchange (HQ20) chromatographic separation of individual holotoxin variants (labels 0 to 4 on peaks correspond to the number of tagged CTB polypeptides in the holotoxin variant) eluted with a salt gradient (dashed line, 0 to 1 M NaCl). (D) Coomassie-stained SDS-PAGE analysis of peaks 0 (lanes 2 to 5), 1 (lanes 6 to 9) and 2 (lanes 10 to 13); size standards (in kDa) are in lane 1. Upper panel, unboiled samples separated on a 10% gel; bracket identifies holotoxins; lower panel, boiled samples on a 15% gel; “A” indicates CTA polypeptide, “B_GSH6” indicates GS-H6-tagged wt CTB polypeptide; and “B_G33D” indicates CTB-G33D mutant polypeptide.

FIG 2

Coomassie-stained SDS-PAGE analysis of all eight purified holotoxin variants. Upper panel, unboiled samples separated on a 10% gel; lower panel, boiled samples separated on a 15% gel. Bio-Rad low range size standards (in kDa) are in lane 1. Five-BS holotoxins with 0, 1, or 2 tagged B subunits (lanes 2, 3, and 4, respectively), a 0-BS holotoxin (5 G33D B subunits, lane 5), or 1- to 4-BS holotoxins (lanes 6, 7, 9, and 8, respectively) are shown. “H” indicates a holotoxin heterohexamer (AB5), “B5” indicates a CTB pentamer, “A” indicates a CTA polypeptide, “B_GSH6” indicates a GSH6-tagged CTB monomer, “B” indicates a CTB monomer, and “wt BS” indicates the number of native GM₁ BS in each holotoxin variant. Numbers on bands in the lower gel show the numbers of the respective monomer in the pentameric B subunit of each holotoxin (the middle band contains GSH6-tagged CTB monomers, and the lower band contains CTB monomers; black numbers designate functional GM₁-binding sites per CTB pentamer, and white numbers designate G33D-containing, non-GM₁-binding sites per CTB pentamer). Amounts of toxin loaded per lane varied from 0.6 µg for lane 6 up to 2.9 µg for lane 2 (2.9, 1.1, 1.15, 0.8, 0.6, 0.85, 2.55 and 1.95 µg for lanes 2 to 9, respectively).

FIG 3

GM₁ ELISA of variant holotoxins. Toxins with from zero (0BS) to five (5BS) BS bound to ELISA plates coated with serial dilutions of GM₁; the graph shows mean signal ± SD for triplicate wells. Wells were coated with serial 2-fold dilutions of GM₁ starting at 7.5 pmol/well (50 µl of 150 nM), and 5 ng of holotoxin was added to each well (approximately 60 fmol). The downward arrow marks the point where toxin and GM₁ are equimolar (59 fmol/well), assuming all GM₁ molecules are bound. Numbers of tagged B subunits in each holotoxin are indicated in parentheses.

FIG 4

Real-time electrophysiological comparisons of each holotoxin variant against wild-type or control-tagged toxins. (A) Comparison of wt holotoxin versus holotoxins with 4, 3, 2, 1, or no native GM₁ BS. Isc, short circuit current in µA/cm². (B) Effect of adding one or two GSH6-tagged CTB subunits to wt holotoxin with 5 native GM₁ BS. (C to F) Normalized data for tag-controlled comparisons of 5-BS holotoxin versus 4-BS (C), 3-BS (D), 2-BS (E), or 1-BS (F) holotoxin variants. Maximum signal for the tagged wt holotoxin in panels C through F was adjusted to 1.00, and all data points in the panel were normalized to that value. Data points show the mean response ± SE; n = 2 to 4 for A; n = 3 to 4 for B; n = 2 for C to F; each study was reproduced at least once.

FIG 5

Electrophysiological comparison of the effect of brefeldin A on single-tagged holotoxins with 5 or 1 native GM₁ BS. Assays were conducted with (open symbols) or without (solid symbols) brefeldin A (10 µM) present. At 90 min, 10 nM vasoactive intestinal polypeptide (VIP) was added to all assays except the 5-BS treatment to show that monolayers remained viable and were responsive to VIP stimulation. Data points show mean response ± SE for duplicate monolayers.