key: cord-0000976-sxdstw4a
authors: Jobling, Michael G.; Yang, ZhiJie; Kam, Wendy R.; Lencer, Wayne I.; Holmes, Randall K.
title: A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway
date: 2012-10-30
journal: mBio
DOI: 10.1128/mbio.00401-12
sha: f8da6bc56b3d7a64f5f0ba9a7928264dc986e43f
doc_id: 976
cord_uid: sxdstw4a

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.

dation by the proteasome due to the paucity of lysine residues in its structure. It then forms an allosterically activated complex by binding to an ADP ribosylation factor (ARF), and it ADP ribosylates the alpha subunit of the stimulatory G protein, leading to constitutive activation of adenylate cyclase. In the human intestinal cell line T84, an increased concentration of cyclic AMP elicits a Clsecretory response which can be measured electrophysiologically in real time as a change in short circuit current, I sc (11) .

A key step in the intoxication process is the transport of the toxin from the cell surface to the ER. How GM 1 confers the specificity for lipid trafficking has not yet been determined. Pentameric binding of GM 1 by the B subunit of CT may itself induce membrane curvature and induce invagination to begin the entry process, as is seen with simian virus 40 (SV40) (12) and Shiga toxin (13) (14) (15) . Interestingly, invasion by SV40 occurs only with GM 1 that has native long-chain acyl groups. Clustering of GM 1 may enable the toxin to associate with lipid rafts that serve as platforms for trafficking of CT through the retrograde pathway or parts of it. Lipid rafts are viewed as highly dynamic microdomains that may self-assemble in membranes from sphingomyelin, cholesterol, glycolipids, and proteins that favor a lipid-ordered microenvironment (reviewed in references 16 to 18). Some functions attributed to lipid rafts may require interactions with protein components or scaffolds to stabilize them, extend their lifetimes, or facilitate their coalescence into larger physiologically significant structures. By binding to and cross-linking five GM 1 molecules, CTB might serve as such a protein scaffold and promote the function of lipid rafts in toxin trafficking. Conversion of PM sphingomyelin to ceramide or acute depletion of membrane cholesterol both prevent endocytosis of CT (19, 20) , consistent with a role for lipid rafts in CT trafficking. A requirement for the lipid raftassociated proteins flotillin 1 and flotillin 2 in a zebra fish model of intoxication by cholera toxin (21) also supports the key role of lipid rafts in trafficking of CT.

Our previous study (22) showed that a mixture of CT holotoxins produced in vivo and having chimeric B pentamers with from zero to two wild-type (wt) GM 1 binding sites (BS) was still capable of intoxicating host cells. Because genetic methods, not chemical modifications, were used to prepare these toxin variants, the structures of their GM 1 BS were fully defined and were known to be either the same as wt sites or inactive. Thus, having at most 2 wt GM 1 BS was sufficient for CT to intoxicate host cells, albeit at reduced efficiency. Left unanswered, however, was the question of whether a holotoxin with only one binding site for the GM 1 receptor can function. A toxin molecule able to bind only a single GM 1 molecule would be completely unable to cluster GM 1 molecules or scaffold them into microdomains and would therefore be unable to induce membrane curvature (2, 23) . We designed the experiments reported here to produce defined holotoxin variants that have from zero to five native GM 1 BS in their B pentamers, and we compared their abilities to bind to GM 1 and intoxicate T84 cells. We found that holotoxins able to interact with only a single GM 1 molecule can nevertheless still complete the intoxication process, demonstrating directly that binding a single GM 1 molecule permits the toxin to enter the host cell, complete the trafficking process, and deliver the toxic CT-A1 fragment to the cytoplasm. However, we also found that eliminating even a single binding site from wt holotoxin produced a detectable attenuation in toxicity.

Production and purification of cholera toxin variants with 0, 1, 2, 3, 4, or 5 native GM 1 binding sites. To produce CT holotoxins with defined combinations of wt and mutant CTB subunits, we made three compatible plasmid constructs encoding either tagged wt CTB, CTB-G33D, or wt CTA and expressed them in the same Escherichia coli strain to produce a holotoxin pool with B pentamers consisting of tagged wt CTB and/or CTB-G33D, from which to purify all six possible holotoxin variants. The tag is a 34-aminoacid peptide (denoted GSH6) which is genetically appended after the Met103 codon of native ctxB and encodes glycosylation (bold) and sulfation sites (underlined) (24) , SSSGGGGSSH-PNNTSNNTSSAEDYEYPS, followed by six His residues. Each plasmid has a different replication origin, antibiotic selection, and combination of promoters ( Fig. 1A ; Table 1 ). The ctxA gene, encoding CTA, is expressed from dual pLac and pBAD promoters. Holotoxins and free B pentamers were purified from whole-cell lysates by metal-ion affinity chromatography on Talon resin, and free B pentamers were removed by passing the eluate pool over cation exchange resin, resulting in binding of the free CTB pentamers to the resin and recovery of holotoxin in the flowthrough (Fig. 1B) . Holotoxins with different numbers of BS were then separated by anion-exchange chromatography (Fig. 1C) . By varying the amount of each inducer, we sought to alter the ratio of wt and mutant B subunits in the holotoxin pool. However, for practical purposes, we found that a single condition of relatively low levels of arabinose (0.0005% to express GSH6-tagged wt-CTB) and high levels of isopropyl-␤-d-thiogalactopyranoside (IPTG) (400 M to express native-size CTB-G33D) gave acceptable yields of holotoxin with homopentameric and singly or doubly tagged heteropentameric B subunits (with zero, one, and two wt BS, respectively) and detectable but lower yields of the triple, quadruple, and homopentameric tagged B subunits as described in detail in the next paragraph. This production strain we designated AMBT (for A, mutant CTB, wt CTB-tagged). Simply by swapping the wt and G33D alleles in the respective CTB-encoding vectors and keeping the same inducer ratios, we were able to express the single or double tagged heteropentameric and homopentameric wt CTB subunits with three, four, and five wt BS, respectively, from the production strain designated ABMT (A, wt CTB, mutant CTBtagged). Using these strategies, from two production strains, we produced all six variant holotoxins, which had from zero to five wt BS and a maximum of two tagged subunits. A third production strain, designated ABBT (A, wt CTB, wt CTB-tagged), in which both the GSH6-tagged and untagged B polypeptides were wt CTB, was used under the same expression conditions to produce control holotoxins with five native BS and zero, one, or two tagged CTB subunits. Figure 1 shows the purification process for the AMBT strain (mutant CTB-G33D, wt CTB tagged). Since CTB pentamers naturally bind to metal affinity resins (25) , cholera toxin variants can be purified to near-homogeneity by a single passage over Talon resin. The imidazole eluate pool from the Talon resin contained a mixture of holotoxin and some free CTB pentamers. These free pentamers were removed by cation exchange chromatography, where at pH 8.0 in 50 mM Tris buffer, holotoxin passed through the column while free pentamers bound and could be eluted subsequently with a salt gradient (Fig. 1B) . The heterogenous mixture of holotoxins was further separated into its components by anion-exchange chromatography. The GSH6 tag not only changed the size of the B monomer but also changed its predicted pI from 8.24 for native CTB to 6.21 for CTB-GSH6. The respective predicted pIs for the CTB-G33D variant changed from 7.31 for the native length to 5.91 for the GSH6-tagged variant. All holotoxins bound to the anion exchange matrix, and at least five peaks could be discerned following elution with a salt gradient (Fig. 1C) . Samples from individual fractions were analyzed by SDS-PAGE either without (Fig. 1D , upper) or after (Fig. 1D , lower) boiling in sample loading buffer. The unboiled samples in lanes 4 to 13 showed a single band corresponding to assembled holotoxin (AB 5 ), with a small amount of contaminating protein of slower mobility in lanes 2 and 3. The major fractions from each peak are homogenous and are consistent with each peak containing a single species CTB-G33D-Linker-His 6 , pAra BAD , Cm r , Ori p15a This study of variant holotoxin, The assembled holotoxins in the unboiled samples bind less SDS than if they were fully denatured and therefore do not migrate in direct proportion to their molecular weights. The relative molecular weight (M r ) for each holotoxin variant is increased by the presence of one or more tagged CTB subunits. Each of these holotoxins completely disassembles into its component polypeptides after boiling (Fig. 1D , lower panel; CTA, 29 kDa; CTB-GSH6-tagged monomer, 14.4 kDa; and native CTB-G33D monomer, 11.5 kDa). The holotoxin in peak 0 ( Fig. 1C ) has homopentameric CTB-G33D subunits and no functional GM 1 BS, while holotoxins in peaks 1 and 2 have a mixture of wt CTB-GSH6 and CTB-G33D pentamers with 1 and 2 GM 1 BS, respectively. The fractions loaded in lanes 2 to 5 contain CTA plus 5 CTB-G33D (peak 0), and those in lanes 6 to 9 contain CTA plus 1 wt CTB-GSH6 and 4 CTB-G33D (peak 1), and those in lanes 10 to 13 contain CTA plus 2 wt CTB-GSH6 and 3 CTB-G33D (peak 2). Only these first three peaks were well enough separated to be purified efficiently from the extract of production strain AMBT. The fractions corresponding to each peak were pooled, dialyzed, and rerun over the same anion-exchange column to achieve a very high degree of purity. To make holotoxins with 3 and 4 native GM 1 BS, an expression and purification run was done using strain ABMT, which produced three peaks similar to those shown in Fig. 1C , containing holotoxins with zero, one, or two GSH6tagged CTB-G33D subunits and five, four, or three native GM 1 BS, respectively. Finally, as noted above, strain ABBT was used for expression and purification of wt control holotoxins, all with 5 GM 1 BS and zero, one, or two GSH6-tagged CTB subunits. Analysis of the eight final highly purified holotoxin preparations by SDS-PAGE and Coomassie blue staining is shown in Fig. 2 . The upper panel shows a gel run with unboiled samples, and the lower panel shows a gel run with samples boiled in sample buffer to dissociate the components. Without boiling, holotoxins with untagged or tagged wt CTB pentamers (Fig. 2 , upper panel, lanes 2 to 4) dissociated into free CTA and stable pentameric B subunits that ran near the 45-kDa marker (lane 1). Interestingly, the presence of one or more CTB-G33D polypeptides increased stability of the heterohexameric AB 5 holotoxins. Consequently, unboiled holotoxins with three or more CTB-G33D polypeptides (Fig. 2 , upper panel, lanes 5 to 7) remained fully associated and ran near the 66-kDa marker (lane 1), whereas unboiled holotoxins with one or two CTB-G33D subunits exhibited partial dissociation into B pentamer and free CTA (Fig. 2 , upper panel, lanes 8 and 9). The high stability of the heterohexameric AB 5 holotoxins containing five, four, or three CTB-G33D polypeptides is also evident in the upper panel of Fig. 1D . As expected, increasing the number of tagged CTB polypeptides resulted in moderate decreases in observed mobilities of the pentamer bands (Fig. 2 , upper panel, compare lanes 2, 3, and 4) and the holotoxin bands (Fig. 2 , upper panel, compare lanes 5, 6, and 7), and increasing the number of CTB-G33D polypeptides resulted in slight increases in mobilities of the holotoxin bands (Fig. 2 , upper panel, compare lanes 6 to 8 and lanes 7 to 9). Upon boiling, all holotoxins dissociated and resolved into their individual polypeptide components (Fig. 2, lower panel) . All holotoxins have one CTA subunit. Holotoxins in lanes 2 and 5 are predicted to have five native CTB monomers; holotoxins in lanes 3, 6, and 8 have are predicted to have one tagged CTB and 4 untagged CTB monomers; and holotoxins in lanes 4, 7, and 9 are predicted to have two tagged and three untagged CTB monomers.

Stoichiometry of the individual polypeptides in each purified holotoxin variant was confirmed experimentally by densitometric scanning of the gel in the lower panel of Fig. 2 . To adjust for differences in loading of the purified holotoxins (ranging from 0.6 g in lane 6 to 2.9 g in lane 2), the observed density for each band was expressed as a fraction of the total density for all bands in the same lane. The observed fractional densities were then compared with the expected values based on the predicted molecular mass of each polypeptide (CTA, 27.2 kDa; wt CTB, 11.6 kDa; and tagged CTB, 15.3 kDa) and the assumption that binding of Coomassie blue is proportional to the mass of each peptide. The results (see Table S1 in the supplemental material) were generally within 15% of expected values, but all observed values for tagged B subunits were higher than expected, suggesting that the tagged subunits bound proportionately more Coomassie blue stain than native CTA or CTB. Corrected for loading differences, the expected stoichiometric ratios for the tagged B subunit bands in lanes 3 versus 4, 6 versus 7, and 8 versus 9 were 1:2, and the corresponding observed ratios were 1:2.1, 1:1.7, and 1:1.9, respectively.

Ganglioside GM 1 -binding activities of cholera toxin variants. Relative binding activity of each holotoxin preparation with from zero to five binding sites (and with zero, one, or two tagged B subunits) to ganglioside GM 1 receptor was measured by ELISA with a fixed amount of toxin (5 ng, 60 fmol) added to individual wells coated previously with serial dilutions of GM 1 , and bound toxin was detected with polyclonal rabbit anti-CTB and horserad- ish peroxidase (HRP)-conjugated secondary antibody. Figure 3 shows that at high GM 1 density, toxins with more than one native GM 1 BS bound almost as well as native cholera toxin. The single-BS holotoxin showed a lower plateau signal at high GM 1 density than holotoxins with more than one BS, which we interpret to be due to a less favorable equilibrium between binding and release for the holotoxin with one BS for GM 1 . With more than one BS, a toxin molecule is expected to exhibit faster initial binding to immobilized GM 1 , multivalent binding as the density of immobilized GM 1 increases, and slower dissociation from GM 1 , resulting in increased avidity of binding. In wells coated with 75 nM GM 1 (50 l, 3.75 pmol), toxins with 2 or more native GM 1 BS bound at more than 90% of the wt level, toxin with a single BS bound significantly less at 60% of the wt level, and toxin with no BS gave a minimal signal (4% or less of the wt level). At lower GM 1 densities, there were significant differences between all variants, and in wells coated with 1.2 nM GM 1 (50 l, 60 fmol), toxins with four or three BS bound at 54 and 49% of wt levels, respectively, and toxins with 2, 1, or no BS bound at 25, 12, and 1% of wt levels, respectively. The amount of GM 1 required to coat each well and give 50% of the maximal signal was calculated to be 65, 100, 150, 240, and 850 fmol per well for wt holotoxin and for holotoxin variants with 4, 3, 2, or 1 wt BS, respectively.

Biological activities of the eight holotoxin preparations (the untagged and singly and doubly tagged native holotoxin controls and the variant holotoxins with zero to four wt BS and one or two tags) were initially tested in an overnight assay on mouse Y1 adrenal cell monolayers, on which cholera toxin causes an easily scorable morphological change (rounding of intoxicated cells). In this assay, approximately 2 ng of unnicked native cholera toxin caused rounding of 75 to 100% of the cells (48 U/100 ng). The G33D holotoxin showed some rounding at only the highest concentration tested (33 ng, extrapolating to less than 1 U/100 ng). All holotoxin variants with one or more BS showed rounding of Y1 cells at between 24 and 48 U/100 ng, showing that a single GM 1 binding site is sufficient to intoxicate mouse Y1 cells at near-wt levels. Failure to detect significantly less toxicity after overnight exposure of mouse Y1 adrenal cells to holotoxin variants with from 1 to 4 GM 1 binding sites suggests that any differences in delivery of the wt CT-A1 fragment from the cell surface to the cytosol by these holotoxin variants were not rate limiting for development of the morphological manifestations of intoxication.

To assess the biological consequences of decreased numbers of BS in a more quantitative manner, we determined the real-time electrophysiological effects of the holotoxin variants on polarized human intestinal cells (T84 cell line) by measuring the short circuit current required to eliminate the potential difference induced by the cAMP-dependent Clsecretory response resulting from CT-A1-mediated ADP ribosylation of Gs␣ and constitutive activation of adenylate cyclase. Figure 4A to 4F shows the results of these experiments. Panel A shows that loss of even a single GM 1 BS attenuated the activity of cholera toxin to some degree, an effect which increased as the number of native GM 1 BS was lost. The time of onset of intoxication was also delayed as the number of native GM 1 BS decreased. Nevertheless, toxin with a single native GM 1 BS had clearly detectable activity. As expected, the G33D holotoxin with no native GM 1 BS had almost no activity over the time frame of the experiment: 1.5% of wt toxin signal over baseline at 90 min and less than 9% of wt signal at 120 min. Since these variants have differing numbers of C-terminal GSH6 tags (zero, one, or two), we also examined the effect that the number of tags had on the activity of wt holotoxin with five BS (Fig. 4B) . The presence of the tags modestly affected both the time of onset of intoxication, a measure of toxin-GM 1 trafficking from the PM to the ER of host cells, and the rate of increase of I sc . The effect was greater for doubly tagged holotoxin, although it eventually showed a similar maximal I sc . To control for these effects of the B-subunit tags, toxins with different numbers of native GM 1 BS but the same number of tags were compared. For these analyses, data were normalized by setting the maximal signal for the five native GM 1 BS toxins in each comparison to 1.00 (Fig. 4C to F) . For the toxin variants with one tag on the B subunit, the holotoxin with four native GM 1 BS exhibited a slight (25%) attenuation of toxicity relative to holotoxin with five native GM 1 BS, seen as a decrease in maximal I sc but with no delay in onset of intoxication (Fig. 4C) . A similar result (30% decrease of maximal I sc ) was seen for comparisons of the holotoxin variants with three or five native GM 1 BS and two CTB-GSH6-tagged subunits (Fig. 4D) . When the number of native GM 1 BS was reduced to two or one in doubly or singly wt B-subunit-tagged holotoxin variants, the peak I sc decreased by more than 50% and also the onset of intoxication was delayed ( Fig. 4E and 4F) , suggesting defects in entry of CT into the cell or transport to the ER (or both).

As an initial step toward investigating whether CT variants with five and one native GM 1 BS trafficked from the cell surface to the ER by the same pathway, we examined the effects of brefeldin A (BFA) on their toxicity for T84 cell monolayers. We found that BFA completely inhibited the I sc (Fig. 5) induced by both single and five native GM 1 BS toxin variants. BFA-treated cells, however, still responded to addition of the cAMP agonist vasoactive intestinal polypeptide (VIP) at 90 min, showing that the toxin-treated cells retained viability and were competent for cAMP-dependent Clsecretion (I sc ). Thus, a toxin variant with a single native GM 1 BS, like native CT holotoxin, traffics through a BFA-sensitive pathway to exert its toxic effects.

Previous studies have shown that holotoxins with reduced numbers of GM 1 BS are still able to intoxicate host cells, albeit with attenuated activity. The initial studies (26, 27) used chemical modification and denaturation-renaturation to generate mixed populations of holotoxins predicted to have one or two (nonnative but active) GM 1 BS. In our previous study (22) , we used genetic methods to create populations of chimeric holotoxins with only one or two completely native GM 1 BS and showed that these chimeras were still capable of intoxicating host cells but with attenuated activity.

More recently, we studied how a membrane lipid might specify trafficking of toxin in the retrograde pathway by using fluorophore-labeled GM 1 and imaging toxin trafficking in live cells (28) . We found that a subset of GM 1 species, those with unsaturated ceramide domains, sorted efficiently from the PM to the trans-Golgi network and the ER. Cross-linking by toxin binding was dispensable for such GM 1 trafficking, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. Our results implicated an endogenous proteindependent mechanism of lipid sorting that is dependent on cer- 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 Ϯ SE; n ϭ 2 to 4 for A; n ϭ 3 to 4 for B; n ϭ 2 for C to F; each study was reproduced at least once. amide structure and could explain how the toxin gains access to the ER of host cells to induce disease (28) .

This work shows that multivalent binding to GM 1 is indeed dispensable for CT toxicity. To do this, we prepared and purified homogenous preparations of holotoxins with defined numbers of GM 1 BS. Monomeric binding of CT to single molecules of GM 1 permits the holotoxin to intoxicate the host cell. Our results confirm and expand our studies on trafficking of the single GM 1 lipids. Conversely, loss of even a single GM 1 BS resulted in a measurable diminishment of toxicity in the T84 line of polarized human intestinal cells. This result is also consistent with findings of our studies of trafficking of the non-cross-linked GM 1 molecules, where we found that GM 1 cross-linking by toxin binding enhanced entry of CT into the ER. Thus, although multivalent binding to GM 1 by CT is fundamentally dispensable, it does have a significant effect on retrograde trafficking from the cell surface to the ER and on toxicity.

One way that multivalent binding could affect CT function would be by enhancing the binding avidity for cell membranes containing GM 1 . In this and in our previous studies of the CT binding site mutants, we observe a loss of avidity for binding GM 1 when the GM 1 binding pockets are mutated. In the current studies, the loss of apparent avidity for the one-binding-site species is 10-fold, but the toxin still binds to GM 1 applied to the well at nanomolar concentrations, suggesting this may not fully explain the strong loss of toxin function.

It is possible that the attenuated toxicity seen with holotoxins with reduced numbers of native GM 1 BS could be enhanced by the presence of one or more CTB-G33D polypeptides that confer increased stability to the holotoxins (and thus might decrease the delivery of the CT-A1 subunit to the cytosol). We raise this idea because we observed that insertion of even a single CTB-G33D monomer into the pentameric B subunit rendered the holotoxin partially resistant to dissociation by SDS, and increasing the number of CTB-G33D monomers resulted in holotoxins that were completely resistant to dissociation by SDS. Conversely, there is no a priori reason to assume that the presence of one or more CTB-G33D polypeptides would affect chaperone-mediated release of CT-A1 from the nicked and reduced holotoxin in the ER, which appears to facilitate CT-A1 retrotranslocation from the ER to the cytosol (29) . Further experiments will be required to test these possibilities.

Another way that multivalent binding to GM 1 might affect CT function would be through reorganization of membrane structure and function induced by scaffolding GM 1 into ceramide-based nanodomains, as suggested by studies in vitro and in vivo (14, 15, (30) (31) (32) (33) . Significantly, studies of the closely related AB 5 subunit Shiga toxin show that multivalent binding to the glycosphingolipid GB3 spontaneously induces high-curvature membrane tubules by coupling the toxin-lipid complex to membrane shape (31) . Multivalent binding of CT to GM 1 in model membranes also induces spontaneous membrane curvature, implying a similar coupling of the toxin-glycosphingolipid complex formation to membrane shape (32, 34, 35) . This could allow partitioning of the CT-GM 1 complex into highly curved sorting tubules of the sorting endosome, which is required for retrograde transport, and explain why the toxins with more than 1 GM 1 binding site are more potent in intoxication. It is also possible that multivalent binding to GM 1 may be needed to activate intracellular signaling pathways that enhance uptake and trafficking, as for Shiga toxin (36) .

Effects either on binding avidity or on membrane structure and trafficking dynamics could underlie how the CTB subunit, and the other AB 5 toxins, evolved as pentameric structures. This and our other recent studies, however, show that the most fundamental function exploited by CT is to coopt an endogenous glycosphingolipid sorting pathway from the PM to the ER that is essential for toxin entry into the cytosol and the induction of disease.

Bacterial strains and construction of expression clones. All chimeric toxins in this study were produced by expression from recombinant plasmids in Escherichia coli BW27784 (araFGH pCP18-araE [37] ). This strain does not metabolize arabinose and constitutively expresses the arabinose transporter, and therefore a uniform degree of induction occurs in all cells of a culture at any arabinose concentration. To produce the expression strains, genes encoding native-length CTB or carboxy-terminally extended CTB tagged with a glycosylation-sulfation signal (24) followed by a hexahistidine peptide were cloned on compatible plasmids with different selectable markers and inducible promoters. These encoded resistance to ampicillin (Ap) with the IPTG-inducible lacUV5 promoter or resistance to chloramphenicol (Cm) and the arabinose-inducible araBAD promoter, along with the regulator araC. Variants of these CTB clones were also made with G33D substitutions that eliminate GM 1 binding. A separate plasmid (pSlacbadCTA) selectable with spectinomycin (Sp) and compatible (with a pSC101 origin) with both ctxB plasmids was used to independently express the ctxA gene under control of both the lac and araBAD promoters. Each expression strain thus contained three plasmids, encoding a native CTB subunit (wt or G33D variant, Ap, and lac promoter), a GSH6-tagged CTB subunit (G33D or wt, Cm, and ara promoter), and the wt CTA subunit (Sp and ara and lac promoters). Details of construction, including full DNA sequences, are available on request. Plasmids created or used in this study are listed in Table 1 . Plasmids pAlacCTB and pAlacCTB G33D encode Ap resistance and the wt and G33D native-length variants of CTB, respectively, under control of the lac promoter and have previously been published as pLMP1 and pLMP148, respectively (38) . Carboxy-terminally linker-hexahistidine-tagged variants of CTB and CTB-G33D were made in a compatible Cm r arabinoseinducible vector, pAR3 (39), using the hexa-His tag from pT7sh6 (40) and a glycine-rich repeat from the M13 phage PIII protein, encoding XX (EX) 4 XDPRVPSS (where X is GGGS) inserted between residues 102 and 103 of CTB. Initial experiments to make wt and CTB-G33D mixed pentamers were done using these plasmids, but we saw significant proteolysis of the polyglycine linker-tagged variants, and subsequent experiments were done with variants that had the linker replaced with the GSH6 tag-pCbadCTBGSH6 and pCbadCTB G33D GSH6.

Toxin expression and purification. A 400-ml LB culture at 30°C was inoculated with a 1/8 volume of an overnight culture with appropriate antibiotic selection (Sp and Ap at 100 g/ml and Cm at 25 g/ml) and grown to an A 600 of 1.2, when it was induced with 400 M IPTG and 0.0005% l-arabinose with incubation continued overnight. Cells were collected by centrifugation, resuspended in 20 ml phosphate-buffered saline (PBS), and lysed by mixing for 20 min at room temperature with 0.5 mg/ml lysozyme and Elugent detergent (EMD Biosciences, Inc., La Jolla, CA) to 2%. Viscosity was reduced by sonication four times (10 s each) on ice, and the lysate was cleared by centrifugation at 15,000 rpm for 20 min in an SS34 rotor. Toxin was purified from the supernatants by Talon chromatography as detailed by the manufacturer (Clontech Laboratories, Inc., Mountain View, CA). Imidazole eluates were dialyzed against 50 mM Tris-HCl (pH 8.0) (buffer A). All chromatographic separations were conducted on an Akta purifier (GE Healthcare Biosciences, Pittsburgh, PA) in 4.6-mm by 100-mm Poros perfusion chromatography columns packed with Poros HS 20 cation exchange medium or Poros HQ 20 anion exchange medium. For cation exchange, the column was equilibrated with 5 column volumes (CV) buffer A and washed with 5 CV buffer A after sample loading. Bound material was eluted with 10 CV of a linear gradient of 0 to 100% buffer B (50 mM Tris-HCl, pH 8.0, 1M NaCl). For anion exchange, the column was equilibrated with 5 CV buffer A, washed with 5 CV of buffer A after sample loading, and was eluted with a 40-CV (initial separation) or 10-CV (second purification) linear gradient of 0 to 100% buffer B. Anion exchange fractions were pooled and concentrated using Microcon Ultracel YM-10 filter devices (EMD Millipore, Billerica, MA).

Ganglioside GM 1 ELISA. Ninety-six-well microtiter plates were coated overnight with 50 l of 2-fold serial dilutions of ganglioside GM 1 (Supelco, Sigma-Aldrich, St. Louis, MO) in PBS, starting at 150 nM, and then blocked with 10% horse serum in PBS. All steps were performed at 37°C for 1 h, after which samples were aspirated off and wells were washed three times with PBS plus 0.05% Tween 20. Each holotoxin was assayed in triplicate by incubating 100 l of 50-ng/ml holotoxin per well (approximately 60 fmol), followed by 1/5,000 rabbit anti-cholera toxin B subunit (B10) and then 1/2,000 HRP-conjugated goat anti-rabbit serum (Thermo Fisher Scientific Inc., Rockford, IL), followed by o-phenylenediamine (OPD) substrate (Sigma-Aldrich), and the reaction was stopped with 3 M HCl. Absorbance was read at 450 nm. Y1 assay. Mouse Y1 adrenal cells (ATCC CCL-79) were grown at 37°C in a humidified 5% CO 2 atmosphere in RPMI medium with 5% fetal bovine serum with 1ϫ penicillin-streptomycin (Life Technologies), and 96-well plates were seeded with 10 4 cells per well. One-hundredmicroliter volumes of 2-fold serial dilutions of toxins were added to semiconfluent monolayers and incubated overnight, followed by scoring for toxin-induced rounding. One unit of toxin was defined as the amount contained in the last dilution giving 75 to 100% rounding of cells.

T84 cells and electrophysiology. Measurements of short-circuit current (I sc ) on monolayers (0.33-cm 2 inserts) of the polarized human intestinal cell line T84 were performed as previously described (41) .

Cholera toxin-A foe & a friend

Lipid-mediated endocytosis

Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum

Transcytosis of cholera toxin subunits across model human intestinal epithelia

Proteindisulfide isomerase displaces the cholera toxin A1 subunit from the holotoxin without unfolding the A1 subunit

The Ero1␣-PDI redox cycle regulates retro-translocation of cholera toxin

A class of mutant CHO cells resistant to cholera toxin rapidly degrades the catalytic polypeptide of cholera toxin and exhibits increased endoplasmic reticulum-associated degradation

Role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation

Cholera toxin is exported from microsomes by the Sec61p complex

Stabilization of the tertiary structure of the cholera toxin A1 subunit inhibits toxin dislocation and cellular intoxication

Proteolytic activation of cholera toxin and Escherichia coli labile toxin by entry into host epithelial cells. Signal transduction by a protease-resistant toxin variant

GM1 structure determines SV40-induced membrane invagination and infection

Shiga toxins-from cell biology to biomedical applications

Induced domain formation in endocytic invagination, lipid sorting, and scission

Lipid consorting mediated by Shiga toxin induced tubulation

Polarized sorting and trafficking in epithelial cells

Lipid rafts, caveolae and GPIlinked proteins

Membrane organization and lipid rafts

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Uncoupling of the cholera toxin-G M1 ganglioside receptor complex from endocytosis, retrograde Golgi trafficking, and downstream signal transduction by depletion of membrane cholesterol

Intoxication of zebra fish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Attenuated endocytosis and toxicity of a mutant cholera toxin with decreased ability to cluster ganglioside GM 1 molecules

Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins

Gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulum

The affinity of cholera toxin for Ni 2ϩ ion

Regeneration of active receptor recognition domains on the B subunit of cholera toxin by formation of hybrids from chemically inactivated derivatives

Interaction of a cholera toxin derivative containing a reduced number of receptor binding sites with intact cells in culture

Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1

Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin

Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis

Shiga toxin induces tubular membrane invaginations for its uptake into cells

Bending stiffness depends on curvature of ternary lipid mixture tubular membranes

Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells

Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins

Dynamic sorting of lipids and proteins in membrane tubes with a moving phase boundary

Internalization of cholera toxin by different endocytic mechanisms

Homogeneous expression of the P BAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter

Construction and characterization of versatile cloning vectors for efficient delivery of native foreign proteins to the periplasm of Escherichia coli

An arabinose-inducible expression vector, pAR3, compatible with ColE1-derived plasmids

Structural characterization of the SARS-coronavirus spike S fusion protein core

Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic

Surprising leads for a cholera toxin receptorbinding antagonist: crystallographic studies of CTB mutants

This work was supported by NIH grant R01 AI31940 to R. K. Holmes and by NIH grants DK48106, DK090603, and DK53056 and Harvard Digestive Disease Center grant DK34854 to W. I. Lencer.