THEME
Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interactions
V. Cholera: invasion of the intestinal epithelial barrier by a stably folded protein toxin

Wayne I. Lencer

GI Cell Biology, Combined Program in Pediatric Gastroenterology and Nutrition, Children's Hospital, Harvard Digestive Diseases Center, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
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CHOLERA TOXIN: ENTRY OF...
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Cholera toxin (CT) produced by Vibrio cholerae is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera. To cause disease, CT enters the intestinal epithelial cell as a stably folded protein by co-opting a lipid-based membrane receptor, ganglioside GM1. GM1 sorts the toxin into lipid rafts and a retrograde trafficking pathway to the endoplasmic reticulum, where the toxin unfolds and transfers its enzymatic subunit to the cytosol, probably by dislocation through the translocon sec61p. The molecular determinants that drive entry of CT into this pathway are encoded entirely within the structure of the protein toxin itself.

GM1; endoplasmic reticulum-associated degradation; lipid rafts; caveolae


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CHOLERA TOXIN (CT) produced by Vibrio cholerae is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera. Cholera is endemic in Asia and Africa, with 5 million cases each year. Eight worldwide pandemics have been documented since the mid-nineteenth century. The disease can cause the death of a previously healthy adult within hours of onset. In 1994, >10% of infected individuals died during the cholera outbreak in Rwandan refugee camps at Goma, Zaire, with one day recording a case fatality rate of 48% (1).


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The genome of one V. cholerae strain (El Tor N16961) has been completely sequenced (5). It consists of two chromosomes. The larger of the two chromosomes includes genes encoding for two essential virulence factors, toxin-coregulated pilus (TCP) and CT. TCP is a type IV pilus required for intestinal colonization (22). The structural genes for TCP are encoded within a pathogenicity island that displays some features of a transmissible genetic element (7). CT is a potent enterotoxin required for the induction of secretory diarrhea. The structural genes for CT are encoded within a lysogenic phage (CTxphi ) integrated into the large chromosome (23). The CTxphi can transfer horizontally to non-toxin-producing strains as long as the recipient expresses TCP. TCP acts as the phage receptor. Thus CTxphi selects only pathogenic Vibrios for infection (23). Two inner membrane proteins, ToxR and TcpP, interact with the cytosolic protein ToxT to coordinately regulate the expression of TCP and CT. ToxR and TcpP induce expression of TCP and CT after sensing as yet unknown factors in the Vibrio's microenvironment during infection. Other potential virulence factors have been identified. These include microbial motility, the secreted proteins HA protease, RTX toxin, Vac a-like toxin, and the CTxphi structural proteins Ace and zonula occludens toxin (ZOT).

Recently, Lee and Camelli and co-workers (8) demonstrated that V. cholerae interacts with the intestinal microenvironment to induce expression of TCP and CT. These investigators used a novel transcriptional reporter system to track the in vivo expression of TCP and CT during infection of the suckling mouse (8). The results of these studies provide evidence for the following model. After entry into the intestinal lumen, the Vibrio senses a luminal factor(s) that induces low-level expression of TCP. Expression of TCP enables the Vibrio to adhere to the intestinal mucosa (presumably the glycocalyx) and then to read the next environmental signal. This second environmental signal depends on microbial adherence to the mucosa and induces in the Vibrio enhanced levels of TCP expression, enhanced intestinal colonization, and finally production and secretion of the enterotoxin CT. Both ToxR and TcpP are critical for these events. However, the identity of environmental factors that sequentially activate ToxR and TcpP during in vivo infections and the origin of the signals (the Vibrio itself, neighboring microbial flora, or the intestinal mucosa of the host) remain unknown.


    CHOLERA TOXIN: ENTRY OF A MACROMOLECULAR PROTEIN INTO HOST INTESTINAL CELLS
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To induce disease, CT released into the intestinal lumen must enter the intestinal epithelial cell at the apical membrane and eventually activate epithelial adenylyl cyclase at the cytoplasmic surface of the basolateral membrane. This event, likely amplified by interaction of toxin with subepithelial enteric nerves and the effect of local neurotransmitter release, leads to the massive intestinal salt and water secretory response characteristic of cholera. The microbe V. cholerae, however, does not invade the intestinal mucosa (15) or directly assist the delivery of toxin into the cell cytoplasm by other mechanisms (such as by type III secretion). Thus the molecular determinants that drive entry of CT into the host intestinal epithelial cell are encoded entirely within the structure of the fully assembled and folded protein toxin itself.

The crystal structures of CT, and the closely related Escherichia coli heat-labile enterotoxin LTI responsible for "traveler's diarrhea," have been solved at 2.2-Å resolution (Fig. 1; Refs. 20, 21). Both toxins belong to the AB5 subunit family that also includes Shiga/verotoxin (responsible for hemolytic uremic syndrome) and pertussis toxin. In CT, five identical peptides (~11 kDa) assemble into a highly stable pentameric ring termed the B subunit (~55 kDa). The B subunit exhibits specific and high-affinity binding to the oligosaccharide domain of ganglioside GM1 and functions to tether the toxin to the plasma membrane of host cells. B subunit binding to GM1 is stoichiometric, with one B subunit pentamer cross-linking five GM1 gangliosides at the cell surface. The specificity and stability of toxin binding to GM1 dictate toxin function, likely by affecting toxin trafficking into the cell (as discussed further below). The single A subunit is comprised of two major structural domains termed the A1 and A2 peptides. The A1 and A2 peptides are linked by an exposed loop containing a serine protease-sensitive "nick" site and a single disulfide bond. The A2 peptide (~5 kDa) tethers the A1 peptide to the B subunit and contains a COOH-terminal KDEL motif (RDEL in LTI) that protrudes below the pentameric B subunit on the side that binds GM1 at the cell surface (20, 21). The KDEL motif is known to be a sorting signal that allows endogenous luminal endoplasmic reticulum (ER) proteins of the eukaryotic cell to be retrieved efficiently from post-ER compartments. The A1 peptide (~22 kDa) is the enzymatically active subunit that must eventually dissociate from the B subunit, translocate across a cellular membrane, and act inside the cell to activate adenylyl cyclase by catalyzing the ADP-ribosylation of the heterotrimeric GTPase Gsalpha .


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Fig. 1.   Crystal structures of cholera toxin (CT) and LTIIb. The base of the B pentamers and the COOH-terminal KDEL motifs of the A subunit face the membrane of target cells. The enzymatic NH2 termini of the A subunits face away. *, Active site. These figures were kindly provided by Ethan Merritt and Wim Hol, University of Washington, Seattle.

To invade the intestinal cell, CT co-opts the machinery for membrane traffic endogenous to the host epithelial cell itself (Fig. 2). CT is not a pore-forming toxin. Rather, CT enters polarized epithelial cells through a complex pathway involving apical endocytosis and retrograde membrane traffic through Golgi cisternae to ER (9, 13, 16). It is currently believed that CT must enter the ER for the A1 peptide to unfold and translocate into the cytosol. After membrane translocation, the A1 peptide may move to the adenylyl cyclase complex on the cytoplasmic surface of the basolateral membrane by diffusion through the cytosol (if the A1 peptide breaks away from the membrane after translocation) or by vesicular transport back out the secretory pathway (if the A1 peptide remains membrane associated). Evidence supporting this working model was recently summarized (10), and the reader is referred to this review for references to the original literature.


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Fig. 2.   Working model for trafficking of CT into polarized cells. The CT holotoxin binds to GM1 in the apical membrane. After endocytosis, the CT-GM1 complex trafficks retrograde through Golgi cisternae into the lumen of the endoplasmic reticulum (ER), where the A1 peptide is unfolded and dissociated from the B pentamer. The unfolded A1 peptide is probably dislocated to the cytosol through the sec61p complex. The A1 peptide may then gain access to ADP-ribosylate its substrate, the heterotrimeric GTPase Gsalpha on the cytoplasmic surface of the basolateral membrane, by diffusion through the cytosol (if the A1 peptide breaks away from the membrane after translocation) or by membrane traffic back out the secretory pathway (if the A1 peptide remains membrane associated). The B subunit is not unfolded in the ER, remains membrane associated (presumably bound to GM1), and moves to the basolateral membrane by trafficking back out the secretory pathway in anterograde vesicles in a process we have termed indirect transcytosis.

The B subunit, unlike the A1 peptide, does not translocate across cell membranes. Rather, the B subunit remains membrane associated (presumably bound to GM1) and eventually moves back out the secretory pathway by vesicular traffic to the cell surface (11). Thus the B subunit (and a small fraction of holotoxin) can move from its original site of binding on the apical (or mucosal) cell surface to the basolateral (or serosal) cell surface by first moving through Golgi cisternae and possibly ER. We have termed this process "indirect" transcytosis (11). This ability of CT to breach the epithelial barrier by crossing through epithelial cells within transport vesicles may contribute to the potent effects of orally delivered CT on mucosal and systemic immune responses.


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In recent years, several laboratory groups made considerable progress in solving the detailed cellular biology exploited by CT to reach the cytosol of target eukaryotic cells. These advances have touched on two main areas of research: 1) the mechanics of toxin sorting and retrograde traffic into Golgi cisternae and ER, and 2) the mechanics of toxin unfolding and membrane translocation of the A1 peptide from the ER lumen into the cytosol.

Mechanics of Toxin Sorting and Retrograde Traffic

GM1 specificity. Retrograde trafficking of CT into the Golgi and ER of target intestinal epithelial cells is not stochastic. For example, CT contains a well-defined ER-targeting KDEL motif that increases the efficiency of toxin function, presumably by facilitating toxin entry into the ER (9). The KDEL sorting signal, however, is not essential for toxicity. These results suggested to us that toxin-binding (or clustering) ganglioside GM1 must be the critical step (necessary and sufficient) for targeting CT into all intracellular compartments required to elicit a cellular response, although the presence of the KDEL motif improved the efficiency of targeting.

We recently tested this idea. Our studies showed that sorting into the Golgi and ER of polarized human intestinal T84 cells depends on toxin binding to the lipid-based membrane anchor provided specifically by the toxin's receptor GM1 (26). Other closely related gangliosides such as GD1a could not substitute for GM1 in this cell type. For these studies, we used the E. coli heat-labile type II toxin LTIIb and recombinant chimeric toxins prepared from the A subunit of CT and the B subunit of LTIIb and vice versa. LTIIb and CT are highly homologous AB5 subunit toxins that exhibit nearly identical enzymatic A subunits (Fig. 1), and both are highly toxic when applied to mouse Y1 adrenal cells. Both toxins contain a COOH-terminal KDEL motif. The B subunits of LTIIb and CT, however, diverge in structure and function, and the B subunit of LTIIb binds ganglioside GD1a and not GM1. Thus the chimeric toxins prepared by mixing the A and B subunits of CT and LTIIb exhibited high-affinity binding to the specific carbohydrate head group of either GD1a (recognized by chimeric toxins containing the B subunit of LTIIb) or GM1 (recognized by chimeric toxins containing the B subunit of CT). Both wild-type and chimeric toxins bound to T84 cells with 10-9 M affinity. However, only CT or toxin chimeras that bound GM1 induced a Cl- secretory response. Wild-type LTIIb and toxin chimeras that bound ganglioside GD1a were inactive. Thus, in polarized intestinal T84 cells, the KDEL motif was not sufficient for toxicity, and toxin binding to GM1 was essential (Table 1).

                              
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Table 1.   Ganglioside structure dictates toxin action and association with lipid rafts

How can a lipid-based membrane anchor such as that provided by the ceramide domain of GM1 impart specificity for protein trafficking? Recently, it was found that in both the human intestinal T84 and Caco-2 cell lines, GM1 functions to concentrate CT in detergent-insoluble, glycolipid-rich apical membrane microdomains (termed "lipid rafts"), and this correlated with toxicity (17, 26). Lipid rafts are distinct membrane structures rich in cholesterol, glycolipids, and certain lipid-modified proteins that function in various cell types as membrane organizing centers for signal transduction, protein and lipid sorting, endocytosis, and transcytosis. Available data indicate that raft structure and function depend on the specific lipids that self-assemble to form the membrane microdomain. On the basis of these studies (summarized in Ref. 3), we now hold the view that CT binding to GM1 represents a form of protein acylation that anchors the toxin to the membrane and acts as the sorting motif for toxin trafficking into Golgi, ER, and possibly the transcytotic pathway of polarized epithelial cells. We also propose that the sorting function of the toxin-GM1 complex depends on association with apical membrane lipid rafts. Binding to lipid rafts may be required to couple the lipid-anchored toxin with the intracellular machinery for protein sorting and vesicular traffic (2, 10, 26).

We have now found that lipid rafts containing the CT-GM1 complex are present in native human intestinal epithelial cells (2) and that lipid rafts isolated from the human intestinal cell line T84 are heterogeneous in structure (and likely function) with respect to ganglioside GM1 content, association with F-actin, and association with the raft-associated protein caveolin-1 (Ref. 2; Badizadegan K, Rodighiero C, Wolf AA, and Lencer WI, unpublished studies). Stable binding to the functional GM1-receptor complex is essential to CT function. Small changes in binding affinity ablate toxicity (Rodighiero C, Hirst TR, and Lencer WI, submitted for publication).

COPI- or rab6-dependent retrograde transport from Golgi to ER. Membranes and soluble proteins move dynamically in the secretory pathway from ER through intermediate compartment to Golgi cisternae and back again. Two mechanistically distinct pathways have now been identified for retrograde trafficking vesicles between these compartments (6, 25). One depends on the assembly of COPI vesicle coats. This is the pathway followed by the KDEL receptor and other ER-resident type I and type II membrane proteins containing dilysine or diarginine sorting motifs. The other depends on the function of the small GTPase rab6. This is the pathway followed by Shiga toxin (25).

Shiga toxin and CT are toxins that have similar molecular structures and bind similar glycolipids as membrane receptors (Shiga toxin binds the glycosphingolipid Gb3, and CT binds GM1). Both toxins require entry into the ER of host cells for bioactivity. Shiga toxin, however, does not contain a KDEL motif, and, when tested in HeLa cells, Shiga toxin was found to move retrograde through Golgi to ER by rab6-dependent vesicle transport (25). The rab proteins are small GTPases that regulate and thus define the specificity of vesicular transport in eukaryotic cells. CT, in contrast to Shiga toxin, contains a COOH-terminal KDEL motif, associates with the KDEL receptor ERD2 in vivo, and was recently found to depend on COPI-coated vesicles for movement retrograde into the ER (12). Because we already know that CT variants lacking a functional KDEL motif can intoxicate host epithelial cells (although less efficiently), it will be interesting to learn whether CT, which anchors itself to the membrane via lipid receptors like Shiga toxin, can also engage rab6-dependent transport vesicles.

Mechanics of Toxin Unfolding and Membrane Translocation

Toxin unfolding and separation of A and B subunits. CT is a stably folded protein. Such stability in protein folding is required for all toxins that must overcome both extracellular and intracellular barriers to bind and enter host cells. CT, however, is not a pore-forming toxin, and exactly how and where in the cell the toxin unfolds to cross cellular membranes remains unknown.

The results of one recent study indicate that the entire A subunit (A1 and A2 peptides together) dissociates from the B pentamer in the Golgi complex and only the A subunit that contains the ER-targeting KDEL motif moves retrograde into the ER (13). This sequence of events, however, seems unlikely given the extensive and stable associations between the A subunit and the B pentamer deduced from the crystal structure (20) and demonstrated biochemically (14). In our own studies on retrograde trafficking and membrane translocation, we recently obtained direct evidence that the B subunit moves into the ER together with the A subunit (Fujinaga Y and Lencer WI, unpublished observations) and in vitro evidence that toxin unfolding depends critically on the function of a redox potential-sensitive chaperone in the ER protein disulfide isomerase (22a). These data support the view that the B subunit-GM1 complex, assisted by the KDEL motif, drives toxin sorting into the ER, where the A subunit likely unfolds and dissociates from the B pentamer.

ER-associated degradation and membrane translocation of A subunit. It has been in the literature for several years that CT, and certain other non-pore-forming toxins such as ricin, Pseudomonas exotoxin A, and Shiga toxin, must enter the ER to unfold and translocate to the cytosol by "dislocation" through the translocon sec61p (4). This idea arises from recent evidence that the biosynthetic pathways of eukaryotic cells are endowed with the ability to identify and eliminate "misfolded," unassembled, or aberrantly modified membrane or secreted proteins by proteosome-dependent degradation in the cytosol (for review and additional references, see Ref. 18). These studies have shown that certain misfolded proteins can move out of the ER using the sec61p complex or a similar protein translocase.

As nascent proteins enter the ER, they are monitored for correct folding, posttranslational modifications, and assembly into hetero- or homo-oligomeric complexes. Proteins which fail this "quality control" surveillance are degraded in the cytosol (termed ER-associated degradation or ERAD), as first shown for the cystic fibrosis transmembrane regulator (24) and subsequently confirmed for at least 19 other proteins. Recognition and targeting of substrates for ERAD can occur entirely within the lumen of the ER. Dislocated proteins may be found freely diffusable in the cytosol or membrane associated after dislocation. Thus the idea that CT may enter the ER as a fully folded luminal protein to opportunistically engage this system for unfolding and membrane translocation is both biologically plausible and attractive (4, 10). In fact, within the last year, Schmitz et. al. (19) published evidence that signal sequence-dependent in vitro translation of CT A subunits into rabbit microsomes led to "dislocation" of the nascent A subunit from the rabbit microsome via sec61p-dependent transport.


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INTRODUCTION
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CHOLERA TOXIN: ENTRY OF...
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REFERENCES

Cholera is endemic in Asia and Africa. The key to understanding cholera and other secretory diarrheas lies within the intestinal enterocyte. To breach the epithelial barrier, CT trafficks as an intact protein through two unique (and possibly interconnected) pathways in polarized cells. One moves retrograde through Golgi cisternae to ER. The other moves across the cell by transcytosis. Trafficking through these intracellular compartments appears to depend on sorting motifs provided by the structure of ganglioside GM1. These membrane dynamics harnessed by CT to enter the epithelial cell, move retrograde into ER, and then across the cell by transcytosis are fundamental to the structure and function of polarized epithelial cells and to their ability to maintain a selective barrier required for efficient solute transport. Furthermore, the mechanisms whereby foreign macromolecules such as CT enter the ER and engage the ERAD system are of central importance to cell biology, microbial pathogenesis, and mucosal immunology.


    ACKNOWLEDGEMENTS

I thank M. R. Neutra and members of the Lencer Lab for critical reading of the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-48106 to W. I. Lencer and DK-34854 to the Harvard Digestive Diseases Center.

Address for reprint requests and other correspondence: W. I. Lencer, Combined Program in Pediatric Gastroenterology and Nutrition, GI Cell Biology, Enders 1220, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 (E-mail: lencer{at}tch.harvard.edu).


    REFERENCES
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Am J Physiol Gastrointest Liver Physiol 280(5):G781-G786
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