Toxicon 39 (2001) 16291635

www.elsevier.com/locate/toxicon

Shiga toxins
K. Sandvig*
Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

Abstract Shiga toxin and Shiga-like toxins belong to the group of protein toxins which have a moiety that binds to the cell surface and another enzymatically active moiety that after entry into the cytosol inhibits protein synthesis enzymatically. The toxins can also cause apoptosis by mechanisms that may be different from the effect on the protein synthesis machinery. Shigella dysenteriae, some strains of Escherichia coli as well as other bacteria can secrete such toxins which cause serious complications during infections. An increasing knowledge about the toxins and their interactions with cells is important both for treatment of disease, and for elucidation of pathways of intracellular transport. q 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Shiga; Toxin; Hemolytic uremic syndrome (HUS); Golgi apparatus; Endocytosis

1. Introduction Shiga toxin and Shiga-like toxins belong to a large family of plant and bacterial toxins that kill cells by rst binding to the cell surface, then they are endocytosed and subsequently an enzymatically active part of the molecule enters the cytosol where it very efciently inhibits protein synthesis and thereby kills the cell (Sandvig and van Deurs, 1996; Falnes and Sandvig, 2000). Shiga toxin is produced by Shigella dysenteriae and increases the severity of disease caused by this bacterium, whereas the Shiga-like toxins, one of which is almost identical to Shiga toxin (see the rst paragraph of Section 2), are secreted by certain strains of Eshcerichia coli (STEC; Shiga-toxin producing Eshcerichia coli). Not only E. coli can produce Shiga-like toxins, but also Citrobacter freundii, Aeromononas hydrophila, Aeromononas caviae, and Enterobacter cloacae have been reported to be able to express the toxins (Paton and Paton, 1998). Shiga toxin genes in both E.coli and S. dysenteriae are generally phage-borne (Unkmeir and Schmidt, 2000). Infections with bacteria producing Shiga toxins are responsible for widespread disease and for the death of a large number of people (Takeda et al., 1993; Kaper, 1998; Uchida et al., 1999; Bower, 1999; Paton et al., 2000; Kitov et al., 2000; Paton and Paton, 1998). Bacteria producing Shiga-like toxins seem to be the most common cause of hemolytic uremic syndrome (HUS), characterized by throm* Tel.: 147-229-34294; fax: 147-225-08692. E-mail address: ksandvig@radium.uio.no (K. Sandvig).

bocytopenia, microangiopathic hemolytic anemia and renal failure. In some cases, even neurological symptoms are seen (Paton and Paton, 1998). Children and elderly most often get this disease (Paton and Paton, 1998). These infections have become an increasing threat to human health also in developed countries where the bacteria secreting Shiga-like toxins are found in different types of food, including milk, apple juice and vegetables (Kaper, 1998; Uchida et al., 1999; Bower, 1999). As described below, our increasing knowledge about the toxins and their interactions with cells has allowed production of molecules that might help us to treat this type of infection. Although the Shiga-like toxins have a similar structure, they do not have the same effect on cells, and we still need more information to fully understand these differences. As the complex picture of the severe complications caused by the toxins is being claried, it becomes clear that a number of cell types in the body can be a target for toxin-induced inhibition of protein synthesis and apoptosis, as well as for toxin-induced cytokine secretion (for review, see Paton and Paton, 1998; Meyers and Kaplan, 2000). Studies of Shiga toxin have also proven useful to elucidate intracellular transport. Shiga toxin was the rst molecule observed to be transported all the way from the cell surface to the Golgi and the ER (Sandvig and van Deurs, 1996), and other toxins are now known to follow this route (Sandvig and van Deurs, 1996; Rapak et al., 1997). Such retrograde transport may be of physiological importance and not used by toxins only. Interestingly, in B-cells there seems to be a Gb3 (CD77)-dependent retrograde transport of

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CD19, which has sequence similarity to the Shiga toxin B-subunit (Khine et al., 1998). Also, as described below, Shiga toxin has not only been used to investigate intracellular routing, but can serve as a tool to characterize different compartments. Future studies of Shiga toxin and Shiga-like toxins are likely to provide us with new therapeutic strategies as well as valuable basic information in cell biology.

3. Entry of Shiga toxins into cells 3.1. Binding of toxin to the cell surface Shiga toxins act, compared to many other protein toxins, on a limited number of cell types (for review, see Sandvig and van Deurs, 1996; Paton and Paton, 1998). First of all, only cells with toxin binding sites respond. The receptor for most of the Shiga toxins is the glycolipid Gb3. One variant toxin, Stx2e also binds to Gb4. Not only the carbohydrate part of the glycolipid molecule is important for toxin binding. The lipid tail also plays an important role for the toxin receptor interaction (Sandvig and van Deurs, 1996; Kiarash et al., 1994; Paton and Paton, 1998). Even when the toxins bind to a certain cell type, this cell might be resistant: a phenomenon that can be explained by lack of toxin transport to the Golgi apparatus and the ER (Sandvig and van Deurs, 1996). Interestingly, there is now evidence that the lipid composition might be important for the sorting of the toxin to the Golgi and the ER (see below). The binding of Shiga toxin to cells and the sensitivity of these cells to the toxin can be regulated by a number of factors (Sandvig and van Deurs, 1996; Paton and Paton, 1998; Hughes et al., 2000; Meyers and Kaplan, 2000). Changes in signal transduction might be important for such regulation since prolonged incubations with cAMP can affect binding and sensitivity, and also butyric acid exposure can induce synthesis of new receptors for Shiga toxins (Sandvig and van Deurs, 1996; Hughes et al., 2000). Importantly, cytokines (e.g. interleukin 1 (IL-1), and tumor necrosis factor (TNF)), induced by the Shiga toxins or by LPS (lipopolysaccharide) during the infection, can induce synthesis of Gb3 in various cell types (Paton and Paton, 1998; Hughes et al., 2000; Meyers and Kaplan, 2000). The signaling mechanisms involved in the induction of Gb3 synthesis by these agents differs, some of them being dependent on protein kinase C (Paton and Paton, 1998). Such induction might play an important role in facilitating the action of Shiga toxins and increase the severe complications during infection with Shiga toxin-producing E. coli. 3.2. Endocytosis and transport to the Golgi apparatus After binding to the cell surface, Shiga toxin is very efciently internalized from clathrin-coated pits (Sandvig and van Deurs, 1996). Transport of the toxinglycolipid receptor complex to the clathrin-coated domain seems to be mediated by the toxin, since the complex is evenly distributed at the cell surface when the toxin is added to cells at low temperature. The mechanism behind the toxin-induced transport is not known, but it seems reasonable to assume that interaction of the toxin molecule with proteins destined to the clathrin-coated pit is involved. There is, however, also a much slower uptake of Shiga toxin from other areas of the plasma membrane. This can be demonstrated by interfering with the clathrin-dependent uptake. After endocytosis, the

2. Structure and function of Shiga and Shiga-like toxins The family of Shiga toxins all belong to the AB family of protein toxins, i.e. toxins with one enzymatically active part (A), and one part that binds to the cell surface (B). Shiga toxins have an enzymatically active A fragment that contains an internal disulde bond and from which the A1 and the A2 subunits are generated by proteolytic processing of the loop area and reduction of the internal disulde bond (see Fig. 1). The A1 fragment inhibits protein synthesis after release in the cytosol by removing one adenenine from the 28S RNA of the 60S ribosomal subunit. The binding moiety (B) consists of ve identical subunits which can all bind to glycolipid receptors at the cell surface. The molecular weight of the intact toxin is about 70 kD, with the A subunit of about 32 kD and each B subunit of 7.7 kD (Paton and Paton, 1998). The crystallographic structure of Shiga toxin is shown in Fig. 1B. Shiga toxins can be classied into different groups (Paton and Paton, 1998): Shiga toxin (Stx) which is produced by Shigella dysenteriae is almost identical to Shiga-like toxin 1 (Stx1), also called VT1, produced by Escherichia coli. Then, there is Shiga-like toxin 2 (Stx2), also called VT2, which has a similar structure, but which has sequence differences that provide this toxin with different properties than Stx1, both with regards to effects on cells as well as immunological properties. Furthermore, several Stx2 variants are known, again with different properties. Both the Stx- and Stx1-A fragment have 315 amino acids, whereas Stx2-A has 318 amino acids. All the B subunits have 89 amino acids.

Fig. 1. Schematic (A) and crystallographic structure (B) of Shiga toxin. The structure has been obtained from the PDB protein data bank (Shiga toxin: 1DM0), and is based on earlier published work (Fraser et al., 1994).

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toxin in some cells is transported to the Golgi apparatus, in addition to following the endocytic pathway to lysosomes. The transport to the Golgi apparatus is as described required for intoxication (Fig. 2), and cells that bind and internalize Shiga toxin, but are resistant to the toxin, seem to lack the Golgi transport of this molecule (Sandvig and van Deurs, 1996). Interestingly, it was shown that the lipid composition of the receptor seems to be important for this transport step, thus demonstrating that not only protein sequences, but also lipids, are important for sorting to the Golgi apparatus. Cells that are resistant to Shiga toxin may become sensitized to the toxin by compounds that change the lipid composition of the cells, and concomitantly, there is an increased transport to the Golgi apparatus. Treatment of cells with butyric acid, which can be found in quite high concentrations in the intestine, has been found to sensitize cells to Shiga toxin. This was rst demonstrated for A431 cells (Sandvig and van Deurs, 1996), and has later been used to sensitize a variety of cell types (Paton and Paton, 1998). Also, cAMP can sensitize cells to Shiga toxin and induce transport to the Golgi apparatus, and a number of the cytokines/growth factors now known to sensitize different cells to this group of toxins might not only induce synthesis of Shiga toxin receptors, but might also change the intracellular routing (see Fig. 2). By which pathway do Shiga toxin and Shiga-like toxins enter the Golgi apparatus? It is well known that there is a pathway to the Golgi apparatus from late endosomes, a pathway that is dependent on the small GTP-binding protein Rab9, and that is responsible for transport of the mannose6-phosphate receptor (Lombardi et al., 1993; Goda and Pfeffer, 1988). Recently, it has been suggested that a Golgi-associated protein TGN38 (Ghosh et al., 1998) as well as the Stx1-B subunit (Mallard et al., 1998) circumvent late endosomes and are transported from sorting endosomes

and directly to the trans-Golgi network, possibly via the endosomal perinuclear recycling compartment (ERC). The Shiga toxin B-subunit was seen in association with clathrincoated buds on endosomes, and these were suggested to be involved in Golgi transport. However, more detailed studies are required to reveal the detailed transport mechanism. Interfering with molecules involved might help in this elucidation, since a toxin molecule might pass through a certain compartment without being detectable by microscopical methods. It might pass quickly through this compartment, or it might be present in concentrations so low that it is undetectable. Several different toxin molecules are known to be present in the ER on their way to the cytosol, but presumably in concentrations so low that, in most cases, they cannot be shown by electron microscopy. For instance, in the case of a modied version of the plant toxin ricin, one can demonstrate that this toxin can be glycosylated by ER enzymes (Rapak et al., 1997), but ricin has so far not been visualized in the ER. Whatever the mechanism of Shiga toxin transport to the Golgi might be, this transport has been used to measure the pH of the Golgi apparatus (Schapiro and Grinstein, 2000). The Stx1-B subunit was used to deliver pH-sensitive probes to the Golgi apparatus. Clearly, toxins are useful for studies of various questions in cell biology. 3.3. Processing and activation of Shiga toxins by furin Shiga toxins contain, between the A1 and the A2 subunits, a loop formed by the internal disulde bond of the A fragment. This loop is very sensitive to cleavage by trypsin, and cleavage has been found to be essential for rapid intoxication of cells (Sandvig and van Deurs, 1996). However, when intact or precleaved toxins are added to cells they are equally toxic to many cell types, and it turns out that there

Fig. 2. Model of Shiga toxin entry into cells.

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is in most cases a rapid cellular processing of the uncleaved toxin by the enzyme furin (Garred et al., 1995). This enzyme recognizes the sequence Arg-X-X-Arg, which is found in the loop region. Cleavage of Shiga toxin by furin can be demonstrated in cell-free systems, and it occurs optimally at low pH. The enzyme furin is found in the TGN as well as in endosomes and might cleave Shiga toxins in both locations. Interestingly, furin-induced cleavage and activation seems to play an important role for different bacterial protein toxins, such as Pseudomonas exotoxin A and anthrax toxin (Gordon and Leppla, 1994). The role of furin in processing and intoxication with Shiga toxin was studied by using Lovo cells which do not contain furin originally, as well as furin-transfected Lovo cells. Processing of Shiga toxin occurs more slowly without furin, and it occurs in a different cellular location, since it was inhibited by the drug brefeldin A which disrupts the Golgi apparatus (Sandvig and van Deurs, 1996). Furthermore, it was inhibited by calpain inhibitors suggesting that this cytosolic enzyme might be involved. If this is the case, it might imply that the whole A fragment can be translocated to the cytosol (see below). 3.4. Retrograde transport to the ER and translocation of toxins to the cytosol How is Shiga toxin transported retrogradely from the TGN to the ER? A well studied retrograde transport system is based on the so-called KDEL receptors that trafc between the ER and the TGN and bring in a retrograde manner proteins containing a lumenal KDEL sequence back to the ER. This retrograde transport occurs via COPI-coated vesicles (Warren and Malhotra, 1998). However, Shiga toxin and the Stxs, in contrast to, for instance, Pseudomonas exotoxin A, do not contain such a sequence (Sandvig and van Deurs, 1996). That the presence or absence of a KDEL sequence actually means that these types of toxins are transported by different routes, was suggested by the nding that expression of lysosome-KDEL which actually causes a redistribution of KDEL receptors in cells as well as microinjection of antibodies to the cytosolic tail of the KDEL receptor protected against Pseudomonas exotoxin A, but not against Stx1 (Jackson et al., 1999). Recent results indicate that Stx1-B is transported from the TGN to the ER by a Rab6-dependent process that is different from the COPI-dependent pathway (White et al., 1999; Girod et al., 1999). Again, the details of this pathway remain to be elucidated. How are the Shiga toxins transported from the ER and into the cytosol? Recently, it has become clear that newly synthesized proteins which do not become correctly folded in the ER, can be translocated backwards across the ER membrane and degraded in the cytosol by the proteasomal system. Such retrograde transport can occur even when the proteins are glycosylated (Suzuki et al., 1998; Cacan and Verbert, 1999). Investigations of this transport have revealed that it occurs through Sec61, i.e. the same translocator by which newly synthesized proteins enter the ER

(Matlack et al., 1998). In the case of the protein toxin ricin, there is now evidence that this translocator is involved in toxin transport (Wesche et al., 1999; Simpson et al., 1999). Interestingly, one of the subunits of Sec61, Sec61a, was coimmunoprecipitated with ricin (Wesche et al., 1999). Similar data have not been published for Shiga toxins, but it is tempting to speculate that these toxins also enter by the Sec61 complex. Which parts of the toxin molecules are translocated to the cytosol? It is clear that the enzymatically active A1 fragment has to enter to exert its effect on ribosomes. After proteolytic activation of Shiga toxin by furin in the endosomes/Golgi apparatus, the A1 fragment might be released from the A2 fragment in the ER by reduction of the disulde bond connecting them, and the enzymatically active part could then become translocated. However, this has not yet been demonstrated. Interestingly, in Shiga mutants that lack the furin-sensitive site there is also a proteolytic cleavage which is important for maximal activity, a cleavage that might be performed by the cytosolic enzyme calpain since inhibitors of this enzyme reduce the processing (Sandvig and van Deurs, 1996). If this processing occurs in the cytosol, the whole A fragment would have to enter. However, these details remains to be claried. Recently, it was also suggested that perhaps even the B fragment might enter the cytosol, since regulated expression of this fragment could induce apoptotic changes (Nakagawa et al., 1999).

4. Effects of Shiga toxins on cells 4.1. Ability of Shiga toxin to inhibit protein synthesis and induce synthesis of cytokines The A subunit of Shiga toxins has, like several other protein toxins, RNA N-glycosidase activity and removes one adenine from adenosine in position 4324 from the 5 0 terminus in 28S ribosomal RNA, thereby inhibiting binding of amino-acyl-tRNA to the 60S ribosomal subunit (Paton and Paton, 1998). This enzymatic activity leads to a general inhibition of protein synthesis, but the toxin can also have other effects. For instance, Stx1 has been reported to have an antiviral activity that requires a catalytically active toxin. Stxs may, therefore, play a role in protecting infected cattle (Ferens and Hovde, 2000). During infection with STEC, there are changes of cytokine levels that can be ascribed to an effect of Shiga toxin on different cell types (Paton and Paton, 1998; Hughes et al., 1998, 2000). Both Stx1 and Stx2 can induce synthesis of interleukin 8 (IL-8) in Caco-2 cells and in T84 cells, and the induction seems to be dependent on the catalytic activity of the A-subunit (Yamasaki et al., 1999). Furthermore, Shiga toxin can induce release of IL-1, IL-6 and TNF from macrophages and human proximal tubule cells (Paton and Paton, 1998; Hughes et al., 1998). Stx1 seems to be able to induce TNF release in human proximal tubule

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cells at concentrations which are so low that protein synthesis is not affected (Hughes et al., 1998). However, the effect on mRNA levels of TNF and secretion of this cytokine was higher at slightly toxic concentrations of Stx1. No similar effects could be observed by addition of cycloheximide, suggesting that the increased release of TNF was specic for Stx1. In contrast, cycloheximide could, like Stx1, induce release of IL-1, suggesting that different mechanisms are involved in Stx-induced cytokine release. 4.2. Toxin-induced apoptosis Shiga toxin and Shiga-like toxins do not only inhibit protein synthesis in cells, they also induce cell lysis characteristic of programmed cell death or apoptosis (Sandvig and van Deurs, 1992; Jones et al., 2000; Suzuki et al., 2000). The toxins induce DNA degradation and release of the cellular content. This process can thereby facilitate proteolytic attack on neighboring cells and contribute to the toxic effect in whole organisms. Several years ago it was shown that 3-methyladenine, an inhibitor of autophagy reduced toxin-induced apoptosis (Sandvig and van Deurs, 1992), and interestingly, even cycloheximide, which by itself blocks protein synthesis, was found to protect against toxin-induced lysis demonstrating that inhibition of protein synthesis in itself is not sufcient to cause the toxin-induced apoptosis (Sandvig and van Deurs, 1992). More recently, it has been shown that apoptosis induced by Stx1 can be associated with enhanced expression of the proapoptotic protein Bax (Jones et al., 2000), and that overexpression of Bcl-2 (see below) protects the cells against lysis. There are, however, differences in the action of Stx1 and Stx2 concerning their action on cells and role in disease. Stx2 seems to be important for STECinduced cell death in the kidneys and the intestine, and a direct interaction between the intracellular protein Bcl-2 and the A subunit of Stx2, but not Stx1, was reported (Suzuki et al., 2000). The protein Bcl-2, which inhibits apoptosis, can be localized to mitochondria, the ER and the nuclear membrane. Interestingly, Stx2, but not Stx1, was reported to be found in the mitochondrial fraction in cells expressing Bcl-2. Although the molecular mechanisms are not known, it was suggested that the interaction between the A subunit and the mitochondrial Bcl-2 mediates caspase 3 activation and Stx2-induced apoptosis (Suzuki et al., 2000). Increased understanding of the activation of apoptosis by toxins and prevention of this process could lead to a better treatment of the infectious disease. 5. Use of Shiga toxins and our knowledge about toxinreceptor interactions in medicine 5.1. Inhibition of Shiga toxin binding to cellsa strategy to prevent cell death Basic knowledge about the toxins and in particular their

interaction with cell surface receptors is now used to develop agents that might block toxin binding to the cell surface and thereby protect people from the toxic action. Recently, Kitov et al. (2000) used the crystal structure of the B subunits of Stx1 in complex with an analog of the carbohydrate part of the receptor to develop a water soluble oligovalent ligand that binds to Stx1 with an extremely high afnity. Cells were protected against both Stx1 and Stx2 by this ligand, and future investigations will show whether this compound will be useful in therapy. Previously, the trisaccharides of the receptor for Stxs have been coupled to the material Chromosorb P and used for therapeutic/diagnostic purposes (Paton and Paton, 1998). Another new approach for therapy was used by Paton et al. (2000) who have produced a bacterium with a Shiga toxin receptor mimic at the cell surface, and again with the same idea, inhibition of binding of Stxs to the cell surface. Studies with mice indicate that the principle works. Different types of vaccination procedures have been suggested (Paton and Paton, 1998), and humanized antibodies have been produced as well (Paton and Paton, 1998). Thus, there are now several promising approaches to the treatment of the toxin-induced effects seen during infection with STEC. 5.2. Targeting of Shiga toxins to specic cell types In the case of other protein toxins belonging to the AB group, a number of these toxins, for instance the plant toxin ricin and the bacterial toxin diphtheria toxin, are being targeted to cells by conjugating the whole toxin, part of the toxin, or a modied toxin, to, for instance, antibodies to cancer cells. To increase the specicity of toxins they can be coupled also to other types of molecules (e.g. to cytokines, peptide hormones or growth factors) that bind to the cell surface. Several encouraging studies have been performed with such constructs (Kreitman, 1999; Frankel et al., 2000). SLTs might also be used for such purposes. Since the Shiga toxin receptor (Gb3) is found on a limited number of cell types, it has also been suggested that this toxin might be used in cancer treatment even without modication (LaCasse et al., 1999). Ex vivo treatment of stem cell grafts from patients with myeloma, lymphoma and breast cancer was suggested (LaCasse et al., 1999). 5.3. Use of toxins as carriers of peptides into cells Other types of toxin conjugates may also turn out to be useful in medicine. Modied toxins are being investigated as tools to promote vaccination against virus and cancer cells (Donnelly et al., 1993; Lee et al., 1998; Noakes et al., 1999; Doling et al., 1999). Several laboratories are now trying to use toxins as vehicle to bring epitopes into the cytosol, epitopes that might then become released and transported into the ER, bound to MHC (major histocompatibility complex) class I and presented at the cell surface. Such experiments have also been shown to work in the

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K. Sandvig / Toxicon 39 (2001) 16291635 suppression of bovine leukemia virus-related spontaneous lymphocyte proliferation. Infect. Immun. 68, 44624469. Frankel, A.E., Kreitman, R.J., Sausville, E.A., 2000. Targeted toxins. Clin Cancer Res. 6, 326334. Fraser, M.E., Chernaia, M.M., Kozlov, Y.V., James, M.N., 1994. Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 A resolution. Nat. Struct. Biol. 1, 5964. Garred, ., van Deurs, B., Sandvig, K., 1995. Furin-induced cleavage and activation of Shiga toxin. J. Biol. Chem. 270, 10817 10821. Ghosh, R.N., Mallet, W.G., Soe, T.T., McGraw, T.E., Maxeld, F.R., 1998. An endocytosed TGN38 chimeric protein is delivered to the TGN after trafcking through the endocytic recycling compartment in CHO cells. J. Cell Biol. 142, 923936. Girod, A., Storrie, B., Simpson, J.C., Johannes, L., Goud, B., Roberts, L.M., Lord, J.M., Nilsson, T., Pepperkok, R., 1999. Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum. Nature Cell Biol. 1, 423430. Goda, Y., Pfeffer, S.R., 1988. Selective recycling of the mannose-6phosphate/IGF-II receptor to the trans-Golgi network in vitro. Cell 55, 309320. Gordon, V.M., Leppla, S.H., 1994. Proteolytic activation of bacterial toxins: Role of bacterial and host cell proteases. Infect. Immun. 62, 333340. Hughes, A.K., Stricklett, P.K., Kohan, D.E., 1998. Shiga toxin-1 regulation of cytokine production by human proximal tubule cells. Kidney Int. 54, 10931106. Hughes, A.K., Stricklett, P.K., Schmid, D., Kohan, D.E., 2000. Cytotoxic effect of Shiga toxin-1 on human glomerular epithelial cells. Kidney Int. 57, 23502359. Jackson, M.E., Simpson, J.C., Girod, A., Pepperkok, R., Roberts, L.M., Lord, J.M., 1999. The KDEL retrieval system is exploited by Pseudomonas exotoxin A, but not by Shiga-like toxin-1, during retrograde transport from the Golgi complex to the endoplasmic reticulum. J. Cell Sci. 112, 467475. Jones, N.L., Islur, A., Haq, R., Mascarenhas, M., Karmali, M.A., Perdue, M.H., Zanke, B.W., Sherman, P.M., 2000. Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family. Am. J. Physiol. Gastrointest. Liver Physiol. 278, G811G819. Kaper, J.B., 1998. Enterohemorrhagic Escherichia coli. Curr. Opin. Microbiol. 1, 103108. Khine, A.A., Firtel, M., Lingwood, C.A., 1998. CD77-dependent retrograde transport of CD19 to the nuclear membrane: functional relationship between CD77 and CD19 during germinal center B-cell apoptosis. J. Cell. Physiol. 176, 281292. Kiarash, A., Boyd, B., Lingwood, C.A., 1994. Glycosphingolipid receptor function is modied by fatty acid content. J. Biol. Chem. 269, 1113811146. Kitov, P.I., Sadowska, J.M., Mulvey, G., Armstrong, G.D., Ling, H., Pannu, N.S., Read, R.J., Bundle, D.R., 2000. Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands. Nature 403, 669672. Kreitman, R.J., 1999. Immunotoxins in cancer therapy. Curr. Opin. Immunol. 11, 570578. LaCasse, E.C., Bray, M.R., Patterson, B., Lim, W.-M., Perampalam, S., Radvanyi, L.G., Keating, A., Stewart, A.K., Buckstein, R., Sandhu, J.S., Miller, N., Banerjee, D., Singh, D., Belch, A.R., Pilarski, L.M., Gariepy, J., 1999. Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence

case of Stx1 where epitopes coupled both to the N- and the C-terminal end can become presented and cause cell killing by cytotoxic lymphocytes (Noakes et al., 1999). Remarkably, even a peptide coupled directly to the B-fragment of Shiga toxin seems to be presented by MHC class I (Lee et al., 1998). It is, however, not known whether this peptide can be transferred from the B-fragment to the MHC class I molecule in the ER, or whether it is released into the cytosol after transfer of the whole construct to this destination. It is, as discussed above (Section 3.4), not clear whether the Bfragment is translocated to the cytosol. 6. Conclusions Detailed knowledge about Shiga toxin and the Shiga-like toxins as well as their interaction with cells has increased our understanding of the role played by these toxins in infectious diseases. It has also provided us with information that has facilitated construction of molecules that may be useful in the treatment of infectious disease. Importantly, studies of the toxin molecules and their intracellular transport have revealed new pathways in cells. In fact, Shiga toxins may not only be a threat to human health, promising results suggest that the unique properties of these molecules can be exploited for different purposes. The toxins are useful as tools in basic cell biology, and can also become important in, for instance, vaccine development.

Acknowledgements The author has been supported by the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, the Novo Nordisk Foundation, the Jahre Foundation, and Jeanette and Sren Bothners legacy.

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