Shiga Toxins: Potent Poisons, Pathogenicity Determinants, and Pharmacological Agents

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doi: 10.1128/ecosal.8.7.8

A. R. MELTON-CELSA, M. J. SMITH, AND A. D. O’BRIEN^*

[SECTION EDITOR, A. D. O’BRIEN]

Posted March 29, 2005

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814

*Corresponding author. Phone: 301-295-3419, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

INTRODUCTION AND HISTORY

The Shiga toxins (Stxs), also known as Vero toxins and previously called Shiga-like toxins, are a family of potent protein synthesis inhibitors made by Shigella dysenteriae type 1 and some serogroups of Escherichia coli that cause bloody diarrhea in humans. In 1903 Neisser and Shiga (92) and Conradi (23) independently identified Stx as the poison produced by S. dysenteriae type 1. Then, nearly 80 years later, a toxin produced by some diarrheagenic E. coli strains was described that could be neutralized by Stx antiserum (97); that toxin was subsequently purified (96). Concurrently, a Vero cell toxin was identified from E. coli strains isolated from stools of patients who developed the hemolytic-uremic syndrome (HUS), a rare but serious sequela that may occur after an episode of infectious diarrhea (58). Konowalchuk et al. (65) had previously coined the term Vero toxin to describe a new E. coli cytotoxin isolated from E. coli O26 strains H19 and H30. The subsequent realizations that Vero toxins and the E. coli toxins neutralized by Shiga antitoxin were the same toxins (98) and that both Vero toxins and Shiga toxin from S. dysenteriae type 1 were associated with HUS (57, 58) led to a resurgence of interest in this group of toxins. Although early reports referred to the toxins as Stx, S. dysenteriae 1-like toxin, Shiga-like toxins, or Vero toxins, a new nomenclature groups all these Stx-related toxins together in the same family (16), and we follow that convention.

STX FAMILY

All members of the Stx family share the following characteristics: (i) they are A:B₅ toxins in which the A subunit contains the active component of the toxin and the pentameric B subunit binds to the target cell; (ii) the ∼32-kDa A subunit is an N-glycosidase that inhibits protein synthesis by removing an adenosine residue from the 28S rRNA of the 60S ribosomal subunit; (iii) the B subunit is a pentamer of identical ∼7.5-kDa monomers that binds to the glycolipid globotriaosylceramide (Gb₃) for all but one member of the Stx family [Stx2e binds preferentially to globotetraosylceramide (Gb₄) rather than Gb₃]; (iv) trypsin or furin asymmetrically cleave the A subunit at a protease-sensitive site into an A₁ subunit and an A₂ peptide that are held together via a disulfide bridge; (v) the A₂ peptide links the enzymatic A₁ subunit to the B pentamer and extends through the pore in the B pentamer; (vi) the stx genes are located in an operon with the A subunit gene proximal to the B subunit gene; (vi) most genes in the stx family are located on a phage or defective phage related to lambda, although a few of the E. coli toxin genes appear to be chromosomally situated (Table 1). The Stx family is further broken down into two groups, Stx/Stx1 and Stx2, which share about 55 to 60% amino acid sequence homology, but antisera raised against one group do not neutralize the toxicity of the members of the other group. The Stx/Stx1 group is also distinguishable from the Stx2 group in that Stx/Stx1 expression is repressed in the presence of iron (17, 27, 152).

Table 1Stx types, distinguishing feature compared with prototype, and location of toxin-encoding operon.

Stx/Stx1 Group

The Stx/Stx1 group consists of a fairly homogeneous group in which the prototype toxin is Stx from S. dysenteriae serogroup 1. The only other Shigella reported to produce Stx was one patient isolate of S. sonnei that contains an stx-encoding phage (134). Some strains of E. coli produce the same toxin, Stx1, which differs at most by a single amino acid from Stx. There are reports of variants of Stx/Stx1 of human or sheep origin that have subtle changes in DNA sequence (104, 106). However, few of the reports have demonstrated that those sequence variations result in a toxin that is distinguishable by immunological or biological means, which is the criterion for naming a new Stx toxin family variant, as was discussed originally at a World Health Organization meeting in Geissen, Germany, in 1991 and reiterated in a letter to the ASM News (16). However, there is sufficient evidence to add two Stx1 variants, Stx1c and Stx1d, to the Stx/Stx1 group. Stx1c is a variant of Stx first identified in sheep and originally called Stx1_OX3 (104) but subsequently found in human isolates (63, 164). Stx1c is immunologically distinguishable from Stx1 in an agglutination assay (164). Two different groups reported another Stx1 variant (Stx1d and Stx1v52), but sequence analysis shows that the two toxins are the same; so, by convention the toxin should be called Stx1d (14, 101). Y. Takeda’s group showed that Stx1d is immunologically and biologically distinct from Stx1 (101). Additionally, they showed that Stx1d is less toxic than Stx1 for Vero cells and mice (101), an observation that may explain why strains that produce Stx1d alone have not been found in patient isolates. Table 1 lists the members of the Stx/Stx1 group and the characteristics that distinguish them from each other. Figures 1 and 2 show a comparison of the amino acid sequences of the A and B subunits, respectively, of the Stx/Stx1 family.

Fig. 1Comparison of the deduced amino acid sequences of the A subunits from Stx1, Stx1c, and Stx1d. The underlined regions indicate the processed signal sequence, dots depict identical residues, and green or red letters highlight conserved or nonconserved residues, respectively. The GenBank accession numbers are as follows: Stx1, M19473; Stx1c, AJ312232; Stx1d, AY170851. The sequences were aligned using the Align Plus 5 program, version 5.03 (Scientific & Educational Software, Durham, N.C.) following the global-ref alignment procedure (88) and the scoring matrix BLOSUM 62 (43).

Fig. 2Comparison of the deduced amino acid sequences of the B subunits from Stx1, Stx1c, and Stx1d. The underlined regions indicate the processed signal sequence, dots depict identical residues, and green or red letters highlight conserved or nonconserved residues, respectively. The GenBank accession numbers are listed in Fig. 1. The sequences were aligned as for the Stx1 A subunit sequences in Fig. 1.

Stx2 Group

The Stx2 group is distinguished from Stx/Stx1 by immunological means. Antisera to Stx/Stx1 do not neutralize the toxic activity of Stx2 for Vero cells and vice versa (136). Certain monoclonal antibodies also differentiate Stx/Stx1 from Stx2 (109, 135). The Stx2 group contains several variants that are well established, Stx2c (73, 124), Stx2d (111) [Stx2d is quite similar to the Stx2_OX3a and Stx2_O111 identified by Paton’s group (105, 107, 108)], Stx2d-activatable (Stx2d_act) [formerly known as SltIIvha or SltIIvhb (52, 79, 80)], and Stx2e (24, 117). Two new Stx2 variants have been proposed, Stx2f (123) [Stx2f is similar to Stx2va (38)] and Stx2g (68). The methods that distinguish the Stx2 variants are outlined in Table 1. Figures 3 and 4 show a comparison of the amino acid sequences of the A and B subunits, respectively, of the Stx2 family. Because there are many groups that study E. coli strains that carry Stxs, several Stx2-type genes have been sequenced and found to vary in a few nucleotides and thus in a few amino acids from the prototypic Stx2. However, such Stx2 toxins should not be named as variants of Stx2 until they are demonstrated to exhibit immunological or biological differences from the prototype toxin.

Fig. 3Comparison of the deduced amino acid sequences of the A subunits from Stx2, Stx2c, Stx2d, Stx2dact, Stx2e, and Stx2f. The underlined regions indicate the processed signal sequence, dots depict identical residues, green or red letters highlight conserved or nonconserved residues, respectively, and a red dash indicates a gap in the sequence. The GenBank accession numbers are as follows: Stx2, X07865; Stx2c, M59432; Stx2d, AF043627; Stx2dact, AF479829 (modified, February 2004); Stx2e, M21534; Stx2f, EAJ10730. The sequences were aligned as for Stx1 sequences in Fig. 1.

Fig. 4Comparison of the deduced amino acid sequences of the B subunits from Stx2, Stx2c, Stx2d, Stx2dact, Stx2e, and Stx2f. The underlined regions indicate the processed signal sequence, dots depict identical residues, green or red letters highlight conserved or nonconserved residues, respectively, and a red dash indicates a gap in the sequence. The GenBank accession numbers are listed in Fig. 3. The sequences were aligned as for Stx1 sequences in Fig. 1.

STXS AS VIRULENCE FACTORS

Evidence That Stxs Act as Virulence Factors

The evidence that Stxs act as virulence factors is fivefold. First, disease from S. dysenteriae type 1 is usually more severe than disease from shigellas that do not produce Stx (1). Second, epidemiological evidence connects the development of HUS and infection with Stx-producing bacteria (1, 40, 57). Third, an stx mutant of S. dysenteriae 1 caused less severe disease in rhesus macaque monkeys than the wild-type parental S. dysenteriae 1 strain (33). Fourth, Levine et al. fed wild-type S. dysenteriae type 1, or noninvasive but toxigenic mutants, or invasive but nontoxigenic mutants of S. dysenteriae type 1 to human volunteers and found that although the organisms had to be invasive to cause disease, the disease course was more severe in volunteers that had received the invasive and toxigenic strain (69). (These studies were performed before the realization that infection with S. dysenteriae type 1 might lead to the HUS.) Fifth, baboons and greyhounds develop HUS or a HUS-like disease when injected directly with Stx1 or Stx2 (31, 129, 140).

Disease Produced by Stx-Producing Organisms

The diseases caused by S. dysenteriae type 1 and Stx-producing E. coli (STEC) have similar attributes and some differences. First, both intestinal pathogens have a low infectious dose (69, 146). Second, both types of Stx-producing organisms cause a bloody diarrhea, although the diarrhea usually also contains mucus in S. dysenteriae type 1-mediated disease. Additionally, perhaps because S. dysenteriae type 1, unlike STEC, invades the colonic epithelium, both fever and cramps are more common in patients infected with S. dysenteriae type 1 than in STEC-infected patients. Finally, infection with Stx-producing organisms leads to the sequela HUS in a certain proportion of patients. The HUS is a serious complication defined by hemolytic anemia, thrombocytopenia, and kidney dysfunction (55). The HUS appears to be mediated by both direct and indirect (through cytokine induction) effects of the toxin [see review (113)].

Relative Toxicity of the Stxs

There are epidemiological data to support the idea that STEC strains that produce Stx2 may be associated with more severe disease than STEC that make Stx1 or both Stx1 and Stx2 (12, 54, 103, 126). Additionally, six types of experimental data also support the hypothesis that Stx2 is more pathogenic than Stx1: (i) Stx2 has a lower LD₅₀ than Stx1 for mice (144); (ii) Stx2 is more toxic in vitro for human renal microvascular endothelial cells, the specific cell type damaged in patients with HUS (76, 114, 144); (iii) pediatric biopsy samples placed in organ culture extrude cells in response to Stx2 but not Stx1 (125); (iv) significantly lower doses of Stx2 than Stx1 cause HUS in the baboon model (129); (v) Stx2 elicits a greater cytokine response than Stx1 from human umbilical vein endothelial cells (78); and (vi) certain E. coli strains that produce Stx2, Stx2c, or Stx2d_act are more virulent in a streptomycin-treated mouse model than are strains that produce Stx1 (71, 79, 82). Strains that make Stx2d_act are the most virulent in the streptomycin-treated mouse model (79). The reason for the relatively higher toxicity of Stx2 as compared with Stx1 may be twofold. First, Stx2 is more stable at acid pH than Stx1 (144), and, second, although Stx2 binds to Gb₃ more slowly than Stx1, its rate of dissociation from Gb₃ is much lower than that of Stx1 (89).

REGULATION OF TOXIN EXPRESSION AND RELEASE

High levels of iron repress expression of Stx and Stx1 at the transcriptional level (17, 159), but no other environmental regulations for any other Stx family members have been described aside from those agents that act as phage-inducers (85, 138, 155, 160, 165). Although it has been known since the 1980s that many of the genes for the stx family reside on lambda-like phages (46, 99, 131), it was not until the sequence of the genome of the prototype Stx1- and Stx2-producer EDL933 (110) became available that the issue of the regulation of stx genes in the context of the host phage was revisited. The renewed interest in the location on the phage genome of the stx genes led to the discovery that stx expression is coregulated with expression of phage lytic genes (91, 157). Furthermore, phage lysis of the bacteria that produce Stx1 increases the quantity of Stx1 released from the bacteria (91, 156).

Several other findings that relate to the localization of stx genes on bacteriophages are notable. First, E. coli strains may be lysogenized with two different stx-encoding phages, and in fact, up to three toxin genes have been found in a single E. coli isolate (36, 71). Second, the phage that carries the stx genes can be both induced and transduced experimentally to another bacterium in the intestines of mice (3, 165). Third, the presence of such phage-susceptible bacteria in a collection of human fecal E. coli isolates suggests that such a mechanism of toxin amplification may be possible in some people who become infected with strains lysogenic for one or more stx-converting phage (37).

TOXIN STRUCTURE

A handful of methods exist for toxin purification (reviewed in reference 100), and the toxins can now even be purchased commercially. However, the Stxs are now classified as select agents, and specific rules govern the distribution of both the toxin and clones of the toxin (20). Stx was crystallized and its structure resolved to 2.5 Å in 1994 by M. James’ group at the University of Alberta (34). The same group solved the Stx2 structure in 2004 (35). A "side view" of the Stx and Stx2 holotoxins shows the A subunit positioned asymmetrically and well above the B pentamer (Fig. 5). The A₂ peptide, which consists of approximately one-sixth of the C terminus of the A subunit, links the majority of the A₁ subunit to the B pentamer. The A₁ and A₂ segments of the A subunit are joined by a disulfide bond, and the loop created between the linked cysteines contains a furin cleavage site that, when cleaved, allows separation of the B pentamer/A₂ peptide complex from the A₁ toxic component. A study with purified toxin subunits confirmed the importance of the A₂ peptide in maintaining holotoxin integrity. That study demonstrated that purified Stx1 A subunit but not purified A₁ could recombine with purified B subunits to form a cytotoxic molecule (7). The crystal structures of Stx showed that a portion of the loop created by the disulfide bridge that links A₁ and A₂ blocks the active site of Stx (34). In contrast, the active site of Stx2 is not blocked by the A₂ peptide (35). The James group identified three other differences between the structures of Stx and Stx2 besides the apparent accessibility of the toxin active site (35). First, the C terminus of the A₂ from Stx2 forms a more ordered structure than the C-terminal tail of the Stx A₂. Second, the C terminus of the Stx2 A₂ is not only more ordered than the end of the Stx A₂ tail, but it also binds at one of the receptor-binding sites in the B subunit, while the end of the Stx A₂ does not. Third, the type 2 Gb₃ binding site in the Stx2 B pentamer assumes a different conformation than that of Stx. Finally, the Stx and Stx2 holotoxin crystal structures confirmed previous findings that the crystallized Stx B pentamer has structural homology with the B pentamer of heat-labile enterotoxin from E. coli (130, 133). A recent study suggests that the stability of the Stx B pentamer relies on the energetically favorable interaction among the noncovalently linked monomers as they fold together and bury the polar side chains of formerly surface-exposed amino acids (112). Furthermore, that study found no evidence of any other forms of the B subunit apart from monomers or pentamers.

Fig. 5Ribbon diagrams of the crystal structures of Stx (left) and Stx2 (right). The A subunits are in red, and the disulfide bridge is shown in yellow. The B subunits are depicted in orange, cyan, green, yellow, and blue. The figure was drawn with the programs MOLSCRIPT (66) and RASTER3D (83). The Protein Data Bank accession numbers are 1R4Q for Stx and 1R4P for Stx2.

TOXIN MOVEMENT FROM THE BACTERIA INTO THE HOST TO THE TARGET CELL

Initial Toxin Movement from the Gut into the Host

Toxin delivery into the host in S. dysenteriae type 1 is most likely aided by the invasiveness of that organism. For the noninvasive STEC, the mechanism whereby the toxin elaborated in the gut traverses the intestinal epithelium, a cell layer not thought to express Gb₃, to attack endothelial cells that express Gb₃ is not well understood. However, Stx1 and Stx2 cross-polarized epithelial cell monolayers probably via both paracellular and transcellular processes to the basolateral surface in vitro, a process enhanced by the concurrent migration of neutrophils in the opposite direction (2, 47, 48). Acheson et al. (2) further found a reduction in the quantity of Stx1 that translocated when the quantity of Gb₃ expressed on the cells was increased by butyric acid treatment, a finding which suggests that the lack of receptors on intestinal cells may actually increase the capacity of the toxin to be delivered from the gut into the host. Figures 6 and 7 depict the pathway that the toxin may take from the bacterium across the intestinal barrier. Once the toxin reaches the bloodstream, it presumably can travel to distant sites that contain Gb₃. The identity of the cell types or blood components that might bind the toxin once it crosses the intestinal barrier and reaches the bloodstream remains a subject of some controversy. Indeed, Stx family members have been shown to affix to lymphocytes, platelets, and red blood cells that have the P1 antigen (the P1 antigen contains Galα1-4Gal, which is the component of Gb₃ to which the Stx B pentamer binds) (11, 39, 54, 59, 139, 141, 142). Direct evidence that Stx travels systemically was found postmortem in a child who died of HUS (150). The distal tubules in the kidneys of that child demonstrated evidence of both Stx1 and Stx2 by immunohistochemical analysis (150).

Fig. 6The flow of Stx from the bacterium across the intestinal barrier. An STEC strain is depicted making phage particles and Stx at the same time. The toxin (shown as a red triangle [A subunit] with a purple star [B subunit]) emerges from the bacterium at the same time as the stx-converting phage (green).

Fig. 7This cartoon shows how Stx may cross the intestinal brush border (gold cells) via a transcellular (blue arrow) process (2, 47), and a paracellular process (black arrow) enhanced by the migration of neutrophils in the opposite direction (48) (not to scale).

Gb₃ Is the Functional Receptor for the Stxs

The receptor for Stx was identified in the mid 1980s as the terminal galactose sugars [Gal(α1-4)Gal] on Gb₃ (53, 70). Although cell surface expression of Gb₃ is required for the cytotoxicity of Stxs, it is not sufficient. In addition to the terminal Gal(α1-4)Gal disaccharide requirement, the fatty acid chain length of Gb₃ also plays a role in cell sensitivity to Stxs (73, 119). The Gb₃ may need to be localized to lipid rafts, although the interpretation of the findings from these studies is confounded by the fact that only the Stx B subunit and not the holotoxin was used in the experiments (60, 93). However, even with the caveat that only the trafficking of the B subunit has been studied in relation to the role of lipid rafts in toxin uptake, the B subunit alone does move intracellularly in a retrograde manner similar to the holotoxin (121). Furthermore, in two cell types that have Gb₃ but are not sensitive to the toxin, monocytes and macrophages, the B subunit did not associate with lipid rafts (30). There appear to be four mechanisms by which cells exhibit resistance to Stxs: cells that have Gb₃ but route the toxin to a cellular compartment other than endoplasmic reticulum (4, 30, 44, 121); cells that lack Gb₃ (154); cells such as polymorphonuclear leukocytes that lack Gb₃ but have the capacity to bind but not internalize toxin (142); and those that lack Gb₃, such as intestinal cells, but have the capacity to internalize and translocate the toxin but somehow still remain resistant (2, 125). Intriguingly, as described above, the latter two mechanisms of cellular resistance to Stx cytotoxicity contribute to the spread of Stx within the host.

Gb₃-Binding Sites on Stx

Several studies address the question of the number of Gb₃-binding sites on Stx (8, 72, 95, 128, 132). These latter studies, taken together, indicate that up to three Gb₃-binding sites exist on each B subunit monomer, although they presumably cannot be occupied concurrently. Two of the Gb₃-binding sites appear to be high-affinity sites, while the third site has a lower Gb₃ affinity (128). All the Gb₃-binding sites occur on the same plane of the pentamer, on the side distal to the A subunit (see Fig. 5).

Intracellular Movement of the Toxin

Once Stx binds to Gb₃, the toxin/receptor complex travels from clathrin-coated pits to the Golgi and then to the endoplasmic reticulum in a retrograde manner by mechanisms that are not fully understood (118, 120) (Fig. 8). That the Stx/Gb₃ complex can move into the cell via clathrin-independent mechanisms has also been reported, but whether this mode of entry leads to cytotoxicity is unclear (61, 93, 122). Once the toxin/receptor complex moves into the cell, the A subunit is probably nicked at its protease-sensitive site by furin in the endosome or Golgi apparatus, if it has not already been nicked by the enzymes found in the intestine. From the endoplasmic reticulum, the A₁ subunit must reach the cytosol where the target ribosomes reside. The Stx A₁ may be unfolded in the endoplasmic reticulum by the enzyme protein disulfide isomerase in preparation for entry into the cytoplasm, as appears to be the case for cholera toxin (67, 148). (Cholera toxin is an A:B₅ toxin that, like Stx, has A₁ and A₂ segments and whose target also exists in the cytosol.) Finally, the movement of Stx from the endoplasmic reticulum to the cytosol, by analogy with ricin, Pseudomonas exotoxin A, and cholera toxin, may be Sec61p dependent (67, 120).

Fig. 8Stx travels though susceptible, Gb₃-positive cells in a retrograde manner. In step 1, the toxin binds to Gb₃, during step 2 the toxin/Gb₃ complex enters the cell through clathrin-coated pits and merges with the Golgi (step 3), and finally, in step 4, just the A subunit launches into the cytoplasm to attack the ribosome. The cell nucleus is shown as a solid black circle (not to scale).

TOXIN MODE OF ACTION

Once it reaches its target ribosome, the A₁ fragment attacks the 28S rRNA, liberating an adenosine residue from a loop known as the S/R loop because it was originally identified as the target of the plant toxins sarcin and ricin. In fact, Stx and ricin cleave the exact same residue from the S/R loop in the ribosome (29). Since Stx, ricin, and some other ribosome-inactivating proteins share the same mode of action, the amino acids in the Stx active site could be predicted and were subsequently confirmed through genetic studies (reviewed in reference 81). The critical active-site residue is the glutamic acid at position 167 of the mature protein (45). However, several other residues play an important role in the full toxicity of Stx, and these include residues Y77, Y114, R170, and W203 (W202 in Stx2) (25, 26, 163).

Although the Stxs are made and produced by bacteria, they do not appear to act against either their host organism or other bacteria under normal circumstances, most likely because the A subunit is secreted from the cytoplasm as soon as it is synthesized and because the holotoxin cannot enter intact bacterial cells. However, the A₁ subunit is as active against bacterial ribosomes as eukaryotic ribosomes in vitro and expression of the Stx A₁ subunit in the absence of A₂ and B from a plasmid was shown to be deleterious to the growth of laboratory E. coli (137).

Many residues in the B subunit are implicated as important for Vero cell cytotoxicity (reviewed in reference 81). The reasons for the impact of B subunit changes on toxicity are probably multifold. First, there are only a limited number of B subunit residues but a relatively large number of Gb₃-binding sites found in the pentamer. Second, at least one residue appears to be involved in the Vero cell cytotoxicity of Stx but not required for binding to Gb₃ (61). And, finally, certain B subunit residues would be required for monomer:monomer association.

Additional Effects of Stxs

Numerous biological activities of the Stxs on intact cells or in whole animals have been reported, including activation or inhibition of apoptosis (reviewed in reference 21), and induction of various cytokines (reviewed in references 113 and 143). One of the most intensively studied properties of Stx in recent times is the capacity of the toxin to induce apoptosis in some cell types, an effect that could result in cellular damage and lesions (21). In contrast, Stx inhibits apoptosis in neutrophils, an effect that may increase the lifespan of those cells and lead to higher levels of inflammation and tissue damage (74). The issue of which cytokines play a role in HUS is unresolved, perhaps because of host variation in cytokine response (113). However, in experimental models, Stx induces the elaboration of tumor necrosis factor (TNF) and interleukin-1 (IL-1) from human macrophages and monocytes (115, 143, 153), and both TNFα and IL-1 enhance the cytotoxicity of Stx toward endothelial cells (75, 145).

POTENTIAL PHARMACOLOGICAL USES OF STXS

Because the expression of Gb₃ is increased in a variety of human tumors, the use of Stx as a treatment against such malignancies is being investigated in vitro and in animal models (5, 42, 51, 116). Successful elimination of some solid tumor grafts in mice has been reported (5, 51). Long before the use of Stx or a modified Stx could be contemplated as a treatment in a patient, the question of whether the intact toxin could conceivably be used in humans, even at sublethal doses, will need to be addressed because of the risk for the HUS.

Other reports that support the idea that Stx could be used as a treatment are from Hovde and colleagues. These investigators found that Stx1 and Stx2 exhibit antiviral activity against peripheral blood mononuclear cells from cattle that are naturally infected with bovine leukemia virus (9, 32). The authors of these reports speculate that transient infection with STEC may actually play a role in limiting the bovine leukemia virus to a "chronic well tolerated disease" (9). However, it should be noted that cattle, unlike humans, do not usually become ill when infected with STEC.

THERAPIES AND VACCINES THAT SPECIFICALLY TARGET STXS

The effectiveness of antibiotic therapy in patients infected with STEC such as O157:H7 as well as the potential risks of such treatment are areas of controversy. Several studies indicate that the course of the diarrhea stage of the disease is unaltered by antibiotic treatment (10, 22, 102). However, of more concern are reports that growing a STEC strain in the presence of certain antibiotics increases the amount of toxin produced by those isolates (56, 77). Additionally, the use of antibiotics in some animal models results in increased pathogenicity by STEC in those animals (reviewed in reference 84). Furthermore, some studies indicate that the HUS rates are higher in patients treated with antibiotics. For example, one prospective study in the United States found a greatly increased risk of HUS (relative risk = 17.3) in children who were treated with antibiotics versus those who did not receive antibiotics (161). However, one retrospective study conducted during the huge 1996 outbreak in Japan indicated that early administration of the antibiotic fosfomycin (which was not used in the U.S. study) resulted in a decreased risk for the HUS (49, 161). Because of the difficulty in treating STEC infections, both vaccines and toxin neutralization approaches have been pursued as alternative therapies or preventative measures.

Several groups anticipate that a therapy that targets the Stxs is an important component of trying to alleviate disease caused by Stx-producing bacteria. Two classes of therapies that target Stxs are being tested: Gb₃ mimics and toxin antibodies. The original receptor mimic, Synsorb-Pk, binds and neutralizes the Stxs in vitro (6) but failed to lessen the severity of the HUS in children in a clinical trial (147). Second generation receptor mimics such as STARFISH (62) and SUPER TWIG (94) bind the Stxs in vitro and in vivo and, unlike Synsorb-Pk (J. Rogers and A. D. O’Brien, unpublished observation), are protective in mouse models (87, 158). Whether the new generation of receptor mimics will perform better than Synsorb-Pk in clinical trials remains to be determined. Three groups have published findings that relate to the use of monoclonal antibodies as passive immunization agents against the Stxs to protect mice from the lethal infection with STEC (28, 86, 127, 162). These passive immunization treatments show promise not only because they can be administered after infection in mice and still demonstrate protection, but also because they can be administered systemically rather than orally like the receptor mimics (28, 127, 162). In addition to these passive immunization approaches, several active immunization strategies that have been pursued with candidate vaccines mostly against either the Stx/Stx1 or Stx2 group make use of mutant toxins, B subunit alone, toxin-specific peptides, Stx-liposome conjugates, or DNA (13, 15, 18, 41, 50, 64, 151). A few of the vaccines have demonstrated protective efficacy in mice (18, 50, 151).

SUMMARY

The Stx family is one of the most potent groups of bacterial toxins known, second only to botulinum toxins (see appendix C in reference 149). The Stxs act as virulence factors for both S. dysenteriae and E. coli and contribute to the disease process initiated by those organisms both directly and indirectly. The serious nature of the disease produced by STEC is further evidenced by the fact that STEC strain O157:H7 was declared a category B biological threat for civilians by the Centers for Disease Control and Prevention (19, 90). Furthermore, the toxin and toxin clones are considered select agents by the Department of Health and Human Services and, as such, the use and distribution of the toxin and toxin clones is governed by the rules developed by that department (20). Additionally, because the Stxs are such potent toxins, attempts to exploit them as cancer chemotherapy agents are underway. Finally, the study of the Stxs have provided insight into the nature of the HUS, the way bacterial virulence factors may be encoded and regulated by phages, and how retrograde transport of molecules occurs. As we continue to study these intriguing toxins in our attempts to eliminate the diseases caused by S. dysenteriae and STEC, we may well discover additional novel facets to the Stxs.

ACKNOWLEDGMENTS

We thank Marie Frasier for providing us with the crystal structure figures of Stx1 and Stx2.

This work was partially supported by Grants H073KD from the Uniformed Services University of the Health Sciences and AI20148-22 from the National Institutes of Health to A. D. O’Brien.

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