A. R. MELTON-CELSA, M. J. SMITH, AND A. D. O’BRIEN*
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.
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).
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 Stx1OX3 (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.
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 Stx2OX3a and Stx2O111 identified by Paton’s group (105, 107, 108)], Stx2d-activatable (Stx2dact) [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.
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).
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 Stx2dact are more virulent in a streptomycin-treated mouse model than are strains that produce Stx1 (71, 79, 82). Strains that make Stx2dact 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).
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).
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.
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.
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).
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.
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).
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.
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