Shigella toxin is a multimeric protein consisting of one A subunit (M,., 32,000) and five B subunits (Mr, 6,500) (1). It is produced by all species of the genus and is believed to play an important role in pathogenesis of clinical shigellosis (2). Shigella toxin is particularly interesting because several different biological effects are mediated by the same highly purified molecule (1, 3). For example, it causes fluid secretion when placed in the lumen of rabbit small intestine (enterotoxicity) (2), but results in delayed limb paralysis and then death if administered parenterally in the same species (neurotoxicity) (4). When directly injected into the vagus nerve trunk of rats, shigella toxin travels in retrograde fashion via the axon to the neuronal cell body, causing dissolution of Nissl substance and death of the neuron (neuronotoxicity) (5). It is also lethal to certain cell culture lines (cytotoxicity) due to inhibition of protein synthesis (6, 7). The latter effect has also been shown in cell-free protein synthesis systems (8). The mechanism appears to be an irreversible and probably catalytic inactivation of the 60 S ribosomal subunit by the toxin A subunit (9).It is not known how shigella toxin produces these various manifestations. It is possible that there are distinctive binding sites on different tissues for the toxin B subunit that result in diverse effects and that the B subunit has multiple binding domains that recognize these distinctive receptors. It is also conceivable that direct binding of the A subunit could cause some of the observed effects. In HeEa cells, the cytotoxic effect of shigella toxin appears to be related to binding of the B subunit to an N-linked glycoprotein on the cell surface that contains oligomeric ~31---~4 linked N-acetyl-D-glucosamine (GIcNAc) I. The evidence for

For over 80 years now, Shigella dysenteriae 1 has been known to produce one of the most potent of the lethal microbial toxins. It was originally called Shiga toxin (after the discoverer of the organism, K. Shiga) and classified as a neurotoxin because it results in a delayed-onset limb paralysis terminating in death when parenterally administered to sensitive animals (reviewed in reference 1). Shigella toxin is also cytotoxic to certain tissue culture cells, as well as enterotoxic (results in fluid secretion) when applied to intestinal mucosa (2-5). Biochemical and immunological evidence indicate that the three biological activities are the properties of the same molecule (3, 5). Its role in the pathogenesis of shigellosis has always been controversial, in part because other species of the genus could not be shown to produce the same toxin. This argument is no longer valid, for both S. flexneri and S. sonnei have been found to produce shigella toxin under appropriate in vitro conditions, and convalescent patients develop an antibody that neutralizes the dysenteriae 1 toxin (6-8). Pathogenic bacteria of other genera have also been found to produce a similar toxin that is neutralized by antibody to shigella toxin. These organisms include a variety ofE. coli serotypes including human enteropathogenic strains, the causative strain of human hemorrhagic colitis (Escherichia coli 0:155), the noninvasive rabbit pathogen RDEC-1, human Salmonella strains, and even Vibrio cholerae (9-11). The cross-reactive toxin has been dubbed "Shiga-like toxin." Since these E. coli strains do not produce the well-known LT or ST toxins and since a mutant strain of V. cholerae deleted of the gene for the production of the ADP-ribosyl transferase enzyme subunit A of cholera toxin (12) causes diarrhea in humans, the shigella (or Shiga-like) toxin may well be a critical virulence factor in diarrheal disease.Because of these observations, there is great interest in this toxin and the immunologically related products of other organisms. Shigeila toxin has recently (9-15) been purified and partially characterized by several laboratories. The

Invest. 1995Invest. . 96:1328Invest. -1335

In the 1980s, Shiga toxin (Stx)-producing Escherichia coli O157:H7 (STEC) was identified as a cause of hemorrhagic colitis in the United States and was found to be associated with hemolytic uremic syndrome (HUS), a microangiopathic hemolytic anemia characterized by thrombocytopenia and renal failure. The precise way that Stxs cause hemorrhagic colitis and HUS is unclear. Stxs have been thought to cause disease by killing or irreversibly harming sensitive cells through a nonspecific blockade of mRNA translation, eventually resulting in cytotoxicity by preventing synthesis of critical molecules needed to maintain cell integrity. Because STEC is noninvasive, we have been exploring the host-toxin response at the level of the gastrointestinal mucosa, where STEC infection begins. We have found that Stx is capable of interleukin-8 (IL-8) superinduction in a human colonic epithelial cell line. Despite a general blockade of mRNA translation, Stx treatment results in increased IL-8 mRNA as well as increased synthesis and secretion of IL-8 protein. Our data suggest that an active Stx A subunit is required for this activity. Ricin, which has the same enzymatic activity and trafficking pathway as Stx, has similar effects. Exploration of the effects of other protein synthesis inhibitors (cycloheximide, anisomycin) suggests a mechanism of gene regulation that is distinct from a general translational blockade. Use of the specific p38/RK inhibitor SB202190 showed that blocking of this pathway results in decreased Stx-mediated IL-8 secretion. Furthermore, Stxs induced mRNA of the primary response gene c-jun, which was subsequently partially blocked by SB202190. These data suggest a novel model of how Stxs contribute to disease, namely that Stxs may alter regulation of host cell processes in sensitive cells via activation of at least one member of the mitogen-activated protein kinase family in the p38/RK cascade and induction of c-jun mRNA. Stx-induced increases in chemokine synthesis from intestinal epithelial cells could be important in augmenting the host mucosal inflammatory response to STEC infection.

Escherichia coli strains producing Shiga toxins (Stx) 1 and 2 colonize the lower gastrointestinal tract in humans and are associated with gastrointestinal and systemic diseases. Stx are detectable in the feces of infected patients, and it is likely that toxin passes from the intestinal tract lumen to underlying tissues. The objective of this study was to develop an in vitro model to study the passage of Stx across intact, polarized cell monolayers. Translocation of biologically active Stx was examined in four cell lines grown on polycarbonate filters. Stx1 translocated across intestinal cell monolayers (CaCo2A and T84 cells) in an energy-requiring and saturable manner, while the monolayers maintained a high level of electrical resistance. Stx1 had no effect on electrical resistance or inulin movement across these cell lines for at least 24 h. Induction of specific Stx receptors with sodium butyrate reduced the proportion of toxin translocated across CaCo2A monolayers but had no major effect on the movement of horseradish peroxidase or [ 3 H]inulin. We have shown that biologically active Stx1 is capable of moving across intact polarized intestinal epithelial cells without apparent cellular disruption, probably via a transcellular pathway. The data also suggest that the presence of Stx receptors on the apical surface of intestinal epithelial cells may offer some protection against the absorption of luminal Stx1.