No abstract

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

We have determined the nucleotide sequence of the sitA and slB genes that encode the Shiga-like toxin (SLT) produced by Escherichia coli phage H19B. The amino acid composition of the A and B subunits of SLT is very similar to that previously established for Shiga toxin from Shigell4 dysenteriae 1, and the deduced amino acid sequence of the B subunit of SLT is identical with that reported for the B subunit of Shiga toxin. The genes for the A and B subunits of SLT apparently constitute an operon, with only 12 nucleotides separating the coding regions. There is a 21-base-pair region of dyad symmetry overlapping the proposed promoter of the sit operon that may be involved in regulation of SLT production by iron. The peptide sequence of the A subunit of SLT is homologous to the A subunit of the plant toxin ricin, providing evidence for the hypothesis that certain prokaryotic toxins may be evolutionarily related to eukaryotic enzymes.

Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC),

Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) can lead to hemolytic-uremic syndrome (HUS) in 5 to 10% of patients. Stx2, one of two toxins liberated by the bacterium, is directly linked with HUS. We have previously shown that Stx-specific human monoclonal antibodies protect STEC-infected animals from fatal systemic complications. The present study defines the protective antibody dose in relation to the time of treatment after the onset of diarrhea in infected gnotobiotic piglets. Using the mouse toxicity model, we selected 5C12, an antibody specific for the A subunit, as the most effective Stx2 antibody for further characterization in the piglet model in which piglets developed diarrhea 16 to 40 h after bacterial challenge, followed by fatal neurological symptoms at 48 to 96 h. Seven groups of piglets received doses of 5C12 ranging from 6.0 mg/kg to 0.05 mg/kg of body weight, administered parenterally 48 h after bacterial challenge. The minimum fully protective antibody dose was 0.4 mg/kg, and the corresponding serum antibody concentration in these piglets was 0.7 g (؎0.5)/ml, measured 7 to 14 days after administration. Of 40 infected animals which received Stx2 antibody treatment of >0.4 mg/kg, 34 (85%) survived, while only 1 (2.5%) of 39 placebo-treated animals survived. We conclude that the administration of the Stx2-specific antibody was protective against fatal systemic complications even when it was administered well after the onset of diarrhea. These findings suggest that children treated with this antibody, even after the onset of bloody diarrhea, may be equally protected against the risk of developing HUS.

In order to investigate the ability of enteropathogenic Escherichia coli (EPEC) to invade epithelial cells, 24 strains of diarrhea-causing E. coli were studied with a HEp-2 cell-gentamicin invasion assay. Invasive ability was expressed as the percentage of the inoculum surviving gentamicin after incubation of bacteria with HEp-2 cells. Geometric mean survival of EPEC strains possessing the EPEC adherence factor (EAF+ EPEC) was 5.177%, which was significantly greater than survival of enteroinvasive E. coli (EIEC) strains (1.871%). EPEC strains lacking EAF (EAF-EPEC), enterotoxigenic E. coli (ETEC), and enterohemorrhagic E. coli (EHEC) were significantly less invasive (geometric mean survival, 0.032%, 0.013%, and 0.009%, respectively). The variation in bacterial recovery was not due to differences in the number of HEp-2 cells remaining attached to the plates, as measured by the retention of crystal violet stain in parallel assays. Transmission electron microscopy confirmed the presence of many intracellular EAF+ EPEC and EIEC, whereas EAF- EPEC, EHEC, and ETEC were found primarily outside the cells. Epithelial cell invasion is an overlooked property of EAF+ EPEC of potential relevance in disease pathogenesis.

Infection with Escherichia coli O157:H7 can lead to hemolytic uremic syndrome (HUS) in some children. Epidemiologic data suggest that Shiga toxin (Stx) 2-producing strains are more frequently associated with HUS than are Stx1-producing strains. Less clear is whether strains that express Stx2 alone are more frequently associated with HUS than strains that express Stx1 and Stx2. Isogenic mutants 933stx1- and 933stx2- were produced from strain 933 (Stx1 and Stx2 producer), and 86-24stx2- was produced from strain 86-24 (Stx2 producer). Neurologic lesions or symptoms developed in 18 (90%) of 20 gnotobiotic piglets orally infected with strain 86-24, in 15 (85%) of 18 infected with mutant 933stx1-, in 9 (31%) of 29 infected with strain 933, in 0 of 5 infected with mutant 86-24stx2-, and in 0 of 6 infected with mutant 933stx2-. It was concluded that strains expressing Stx2 alone are more neurotropic for piglets when fed orally than are those strains expressing Stx1 and 2, whereas Stx1-producing strains induce only diarrhea. It is also conceivable that strains that produce Stx2 may constitute a significant predictive risk factor for HUS in humans.