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Shigella toxin reproduces the major manifestations of shigellosis in ligated intestinal loops from adult rabbits and binds to a microvillus membrane (MVM) glycolipid receptor, globotriaosylceramide (Gb3). Because neonatal human shigellosis is uncommon, we used the animal model for obtaining MVMs from rabbits of different ages to determine the presence of toxin receptors and Gb3 and to measure the fluid secretory response to toxin in ligated ileal loops. A single class of MVM receptors for 125I-labeled shigella toxin, first detected at 20 d of age, reached adult levels by 24 d (n = 1.7-23.8 X 10(10)/micrograms of protein; K = 1.1-3.8 X 10(9) M-1). Binding was specific for toxin subunit B. A toxin binding MVM glycolipid, identified as Gb3, was detected in animals greater than or equal to 16 d of age by high-performance thin-layer chromatography and autoradiography. Fluid secretion in response to shigella toxin in ligated small bowel loops occurred in temporal relation to the appearance of Gb3, a result thus indicating the involvement of Gb3 in mediating the toxin effects.

Binding kinetics of Shiga toxin to HeLa CCL-2 cells and to cell lines cloned by limiting dilutions were determined. Lines with a wide range of sensitivity to Shiga toxin were obtained. Binding data, analyzed by a computer-based Scatchard model program, revealed two classes of binding sites, one of low affinity and high capacity and one of high affinity and low capacity. The number of high affinity, but not low affinity, sites present on the clones correlated with their sensitivity to toxin. Tunicamycin-treated CCL-2 cells became resistant to Shiga toxin in parallel with a reduction in the capacity of the high-affinity site. Cell content of Gb3, the glycolipid receptor for Shiga toxin, decreased as the sensitivity of the cells diminished. These data show that a minority of Shiga toxin binding sites of HeLa cells are involved in the cytotoxic response and suggest that Gb3 is the high-affinity functional cytotoxin receptor.

Two strains of Shigella flexneri and one of Shigella sonnei were studied for toxin production in vitro. All of the three strains produced a cell-free cytotoxin that showed marked similarity to that produced by Shigella dysenteriae 1. Each toxin eluted in two distinct peaks on chromatography with Sephadex G-150, was destroyed by heating at 90 C for 30 min, and was neutralized by S. dysenteriae 1 antitoxin. Patients with infections due to S. flexneri and S. sonnei developed antibody that neutralized S. dysenteriae 1 toxin in vitro. In three of seven positive sera studied by sucrose density-gradient ultracentrifugation, antibody activity was associated only with the IgM fraction. The time course of the antibody response resembled that found in infections due to S. dysenteriae 1, in which an IgM antitoxin antibody has also been described. Since three species of Shigella have now been shown to be toxigenic, it is possible that bacterial toxin may play a role, along with bacterial invasion, in the pathogenesis of infections due to S. flexneri and S. sonnei, as well as those due to S. dysenteriae 1.

Pathogenic mechanisms in infectious diseases often involve specific receptor-ligand interactions of cells and soluble molecules. To further elucidate structure-function relations for shigella toxin receptors, we studied binding of purified 125I-labeled toxin and biologic response under various conditions in an experimental model using HeLa cells. Response to toxin was reversibly inhibited by treatment of cells with trypsin or tunicamycin, an inhibitor of glycoprotein synthesis that also significantly inhibited toxin binding, a result indicating that the receptor is an N-linked glycoprotein. Removal of terminal beta-linked galactose from the HeLa cell surface with beta-galactosidase increased toxin binding and activity, and it also potentiated the effects of lysozyme and wheat-germ agglutinin, which recognize oligomeric beta 1----4-linked N-acetyl-D-glucosamine and inhibit toxin activity as well. Incubation of cells with beta-N-acetylglucosaminidase, which cleaves terminal beta-linked N-acetyl-D-glucosamine, inhibited toxin activity. Effects of beta-galactosidase were reversed by readdition of galactose to cell-surface oligosaccharide acceptors. The data demonstrate that alterations of a single sugar on cell-surface glycoproteins may have a dramatic effect on receptor activity and indicate that shigella toxin is a sugar-binding protein with specificity for beta 1----4-linked N-acetyl-D-glucosamine.

Shigella toxin interacts with HeLa cells by binding to a glycoprotein cell surface receptor and, after a lag period, inactivates the 60S ribosomal subunit and inhibits protein synthesis. The lag period may be due to energy-dependent translocation of the toxin to the interior of the cells. This step was investigated with agents known to inhibit glucose metabolism, mitochondrial energy production, or macromolecular synthesis and with drugs that alter the cytoskeletal system or lysosomes. Metabolic inhibitors reduced both cytotoxicity and binding of the toxin. All agents tested except actinomycin D also diminished endocytosis. Effects on cytotoxicity were partially reversed in the presence of the membrane permeabilizer dimethylsulfoxide. Various cytochalasins, colchicine, vinca alkaloids, chloroquine, and steroids also reduced the activity of the toxin. These diverse agents are probably acting on a common pathway affecting the internalization and/or intracellular processing of shigella toxin. Translocation of this toxin apparently occurs by endocytosis from the cell surface.

The biological activity of the enterotoxin of Shigella dysenteriae 1 was compared with that of a well-studied 20-year-old partially purified preparation of neurotoxin from the same organism. Enterotoxicity, neurotoxicity, and cytotoxicity were present to an equivalent extent in both preparations. Human convalescent antisera and experimental rabbit antisera had equal toxin-neutralizing antibody to the cytotoxic activity in these toxin preparations. Multiple protein bands were present in each toxin studied. Two separate HeLa cell fractions could be obtained by Sephadex gel filtration chromatography, isoelectric focusing in a sucrose gradient, and polyacrylamide gel electrophoresis. Only one of these fractions (isoelectric at pH 7.2) was associated with enterotoxicity and neurotoxicity. The second smaller-molecular-weight fraction, which was isoelectric at pH 6.1, possessed only cytotoxic activity. These data suggest that Shiga enterotoxin and neurotoxin are closely related proteins and, indeed, may be identical. The nature of the cytotoxin with pH 6.1 is not clear, although it may be a subunit of the larger toxin that is capable of acting directly on the HeLa cell.

Background: Shiga toxin (Stx) is the primary virulence factor of Stx-producing Escherichia coli (STEC). STEC can produce Stx1a and/or Stx2a, which are antigenically distinct. However, Stx2a-producing STEC are associated with more severe disease than strains producing both Stx1a and Stx2a.Methods and Results: To address the hypothesis that the reason for the association of Stx2a with more severe disease is because Stx2a crosses the intestinal barrier with greater efficiency that Stx1a, we covalently labeled Stx1a and Stx2a with Alexa Fluor 750 and determined the ex vivo fluorescent intensity of murine systemic organs after oral intoxication. Surprisingly, both Stxs exhibited similar dissemination patterns and accumulated in the kidneys. We next cointoxicated mice to determine whether Stx1a could impede Stx2a. Cointoxication resulted in increased survival and an extended mean time to death, compared with intoxication with Stx2a only. The survival benefit was dose dependent, with the greatest effect observed when 5 times more Stx1a than Stx2a was delivered, and was amplified when Stx1a was delivered 3 hours prior to Stx2a. Cointoxication with an Stx1a active site toxoid also reduced Stx2a toxicity.Conclusions: These studies suggest that Stx1a reduces Stx2a-mediated toxicity, a finding that may explain why STEC that produce only Stx2a are associated with more severe disease than strains producing Stx1a and Stx2a. [ABSTRACT FROM AUTHOR]

Background A Shiga toxin type 2a (Stx2a)–producing enteroaggregative Escherichia coli (EAEC) strain of serotype O104:H4 caused a large outbreak in 2011 in northern Europe. Pathogenic mechanisms for this strain are unclear. We hypothesized that EAEC genes encoded on the pAA virulence plasmid promoted the translocation of Stx2a across the intestinal mucosa. Methods We investigated the potential contribution of pAA by using mutants of Stx-EAEC strain C227-11, either cured of the pAA plasmid or deleted for individual known pAA-encoded virulence genes (ie, aggR, aggA, and sepA). The resulting mutants were tested for their ability to induce interleukin 8 (IL-8) secretion and translocation of Stx2a across a polarized colonic epithelial (T84 cell) monolayer. Results We found that deletion of aggR or aggA significantly reduced bacterial adherence and (independently) translocation of Stx2a across the T84-cell monolayer. Moreover, deletion of aggR, aggA, sepA, or the Stx2a-encoding phage from C227-11 resulted in reduced secretion of IL-8 from the infected monolayer. Conclusions Our data suggest that the AggR-regulated aggregative adherence fimbriae I enhance inflammation and enable the outbreak strain to both adhere to epithelial cells and translocate Stx2a across the intestinal epithelium. [ABSTRACT FROM AUTHOR]

Background. Shiga toxin (Stx)-producing Escherichia coli (STEC), especially O157:H7, cause bloody diarrhea, and in 3%-15% of individuals the infection leads to hemolytic uremic syndrome (HUS) or other complications. Use of antibiotics to treat STEC infections is controversial. Here, we describe the use of piglets to evaluate the efficacy and mechanism of action of antibiotics in these infections. Methods. The effects of 2 antibiotics on STEC toxin production and their mechanisms of action were first determined by enzyme-linked immunosorbent assay and subsequently evaluated clinically in the gnotobiotic piglet infection model. Results. In vitro treatment of clinical and isogenic strains with ciprofloxacin increased the production of Stx2 via phage induction but not the production of Stx1. Azithromycin caused no significant increase in toxin production. After treatment with ciprofloxacin, infected piglets had diarrhea and the severe fatal neurological symptoms associated with Stx2 intoxication. Characteristic petechial hemorrhages in the cerebellum were more severe in ciprofloxacin-treated animals than in control animals. In contrast, azithromycin-treated piglets survived the infection and had little or no brain hemorrhaging. Conclusions. The increased in vitro toxin production caused by ciprofloxacin was strongly correlated with death and an increased rate of cerebellar hemorrhage, in contrast to the effect of azithromycin. The piglet is a suitable model for determining the effectiveness and safety of antibiotics available to treat patients. [ABSTRACT FROM AUTHOR]

P1 and ABO antigens and bacterial stx genotypes might influence the risk of developing hemolytic uremic syndrome (HUS) after Escherichia coli O157:H7 infections. We determined ABO status and P1 antigen expression in 130 infected and 17 uninfected children, and we determined the stx genotype of the infecting isolate. P1 expression was weakly and directly related to HUS risk (P=.04), but this risk did not extend to the group with the greatest P1 expression. P1 expression remained constant as HUS evolved. The ABO frequency was similar in all groups. These associations were not affected by the stx genotype. stx1(-)/stx2(+) E. coli O157:H7 strains were more commonly associated with HUS than were stx1(+)/stx2(+) strains (P=.21), and 1 child with HUS was infected with a rare stx1(+)/stx2(-) isolate. In the present study, ABO antigens and stx genotypes were not major determinants of the outcome of E. coli O157:H7 infections, and P1 expression did not protect against the development of HUS. [ABSTRACT FROM AUTHOR]

Focuses on a study which determined the physical, biologic and immunologic properties of the recombinant Shiga Toxin 2 B subunit. Materials and methods; Results; Discussion.

The role of serum antitoxic antibody in protection against the dysentery caused by Shigella dysenteriae type I (Shiga's bacillus) was studied in monkeys fed 1010 virulent organisms after parenteral immunization with a formalin-inactivated Shiga toxoid preparation standardized in mice. Two 125-µg doses of toxoid adsorbed on aluminum hydroxide adjuvant and given 14 days apart provided mice with a 54-fold increase in resistance to parenteral toxin. In rhesus monkeys (Macaca mulatta), the same regimen of toxoid permitted the safe parenteral administration of toxin in incremental doses ranging from 100 to 1,000 mouse 50% lethal doses and resulted in correspondingly high titers of antitoxin in serum. Nevertheless, the immunized monkeys responded to orally administered Shiga bacilli by development of diarrhea and dysentery that was as severe as (or more severe than) the response of unimmunized controls. The failure of extraordinarily high levels of circulating antibody to protect against experimental shigellosis suggests that the intestinal mucosal sites of toxinmediated response are beyond the reach of systemic antitoxin. [ABSTRACT FROM PUBLISHER]

A semicontinuous fermenter system was developed in which broth culture filtrates of Shigella dysenteriae type 1 yielded substantial amounts of exotoxin. Biologic activity of the exotoxin was characterized by means of three assays: the rabbit ileal loop for fluid evocation (enterotoxicity), mouse lethality after parenteral injection (neurotoxicity), and HeLa cell toxicity in vitro (cytotoxicity). Although the culture filtrate was highly active, disc electrophoresis revealed that the toxin is a minor component of the mixture of proteins in the crude preparation, and that the minor representation contrasts with the relative prominence of exotoxins in cultures of other bacteria, such as Vibrio cholerae, Corynebacterium diphtheriae, and Clostridium tetani. Filtrate toxin, when purified by isoelectric focusing in polyacrylamide gel, was found to contain two toxic moieties. One was resolvable as a single band with an isoelectric point (PI) of 7.25, a molecular weight of 72,000, and all three types of biologic activity. The second moiety was isoelectric at pH 6.00, contained two subcomponents, and further contrasted with the pI 7.25 toxin by being more cytotoxic while being devoid of enteroneurotoxic activity. [ABSTRACT FROM PUBLISHER]

The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal cortical epithelial cells (HRCEC) in primary culture was investigated. HRCEC express CD24, the marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs. Binding of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs. Treatment of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing antibody specific for Stx. DNA fragmentation was found to be accompanied by Stx-mediated cell death in HRCEC, indicating that apoptosis was part of the process. These data and previous reports indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E. coli infection. [ABSTRACT FROM PUBLISHER]

This study explored the role of inducible nitric oxide (NO) synthase (iNOS) in an infant rat model of group B streptococcal meningitis. Brain iNOS activity increased during meningitis (P < .001), and iNOS was detected by immunocytochemistry in the walls of meningeal vessels and cells of the cerebrospinal fluid (CSF) inflammation. Animals treated with iNOS inhibitor aminoguanidine (AG; 130 mg/kg every 8 h) had reduced NO production (P < .05), higher CSF bacterial titers (P < .05), and increased incidence of seizures (P < .01) compared with untreated infected animals. AG also increased areas of severe hypoperfusion in the cortex (31% ± 14% in controls vs. 56% ± 16% in AG; P < .01) and the extent of cortical neuronal injury, both when administered at the time of infection (P < .05) and in established meningitis (P < .02). Thus, NO produced by iNOS may be beneficial in this model of experimental meningitis by reducing cerebral ischemia. [ABSTRACT FROM PUBLISHER]

An approach for studying neurotoxicity of bacterial products is presented. Pentylenetetrazol, a convulsant drug, was injected into mice, and increased sensitivity to pentylenetetrazol was used as an indicator of neurotoxicity. The preinjection of sonicates of Shigella dysenteriae 60R or Escherichia coli H30 (producing Shiga toxin or Shiga-like toxin I, respectively) enhanced the response of mice to pentylenetetrazol within 6 h. This was indicated by a higher mean convulsion score, increased number of mice responding with convulsions, and induction of seizures in animals pretreated with a subepileptic dose of pentylenetetrazol. Preinjection of purified Shiga toxin significantly changed the response to pentylenetetrazol only when coadministered with bacterial lipopolysaccharide (LPS); mean convulsion scores were 1.6 and 0.9 for the Shiga toxin-LPS group and controls, respectively. LPS alone did not affect sensitivity to pentylenetetrazol. These results suggest that Shiga toxin and LPS together induce neurologic disorders early in the course of infection. [ABSTRACT FROM PUBLISHER]

To compare the sensitivity a new ELISA for IgM antibodies to Borrelia burgdorferi that uses a recombinant outer surface protein C (rOspC) with those of a whole cell (WC) ELISA and an immunoblot assay for the diagnosis of early Lyme disease, serum specimens from 82 consecutive patients with physician-documented erythema migrans were analyzed. To compare the specificities of the three assays, serum specimens from 50 patients without a history of Lyme disease and from an area in which B. burgdorferi is not endemic were analyzed. The sensitivities of the WC ELISA, immunoblot assay, and IgM rOspC ELISA were 28%, 29%, and 46%, respectively, while the specificities were 100%, 100%, and 98%, respectively. The IgM rOspC ELISA is a convenient, readily automated, easily standardized serologic test that is significantly more sensitive for the diagnosis of early Lyme disease than either we ELISA or immunoblot assay. [ABSTRACT FROM PUBLISHER]

The effects of Shiga-like toxins on the morphology and protein synthetic capability of HeLa cells in tissue culture could be prevented by brefeldin A, an inhibitor of certain intracellular Golgi functions. Brefeldin A was without effect on the binding of Shiga-like toxin to cells. These results provide evidence that the Shiga-like toxins are first processed by the Golgi before moving to their 60S ribosome target site. [ABSTRACT FROM PUBLISHER]

Shiga toxin recognizesa galactose-α1→4-galactose terminal glycolipid,globotriaosylceramide (Gb3), in sensitive mammalian cells and is translocated by endocytosis to the cytoplasm, where it blocks protein synthesis. To determine if Gb3 is both required and sufficient for toxicity, Gb3 content in cells was altered by blocking key biosynthetic or degradative path enzymes with specific inhibitors. The resulting decrease or increase in cellular Gb3 was associated with a decrease or increase in binding of and response to Shiga toxin. Toxin-resistant Gb3-deficient variants of sensitive cells fused with liposomes containing Gb3 but not globotetraosylceramide (Gb4) became susceptible, whereas fusion of Gb3 liposomes to naturally resistant Gb3-deficient CHD cells increased toxin binding but not cytotoxicity. These data demonstrate that Gb3 is required, but not sufficient, for the action of Shiga toxin and suggest the existence of a toxin translocation mechanism linked to surface glycolipids that is not expressed in CHD cells. [ABSTRACT FROM PUBLISHER]

Human milk antibody and nonantibody factors are thought to be important in protecting infants from diarrheal diseases. The nonantibody factors include host receptor analogues that bind to specific pathogen virulence factors, thereby inhibiting these bacterial products from binding to their intestinal target receptors. Globotriaosylceramide (Gb3), a glycolipid known to bind to both Shiga toxin and Shiga-like toxins, was found to occur in human milk with an average concentration of 73 nM (77 µg/L) for Gb3 containing hydroxylated fatty acids and 50 nM(53 µg/L) for Gb3 containing nonhydroxylated fatty acids. This human milk Gb3 was found to bind to Shiga toxin, consistent with the hypothesis that Gb3 could contribute to the protective effect of human milk against infantile diarrheas associated with Shiga or Shiga-like toxins. [ABSTRACT FROM PUBLISHER]

Shiga-like toxin (SLT)-1 and SLT-II/lIc bound to Synsorbs containing synthetic αGal(1–4)βGal (P1 disaccharide), αGal(1–4)βGal(1–4)βGlcNAc (PI trisaccharide), or αGal(1–4)βGal(1–4)βGlc (Pk trisaccharide) sequences but not to Synsorbs containing αGal(1–3)βGal, αGal(1–3) αGal(1–4)βGlcNAc, or the hydrophobic oligosaccharide linkage arm. SLT-I had a preference for Synsorbs containing trisaccharides, whereas SLT-II/IIe binding was less selective. 125I-labeled SLT-I remained bound to Pk trisaccharide Synsorb in the presence of lactose, galactose, or EDTA but was partially released by acetic acid, guanidine HCl, or a 10% solution ofSDS. Vero cells coincubated with Pk trisaccharide Synsorb and SLT 1 extract were protected from this toxin, whereas Pk trisaccharide Synsorb was much less efficient at neutralizing SLT-II/IIe activity in Vero cell coincubation experiments. The SLT-lIe component was not responsible for the inefficient neutralization. Results suggest that synthetic oligosaccharide sequences related to the P blood group antigens coupled to inert matrices could be useful for rapid diagnosis or possibly therapeutic intervention in enterohemorrhagic Escherichia coli infections. [ABSTRACT FROM PUBLISHER]

Infection with Shiga toxin- and Shiga-like toxin-producing strains of Shigella dysenteriae and Escherichia coli, respectively, can progress to the hemolytic-uremic syndrome. It has been hypothesized that circulating Shiga toxin, Shiga-like toxins, and endotoxins may contribute to the disease by directly damaging glomerular endothelial cells. The effects of these toxins on HeLa, Vero, and human vascular endothelial cells (EC) were examined. Confluent EC were sensitive to Shiga toxin but were at least 106-fold less sensitive to the toxins than were Vero cells. Shiga toxin was the predominant cytotoxic factor. Lipopolysaccharides were not cytotoxic and did not augment Shiga toxin-mediated toxicity. Lower doses of Shiga toxin caused cytotoxicity when coincubated with tumor necrosis factor. The relative resistance of EC to Shiga toxin and Shiga-like toxins may be due to reduced toxin binding, as low levels of globotriaosylceramide (Gb3) , the toxin-specific receptor, were found in EC membranes. [ABSTRACT FROM PUBLISHER]

The neurologic symptoms in human shigellosis have often been attributed to Shiga toxin, although its exact role has not been determined. By use of a [3H] thymidine-labeled HeLa cell assay, cytotoxic activity was demonstrated in stool but not cerebrospinal fluid or serum from five patients with shigellosis presenting with seizures or encephalopathy. Bacterial isolates produced 16.0–88.2 CD50 (50% cytotoxic dose) of cytotoxin/mg of protein. The toxin activity in stool and the cytotoxic activity of the isolates were not neutralized by antiserum to purified Shiga toxin. DNA hybridization studies showed that Shigella isolates from these patients lacked the structural genes for Shiga toxin. The cytotoxin produced was also distinct from Shiga-like toxins I and II. Sonicates of the Shigella strains injected intraperitoneally into mice caused lethargy and lethality. The toxin activity was heat-labile and sensitive to trypsin, indicating that its active component is protein. Ultrafiltration and gel filtration chromatography showed a molecular mass of 100–125kDa. Thus Shiga toxin production is not essential for the development of neurologic manifestations of shigellosis; other toxic products may play a role. [ABSTRACT FROM PUBLISHER]

Shiga toxin from Shigella dysenteriae type 1 strains and Shiga-like toxins (SLT)I and II from Escherichia coli bind to terminal α-D-Galp-(1→4)-D-Galp containing g1ycolipids. Hydatid cyst fluid isolated from sheep infected with Echinococcus granulosus contains a glycoprotein (P1gp) with a terminal α-D-Galp-(1→4)-D-Galp disaccharide. Preparations of Pigp were shown to interact directly with Shiga toxin and to inhibit the binding and cytotoxicity of Shiga toxin to HeLa cells. A sandwich ELISAwas developed using preparations of P1gp as the toxin capture molecule, which, with an appropriate polyclonal antibody, was capable of detecting as little as 80 pg/well Shiga toxin and 132 pg/well SLT-II. Thus, the Pjgp-toxin interaction forms the basis for a simple antigen-capture ELISAthat may be useful clinically for the rapid detection and quantitation of Shiga and Shiga-like toxins. [ABSTRACT FROM PUBLISHER]

The humoral immune response to both Campy/obacter jejuni cell surface antigens and to potential toxins of the organism was studied in 64 adults with inflammatory diarrhea. In an enzyme-linked immunosorbent assay (ELISA) for surface antigens, 17 (71%) of 24 persons with Campylobacter enteritis showed seroconversion in more than one immunoglobulin class, versus only 2 (5%) of 40 patients with non-Campylobacter enteritis. In a GM1 ganglioside-based ELISA for detecting serum IgG to cholera-like enterotoxin, only one patient studied showed seroconversion to the enterotoxin. Of 22 Campylobacter isolates studied for production of cholera-like toxin, none of the supernatants from the Campylobacter strains were positive. Supernatants were also tested for enterotoxin and cytotoxic activity on Chinese hamster ovary cells; all isolates were negative for enterotoxin activity. In contrast, cytotoxin was produced by 7 (32%) isolates but was usually low-level and was not neutralized by patient's serum. These findings indicate that production of cholera-like toxin and cytotoxin by Campylobacter strains in the United States occurs in few strains and that host immune response is absent; their biologic significance in the pathogenesis of Campylobacter infections remains unclear. [ABSTRACT FROM PUBLISHER]

Binding kinetics of Shiga toxin to HeLa CCL-2 cells and to cell lines cloned by limiting dilutions were determined. Lines with a wide range of sensitivity to Shiga toxin were obtained. Binding data, analyzed by a computer-based Scatchard model program, revealed two classes of binding sites, one of low affinity and high capacity and one of high affinity and low capacity. The number of high affinity, but not low affinity, sites present on the clones correlated with their sensitivity to toxin. Tunicamycin-treated CCL-2 cells became resistant to Shiga toxin in parallel with a reduction in the capacity of the high-affinity site. Cell content of Gb3, the glycolipid receptor for Shiga toxin, decreased as the sensitivity of the cells diminished. These data show that a minority of Shiga toxin binding sites of HeLa cells are involved in the cytotoxic response and suggest that Gb3 is the high-affinity functional cytotoxin receptor. [ABSTRACT FROM PUBLISHER]

We measured the cytotoxic activity of 119 strains of Shigella by using a quantitative [3H]thymidine-labeled HeLa cell assay. We assayed 13 strains of Shigella dysenteriae 1; 18 strains of S. dysenteriae types 2 and 3; and 88 strains of Shigella sonnei, Shigella flexneri, and Shigella boydii. Strains of S. dysenteriae 1 demonstrated high levels of cytotoxicity (geometric mean, 105.04 CD50/mg of protein; range, 103.95–106.10). Cytotoxic activities of the non-type 1 strains of S. dysenteriae and of the other Shigella serogroups were ∼1/1,000 that of the S. dysenteriae 1 strains (range, 101.09–103.11 CD50/mg of protein). Neutralization of cytotoxicity by using rabbit antiserum to purified Shiga toxin revealed that in all strains of S. dysenteriae 1, ⩾99.5% of cytotoxic activity was attributable to Shiga toxin. In contrast, 88 of the other Shigella strains produced only nonneutralizable cytotoxic activity. Six of 18 strains of non-type 1 S. dysenteriae and 12 of 88 strains from other Shigella serogroups produced both Shiga toxin and nonneutralizable toxin. [ABSTRACT FROM PUBLISHER]

The relation between in vitro production of HeLa cell cytotoxin by strains of Shigella and clinical symptomatology was determined for 35 travelers from the United States who developed shigellosis in Guadalajara, Mexico. There were 25 patients with Shigella sonnei, eight with Shigella flexneri, one with Shigella boydii, and one with Shigella dysenteriae. These strains were evaluated for in vitro production of cytotoxin. The amount of cytotoxin did not correlate with the number of stools passed, the severity of abdominal pain, or the presence of nausea or vomiting. However, patients with strains of Shigella that produced more cytotoxic activity were more likely to have fever (P < .02) and occult blood in their stools (P < .004). The cytotoxicity produced by 30 (86%) strains could not be neutralized with rabbit antiserum to purified, formaldehyde-treated Shiga toxin from S. dysenteriae type 1 strain 60 R; the cytotoxicity of five (14%) of the strains was partially neutralized. When only nonneutralizable cytotoxin was considered, the presence of fecal leukocytes (P < .04), as well as of occult blood (P < .002) and fever (P < .02), correlated with the amount of cytotoxin. The amount of nonneutralizable cytotoxin produced by shigella strains was related to the clinical findings. This cytotoxic activity was infrequently attributable to “Shiga toxin.” [ABSTRACT FROM PUBLISHER]