Shiga-like Toxin Type Research Articles

Safe and Effective Means of Detecting and Quantitating Shiga-Like Toxins in Attomole Amounts

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ~ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

The Crystal Structure of Shiga Toxin Type 2 with Bound Disaccharide Guides the Design of a Heterobifunctional Toxin Inhibitor

Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.

Phospholipase D1 Is Required for Regulated Secretion of VWF Induced by the B-Subunits of Shiga-Like Toxins 1 and 2

AbstractAbstract 2113Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome (D+HUS) by damaging renal microvascular endothelium. Stx is composed of an active (A) N-glycosidase subunit that is responsible for cytotoxicity, and 5 binding (B) subunits that interact with cell surface Gb3 and promote Stx endocytosis. We have demonstrated that catalytically inactive pentameric B5 subunits of Shiga-like toxin type 1 and 2 (Stx B5) are sufficient to stimulate the acute secretion of von Willebrand factor (VWF) from human endothelial cells and can cause thrombotic microangiopathy in Adamts13–/– mice. Because our previous observations indicated that Stx1 B5 and Stx2 B5 exert distinct effects on Ca2+ and cAMP signaling pathways, we investigated the role of alternative signaling components in Stx B5-induced VWF exocytosis. Incubation of human umbilical vein endothelial cells (HUVECs) with Stx1 B5 (5 nM), Stx2 B5 (5 nM) or histamine (100 μM) caused a time-dependent increase in phospholipase D (PLD) activity that was maximal at 10 minutes after exposure to agonists. For HUVECs under static conditions, inhibition of PLD with n-butanol, or shRNA mediated PLD1 knock down, abolished Stx1 B5- or Stx2 B5-induced acute VWF secretion assayed by ELISA of conditioned media. To assess the stimulated secretion of cell-associated VWF strings under fluid shear stress, HUVECs were perfused in a flow chamber with fluorescently labeled anti-VWF antibody. When Stx1 B5 or Stx2 B5 (5 nM) was added to the perfusate, maximal induction of VWF strings was observed within 5 minutes by immunofluorescence video microscopy. However, tert-butanol, a structural analog of n-butanol that does not inhibit PLD activity, had no effect on VWF secretion or VWF string formation. In addition, treatment of HUVECs with Stx1 B5 or Stx2 B5 triggered actin stress fiber formation (assayed by fluorescence microscopy of Alexa Fluor 488-phalloidin-treated cells), enhanced monolayer permeability (assayed by the penetration of FITC-dextran in transwell chambers), and increased the level of GTP-bound RhoA (assayed by pull-down with Rhotekin-RBD protein coupled to agarose beads). These data indicate that B5 subunits of both Stx1 and Stx2 activate RhoA. Interestingly, Stx1 B5-induced PLD activation and VWF secretion were significantly reduced by pretreatment of HUVECs with the PKC-α inhibitor Go6976 (2 μM) or by shRNA mediated PKC-α knock down, but not by pretreatment with the Rho inhibitor exoenzyme C3 (1 μM) or Rho kinase inhibitor Y27632 (20 μM). Conversely, Stx2 B5-induced PLD activation and VWF secretion were reduced by these Rho/Rho kinase inhibitors, but not by inhibition of PKC-α with Go6976 or by PKC-α shRNA knock down. In addition, transfection of HUVECs with a plasmid encoding dominant negative RhoA (T19N) reduced Weibel-Palade body exocytosis induced by both Stx1 B5 and Stx2 B5. These data indicate that the B5 subunits of both Stx1 and Stx2 activate RhoA and induce acute VWF secretion in a PLD1 dependent manner. However, Stx1 B5 activates PKC-α mediated, Ca2+-dependent signaling, whereas Stx2 B5 preferentially activates RhoA mediated, Ca2+-independent signaling. Disclosures:No relevant conflicts of interest to declare.

Key genes and proteins involved in CTCM-reducing microvascular endothelial cell permeability induced by SLT-IIv using gene chips and DIGE

An Affymetrix mouse genome array and differential in-gel electrophoresis (DIGE) techniques were used to investigate the pharmacological mechanisms of a mixture of herbs, designated CTCM, a compound of traditional Chinese medicine, for the treatment of increased permeability in mouse intestinal microvascular endothelial cells (MIMECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMECs were challenged with 10 μg/ml SLT-IIv for 12 h and then treated with CTCM at a concentration of 200 μg/ml for 12 h. Total RNA and proteins from each treatment group were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip® Mouse Genome 430 2.0 microarray and DIGE. The results obtained demonstrated that there were one genes downregulated and one genes upregulated, one protein downregulated and four proteins upregulated in the SLT-IIv group compared to the control group. In the CTCM group, four genes were upregulated, three genes were downregulated, a single protein was downregulated and a single protein was upregulated when compared to the control group. When the CTCM-treated group was compared to the SLT-IIv group, expression of one gene was found to be increased, and all other genes were decreased, with five proteins downregulated. Analysis of the data suggested that CTCM specifically and effectively reduced microvascular endothelial cell permeability to SLT-IIv in the treatment of pig edema disease. In the CTCM-treated group, hspa9 expression was increased in both gene chip and DIGE analysis, so it may be a key protein in reducing cell permeability and utilized in medical treatments.

Down-regulation of cytochrome P450 proteins and its activities by Shiga-like toxin II from Escherichia coli O157:H7

Escherichia coli O157:H7 infection frequently induces clinical complications such as hemolytic uremic syndromes and intestinal dysfunctions. These changes could alter the disposition of drugs, consequently changing their efficacy. However, the possible changes of drug-metabolizing activities by E. coli O157:H7 infection have not been addressed. Thus, we have investigated the effect of Shiga-like toxin type II (SLT-II), derived from E. coli O157:H7, on the hepatic cytochrome P450 (CYP) content and its activity in rats. SLT-II (2 μg per animal, i.v.) time-dependently decreased total CYP content and the contents of CYP2C11 and CYP3A2 in hepatic microsomal preparations up to 24 hr following injection. Consistently, SLT-II time-dependently decreased CYP activity in vivo, as represented by systemic clearance of antipyrine. An inhibitor of inducible nitric oxide synthase, S-methylisothiourea, restored the decreased systemic clearance of antipyrine by SLT-II, suggesting the involvement of the overproduction of nitric oxide by SLT-II. Moreover, dexamethasone restored the decreased systemic clearance of antipyrine by SLT-II. In the hepatic microsomal preparation, dexamethasone restored the SLT-II-induced decrease of CYP3A2 whereas S-methylisothiourea did not affect both CYP subtypes. Taken together, these results suggest that SLT-II might alter hepatic drug-metabolizing function during E. coli O157 infection and that more than one cytokines induced by SLT-II, including nitric oxide, might make a critical contribution to the decrease of CYP content and its activity.

Frequency of Escherichia coli isolates from pigs suffering from different manifestations of "postweaning coli complex" (PWCC) in agricultural Hungary / Romania / Serbia

Gross postmortem pathologic-anatomical examination and polymerase chain reaction analyses for fimbriae (F), heat-stable (ST) and heat-labile (LT) enterotoxin and verotoxin (Shiga-like toxin (SLT) type II) were performed for 351 weaned piglets originating from units in agricultural Hungary Romania / Serbia. The majority of isolates (94,87%) carrying enterotoxin genes also carried genes for one of the fimbrial adhesins. The predominant genotypes were F4 LT STb and F18 Sta STb SLT respectively. This genetic combination may be explained by regional clonal expansion. Sixty-nine (20%) animals were diagnosed with postweaning diarrhea, thirty five (10%) with edema disease, 240 (68%) with postweaning wasting and 7 piglets (2%) exhibited hemorrhagic gastroenteritis. Postweaning wasting was the major cause of losses.

Purification of Shiga-like toxin 1 by pigeon egg white glycoproteins immobilized on Sepharose gels

The galabiose structure Galα1-4Gal is rarely found in natural glycoproteins, but is abundantly present in pigeon egg white proteins as Galα(1-4)Galβ(1-4)GlcNAc termini. Pigeon ovalbumin, ovomucoid, or the whole egg white were immobilized on periodate-oxidized Sepharose CL-6B gels by reductive amination. These gels were found to bind Shiga-like toxin type 1 (SLT-1) specifically and efficiently. SLT-1 was eluted from the gel beads with 0.5 M melibiose, which was more efficient and milder than elution with 4.5 M MgCl 2. SLT-1 was purified to homogeneity from the crude extract of Escherichia coli SLT100 expressing SLT-1 by a single affinity chromatographic step in 83–88% yield. The capacity of the gel was estimated to be ca. 1 mg toxin/ml gel. Interestingly, SLT-2 was not bound by these affinity gels containing Galα1-4Galβ1-4GlcNAc termini. Since SLT-2 has been shown to bind to Galα1-4Galβ1-4Glc-terminating compounds, our results suggest that Glc in globotriose moiety is important for binding SLT-2, and replacing the Glc with GlcNAc in this triose renders it ineffective for binding SLT-2.

Different kinetic of antibody responses following infection of newly weaned pigs with an F4 enterotoxigenic Escherichia coli strain or an F18 verotoxigenic Escherichia coli strain

To develop a vaccine against Escherichia coli-induced post-weaning diarrhea and edema disease, insights in the induction of the protective immune response following infection with these pathogenic E. coli is needed. Therefore, the fimbriae-specific antibody response of newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-IIv) producing F18 + verotoxigenic E. coli (VTEC) (strain 107/86) was compared with the response following an infection with LT producing F4 + enterotoxigenic E. coli (ETEC) (strain GIS 26). F4 + ETEC were able to colonize the gut very soon after infection, as peak excretion of F4 + E. coli bacteria was seen 2 days post-infection (dpi), but had already disappeared 7 dpi. On the other hand, F18 + VTEC infection resulted in a slower colonization of the gut as the peak excretion of F18 + E. coli was observed between 3 and 5 dpi, but this colonization remained longer as F18 + E. coli were detected till 9 dpi in feces. Furthermore, this fast colonization pattern of F4 + ETEC is accompanied with the presence of F4-specific antibodies in mucosal tissues and serum from 4 dpi onward, with maximal amounts of F4-specific IgA in the jejunal lamina propria and serum 7 dpi. In contrast, F18-specific IgA was only readily detected in the jejunal lamina propria 15 dpi and showed a maximum serum titer 21 dpi. Besides this faster induction and higher antibody response, the switch from IgM to IgA and IgG was also earlier following the F4 + ETEC infection.

Prevalence of Enteropathogenic Escherichia coli in Naturally Occurring Cases of Poult Enteritis–Mortality Syndrome

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.

Synthesis and structure–activity relationships of di- and trisaccharide inhibitors for Shiga-like toxin Type 1

The syntheses of galabiose and Pk-trisaccharide analogues in which selected hydroxy groups are replaced by O-methyl, amino deoxy, acetamido deoxy, and carboxyalkyl groups are reported. The ability of these inhibitors to block E. coli verotoxin 1 binding to its mammalian cell-surface receptor are evaluated by a solid-phase competition assay. The synthesis of a biotinylated glycoconjugate for this assay is described, wherein a Pk-trisaccharide tether derivative 70 is constructed and covalently attached to bovine serum albumin followed by biotinylation. Galabiose derivatives 4 and 5 that contain a carboxymethyl or carboxyethyl substituent at O-2 of the β-galactose residue show 15–20-fold activity gains over the methyl glycoside of galabiose. This enhanced activity is not observed for the corresponding carboxymethyl-substituted Pk-trisaccharide analogue 13. The inhibition data are rationalized with the solved crystal structure for verotoxin 1 complexed with a Pk-trisaccharide analogue and provide insight for the design of dimeric inhibitors that can exploit the unique binding-site distribution of the toxin’s B subunit. This discussion provides a further example of the important role played by ordered water molecules in sugar–protein complexes.

Binding of Avian Ovomucoid to Shiga-like Toxin Type 1 and Its Utilization for Receptor Analog Affinity Chromatography

Development of a simple and efficient purification procedure for Shiga-like toxin I (Stx1) was attempted. Since it has been suggested that pigeon egg white ovomucoid carries a P1 antigenic determinant, we examined its ability to bind Stx1. The ovomucoid glycoprotein fraction (GPro) was prepared from pigeon egg white by acetone precipitation, and a portion of the GPro was treated with pronase to obtain the glycopeptide fraction (GPep). When both GPro and GPep were coupled to CNBr-activated Sepharose 4B and subjected to affinity chromatography, Stx1 specifically bound to both columns. The Stx1 eluted with a buffer containing 4.5 M MgCl2 was shown to be highly purified to homogeneity by polyacrylamide gel electrophoresis under denatured condition; only two protein bands with molecular weights of 32,000 and 8000, which correspond to the A and the B subunits of Stx1, respectively, were recognized. The purified toxin showed cytotoxicity on Vero cells with a specific activity of approximately 6 × 108 CD50/mg protein; almost 100% of the activity was recovered from Escherichia coli cell lysate. We propose that the utilization of avian ovmucoid for the affinity chromatography provides a potentially simple, convenient, and widely available method to purify Shiga-like toxins.

Genes for tRNA(Arg) located in the upstream region of the Shiga toxin II operon in enterohemorrhagic Escherichia coli O157:H7.

We found two genes for tRNA(Arg) in the region upstream of genes for Shiga-like toxin type II (SLT-II) in Escherichia coli O157:H7. The two encoded forms of tRNA(Arg) recognize rare codons in E. coli K12 but these rare codons occur in the toxin genes at high frequency.

Exocytotic secretion of toxins from macrophages infected with Escherichia coli O157.

This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterohemorrhagic Escherichia coil (EHEC) O157:H7. Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro. The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II (SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in the culture medium. Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes. During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II. The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained. Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium. Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157. The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages.

Survival rate of mice after transient colonization with Escherichia coliclones carrying variant Shiga-like toxin type II operons

We have previously employed a streptomycin-treated mouse model to demonstrate that Escherichia coliclones expressing particular variant Shiga-like toxin type II operons differ in oral virulence, as judged by median survival time. Differences in virulence were not seen between all toxin variants, including two which differed significantly in cytotoxicity for Vero cells. In the present study, we have modified the animal model by withdrawing antibiotic selection and reintroducing normal mouse intestinal flora at various times after oral challenge with three different variant SLT-II-producing clones. This resulted in a transient colonization more akin to that seen in natural human infections. This has enabled detection of significant differences in survival rate between mice challenged with E. coliclones producing different SLT-II variants, which were not observed when colonization was maintained at high levels.

Differentiation-associated toxin receptor modulation, cytokine production, and sensitivity to Shiga-like toxins in human monocytes and monocytic cell lines

Infections with Shiga toxin-producing Shigella dysenteriae type 1 or Shiga-like toxin (SLT)-producing Escherichia coli cause bloody diarrhea and are associated with an increased risk of acute renal failure and severe neurological complications. Histopathological examination of human and animal tissues suggests that the target cells for toxin action are vascular endothelial cells. Proinflammatory cytokines regulate endothelial cell membrane expression of the glycolipid globotriaosylceramide (Gb(3)) which serves as the toxin receptor, suggesting that the host response to the toxins or other bacterial products may contribute to pathogenesis by regulating target cell sensitivity to the toxins. We examined the effects of purified SLTs on human peripheral blood monocytes (PBMn) and two monocytic cell lines. Undifferentiated THP-1 cells were sensitive to SLTs. Treatment of the cells with a number of differentiation factors resulted in increased toxin resistance which was associated with decreased toxin receptor expression. U-937 cells, irrespective of maturation state, and PBMn were resistant to the toxins. U-937 cells expressed low levels of GB(3), and toxin receptor expression was not altered during differentiation. Treatment of monocytic cells with tumor necrosis factor alpha (TNF-alpha) did not markedly increase sensitivity or alter toxin receptor expression. Undifferentiated monocytic cells failed to synthesize TNF and interleukin 1beta when treated with sublethal concentrations of SLT type I (SLT-I), whereas cells treated with 12-0-tetradecanoylphorbol-13-acetate acquired the ability to produce cytokines when stimulated with SLT-I. When stimulated with SLT-I, U-937 cells produced lower levels of TNF than PBMn and THP-1 cells did.

Purification of Recombinant Shiga-like Toxin Type I B Subunit

Shiga-like toxin type I (SLT-I) is a cytotoxin produced by certain strains ofEscherichia coli.SLT-I is a bipartite molecule comprised of A (SLT-IA) and B (SLT-IB) subunits. In holotoxin, five B subunits are arranged in a pentameric ring and bind to globotriaosylceramide receptors on the surface of susceptible eukaryotic cells. The SLT-IB pentamer is noncovalently associated with one A subunit that hasN-glycosidase activity and ultimately causes the death of targeted cells. Using a previously described overexpression vector, plasmid SBC32, we developed a two-step procedure for the purification of biologically active recombinant SLT-IB. Periplasmic proteins were extracted fromE. coliJM105(pSBC32), fractionated by ammonium sulfate precipitation, and separated by isoelectric focusing in a pH 3–10 gradient. SLT-IB was present in fractions with pH values between 5.0 and 6.0, consistent with its reported pIof 5.8. SLT-IB was purified to homogeneity in a second step by native polyacrylamide gel electrophoresis. Purified SLT-IB was characterized for biological and biochemical activity. When analyzed by native polyacrylamide gel electrophoresis, the majority of SLT-IB had an apparent molecular weight of 38,900, consistent with a pentameric subunit association. Chemical cross-linking of SLT-IB with disuccinimidyl suberate resulted in species with molecular weights corresponding to dimeric, trimeric, tetrameric, and pentameric forms of B subunit. SLT-IB was not cytotoxic to Vero cells at concentrations as high as 10 μg/ml and protected Vero cells from native SLT-I. Purified SLT-IB maintained its ability to associate with SLT-IA to form holotoxin that exhibited toxicity similar to native toxin.

Increased oral virulence of Escherichia coli expressing a variant Shiga-like toxin type II operon is associated with both A subunit residues Met4 and Gly102

We have previously demonstrated that Escherichia coliDH5α clones expressing closely-related Shiga-like toxin type II operons (designated SLT-II/OX3b and SLT-II/O48) had similar cytotoxicity for Vero cells, but differed in oral virulence for streptomycin-treated mice. Studies with chimeric toxin operons indicated that increased virulence was associated with the A subunit of SLT-II/OX3b, which differs from that of SLT-II/O48 by two amino acids (at positions 4 and 102). In the present study, we have constructed a series of additional chimeric derivatives of the SLT-II/OX3b and SLT-II/O48 operons and assessed the effect of single A subunit amino acid substitutions on oral virulence. Maximal virulence, as judged by median survival time after oral challenge, was associated only with the combination of Met4 and Gly102, as found in the A subunit of SLT-II/OX3b.

Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis.

Shiga toxin (STX), a bacterial toxin produced by Shigella dysenteriae type 1, is a hexamer composed of five receptor-binding B subunits which encircle an alpha-helix at the carboxyl terminus of the enzymatic A polypeptide. Hybrid toxins constructed by fusing the A polypeptide sequences of STX and Shiga-like toxin type II were used to confirm that the carboxyl terminus of the A subunits governs association with the B pentamers. The alpha-helix of the 293-amino-acid STX A subunit contains nine residues (serine 279 to methionine 287) which penetrate the nonpolar pore of the B-subunit pentamer. Site-directed mutagenesis was used to establish the involvement of two residues bordering this alpha-helix, aspartic acid 278 and arginine 288, in coupling the C terminus of StxA to the B pentamer. Amino acid substitutions at StxB residues arginine 33 and tryptophan 34, which are on the membrane-contacting surface of the pentamer, reduced cytotoxicity without affecting holotoxin formation. Although these B-subunit mutations did not involve receptor-binding residues, they may have induced an electrostatic repulsion between the holotoxin and the mammalian cell membrane or disrupted cytoplasmic translocation.

Heterogeneity of the amino-acid sequences of Escherichia coli shiga-like toxin type-I operons

PCR was used to amplify approx. 1470-bp segments of DNA containing complete Shiga-like toxin type I ( sltI) operons from Escherichia coli strains belonging to serotypes O48:H21, O111:H − and OX3:H8. These fragments were cloned and DNA sequence analysis identified several variations, as compared with published sltI sequences. All three sltI genes analysed were more closely related to Shiga toxin-encoding genes ( sht) of Shigella dysenteriae type 1, than to previously published E. coli phage-encoded sltI genes. The greatest deviation in deduced amino acid (aa) sequence was observed in the S1tI protein from the OX3:H8 strain, which differed from the phage 933J-encoded SltI by 9 aa in the A subunit and 3 aa in the B subunit.

Interaction of the Shiga-like toxin type 1 B-subunit with its carbohydrate receptor.

A study of the binding of the Shiga-like toxin 1 (SLT-1) to the P(k) trisaccharide [methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D- glucopyranoside] and its constituent dissacharides was carried out. The trisaccharide represents the carbohydrate recognition domain of the neutral glycolipid receptor of the SLT-1, globotriosylceramide (GbOse3). The binding constant for soluble trisaccharide to the soluble pentameric B-subunit is weak, with a K(a) of (0.5-1) x 10(3) M-1 for B-subunit monomer. Scatchard analysis of the binding data indicates five identical non-interacting carbohydrate binding sites per B-subunit pentamer and no cooperativity in binding. Despite weak binding (delta G = -3.6 kcal mol-1), the enthalpy of binding (delta H = -12 kcal mol-1) and the change in molar heat capacity accompanying binding (delta C(p) = -40 eu) are comparable to other protein-carbohydrate interactions. Dynamic light scattering studies indicate that carbohydrate binding induces protein aggregation. At carbohydrate concentrations where > 90% of B-subunit monomers are bound, the far-UV CD spectra were unchanged, whereas a change in the near-UV CD, maximal near 270 nm, titrated to give an apparent binding constant in good agreement with that obtained by isothermal microcalorimetry. Steady-state fluorescence and fluorescence lifetime measurements indicated that the environments of the central tryptophans are perturbed during saccharide binding, and the changes correlate with the extent of protein aggregation. On the basis of the thermodynamics of binding, optical spectroscopy, and binding-induced aggregation, we propose a model of SLT-1-membrane interaction that relies on protein-carbohydrate interaction for specificity and protein-lipid interaction for tight binding.