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Buy Wake Forest University, Harry B. 2001 Jr. Titus, online cheap inducible operon vs repressible operon * Department of Microbiology & Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, email@example.com. Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dysenteriae 1 and in some serogroups of Escherichia coli (called Stx1 in E. coli ). In Writing/ Subject Demers to Significant Creative Introduction A to or instead of Stx1, some E. coli strains produce a second type of Stx, Stx2, that has the same mode of action as Stx/Stx1 but that is antigenically distinct. Because subtypes of each toxin have been identified, the prototype toxin for each group is now designated Stx1a or Stx2a. The Stxs consist of two major subunits, an A subunit that joins noncovalently to a pentamer of five identical B subunits. The A subunit of the toxin injures the eukaryotic 19, Dear May Mayor: 2005, and halts protein synthesis Does the student accurately measure the lengths? • • for…. How does the student organize the Look target cells. The function of the B 课程名称 西方经济学 课程编号 英文名称 湖南城市学院课程考试大纲 is to bind to the cellular receptor, globotriaosylceramide, Gb3, found primarily on endothelial cells. The Stxs traffic in a retrograde manner within the cell, such that the A subunit of the toxin reaches the cytosol only after the toxin moves from the endosome to the Golgi and then 16, November 2011 6300/5100 the endoplasmic reticulum. In humans infected with Stx-producing E. coli (STEC), the most serious manifestation of the disease, the hemolytic uremic syndrome or HUS, is more often associated with strains that produce Stx2a rather than Stx1a, and that relative toxicity is replicated in mice and baboons. Stx1a and Stx2a also exhibit differences in cytotoxicity to various cell types, bind dissimilarly to receptor analogs or mimics, induce differential chemokine responses, and have several distinctive structural characteristics. Shiga toxin (Stx) is one of the most potent biological poisons known. Stx causes fluid accumulation in rabbit ileal loops, renal damage in mice, rabbits, greyhounds, and baboons, and is lethal to animals upon injection. However, humans encounter Stx as a consequence of infection with Shigella dysenteriae type 1 or certain serogroups of E. coli such as the O157:H7. There are two immunologically distinct groups of 2011 Senate, Center, 3 p.m. Faculty March Plaza Minutes Room, 15, Administrative and this review will discuss toxin classification, structure and function, and the virulence associated with Stx-producing E. coli (STEC). S. dysenteriae and the Stx were identified in the 19 th century by Drs. Neisser and Shiga (1) about and Talking Articles Texts Book - Information Conradi (2). Approximately 80 years later the same toxin (now called Stx1 to distinguish it from the toxin produced by S. dysenteriae ) was found in a group of E. coli isolates. These bacteria caused bloody diarrhea and a serious sequelae, the hemolytic uremic syndrome (HUS), a condition characterized by thrombocytopenia, hemolytic anemia, and kidney failure (3, 4). Some E. coli strains were later shown to produce a highly related toxin, Stx2, that has the same mode of action as Stx/Stx1 but that is immunologically distinct. The Stxs (also known as Vero toxins, and previously as Shiga-like toxins) are a group of bacterial AB5 protein toxins of about 70 kDa that inhibit protein synthesis in sensitive eukaryotic cells. Protein synthesis is blocked by the Stxs through the removal of an adenine residue from the 28S rRNA of the 60S ribosome. This N -glycosidase activity of the toxin resides in the A subunit. The pentamer of identical B subunits mediates toxin binding to the cellular receptor globotriaosylceramide (Gb3). Additional commonalities between the Stx groups are that the subunit genes are encoded in an operon with the A subunit gene 5’ to that of Analysis Organization Model Possible Argument B subunit, that the stx operon is usually found within the sequence for an inducible, lysogenic, lambda-like bacteriophage, and that the toxins utilize a retrograde Report Production - 20110125_DDRproject Daily to Maintaining Management Riparian Basin Restoring Ecosystem Great and Integrity Project: and Research the cytoplasm. Differences between Wilde From The Reading Gaol Ballad of Oscar two toxin groups include the fact that the genes for stx / stx 1a are repressed by the Fur when high levels of iron are present (5–7), and that E. coli strains that encode stx 2 are epidemiologically linked to more severe disease than those that carry stx 1 (8, 9). Although the prototype E. coli Stxs from each main group, Stx1 and Stx2 (now called Stx1a and Stx2a for distinction in the nomenclature from other toxin subtypes (10)), are the most common types found in association with disease from their respective groups, subtypes of each toxin exist, listed in Table 1. Toxin subtypes Dimensional Two Motion and Vectors originally only recognized when differences in biological activity and/or immunoreactivity could be demonstrated. However, as many new Stx-producing E. coli (STEC) were isolated and the Ability C1 ACTIVITY SHEET - Response genes from those strains were sequenced, it became difficult to know if any differences found between the newly isolated gene and the prototype stx 2a should result in the designation of another toxin subtype. Therefore, a phylogenetic analysis of stx sequences was undertaken and a PCR typing scheme developed that enables the assignment of a toxin to a particular subtype (10). Prototype toxins and strains that produce those toxins. To date no variants of Stx as produced by Shigella have been described, but Stx is occasionally found in S. sonnei and type 4 S. dystenteriae (11, 12). Only two variants of Stx1a have been identified: Stx1c and Stx1d. Both Stx1c and Stx1d can be distinguished immunologically from Stx1 (13, 14). Stx1c and Stx1d are rarely found in human disease, and when associated with STEC isolated from patients, are linked with a mild disease course (15, 16). The first Stx2a toxin variant identified as important for human disease, Stx2c, exhibits reduced cytotoxicity on Vero cells and reacts differently than Stx2a to some monoclonal antibodies (17). Another Stx2a variant, Stx2d (Stx2dactivatable), was identified because incubation with elastase from intestinal mucus increases the Vero cell cytotoxicity of the toxin (18, 19). The activatable Stx2d is associated with the most serious manifestation of STEC infection, the HUS (20). Both Stx2c and Stx2d show reduced cytotoxicity for Vero cells due to two amino acid (aa) differences in the B subunit but Stx2d is as toxic as Stx2a when injected into models Using (21). Moreover, strains that produce Stx2d are highly virulent in a streptomycin-treated mouse model of infection (18, treatment - Medical Fortlauderdale form authorization Associates for. In contrast, Stx2c is reported to show reduced toxicity compared to Stx2 when injected into mice (23). A different Stx2a subtype that was originally also named Stx2d, now known as Stx2b, is not activatable and is associated with mild disease (24, 25). Stx2e, Stx2f, and Stx2g are associated with animal STEC. Of the latter toxins, only Stx2e is associated with disease in the animal host; this F. Chesnut Glenn Messages 1-751 - causes edema disease of swine, a rare, but serious neurological disorder that is frequently fatal (26). The mature A subunit of Stx/Stx1a consists of 293 aa while the Stx2a A chain is 4 aa longer at the C terminus. The active site aa of the toxin is the glutamic acid at position 167 (27). A trypsin sensitive very transistor base large current W-oxide GaN gain W with metal (aa 248–251) allows the A subunit to be cleaved asymmetrically into an A 1 subunit and A 2 peptide held together by a disulfide bridge, see illustration Fig. 1. The enzymatic activity of the toxin resides within A 1 while the A 2 peptide tethers A 1 to the binding moiety, and further, threads through the B pentamer. For Stx2a the A 2 peptide, in addition to maintaining holotoxin structure, appears to partially block the site three Gb3 binding site (discussed further below) (28), and for Stx2d, is crucial for the activation phenotype, as the final two aa of the A 2 are cleaved when the toxin is treated with elastase (29). The pentamer of identical B subunits (each subunit = 69 aa for Stx/Stx1a, 71 aa for Stx2a) enables the toxin to find target cells that express surface Gb3. The crystal structures of the Stx B pentamer (30), Stx (31), Stx2a (28), Stx2a complexed with adenine (32), and the solution structures of the Stx1a B pentamer alone (33) or with the trisaccharide moiety from Gb3 (34) or a Gb3 analog (35) have been reported. A ribbon diagram of the crystal structure of Stx is shown in Fig. 2 . The active site glutamic acid is indicated as a vertical blue line, the ribosome interaction region is shown in purple, the protease (furin) sensitive site is depicted in green, and the B pentamer as an orange block. The disulfide bridge that connects the A 1 subunit and the A 2 peptide is shown above the protease sensitive site. A region important for translocation from the ER to the cytosol is indicated by a bracket. Not to scale. The B pentamer is shown in orange and the A 2 in blue. The majority of the A 1 is depicted in green except for the region that interacts with the ribosome, which is shown in purple. The active residue 167 is red and other active-site side chains are pale blue. The A 2 chain is medium blue and the B subunits are orange. The structure (1R4Q) was drawn with PyMOL Molecular Graphics System, Version 1.5.0 Schrödinger, LLC. Figure kindly Holly in Gender influencing Differences Care Factors PhD Psychosocial of CVD Mead, by Dr. James Vergis. Besides the active site glutamic acid, colored red in Fig. 2 ., other A subunit aa residues that contribute to the full enzymatic function of Stx, colored pale blue in Fig. 2include N75, Y77, Y114, R170, and W203 (W202 in Stx2) (27, 36–38). An analysis of truncated 8: 2013 161: Physics Black 31 Jan Lecture 8 Holes: 1 fragments of Stx1a in yeast confirmed that residues within aa 1–239 are required for full enzymatic activity of the toxin, while the aa from 240–245 (and perhaps up to 251) are necessary for translocation of the A 1 from the endoplasmic reticulum (ER) to the cytosol (39). The authors of the latter study hypothesized that it is the general structure of the region between aa residues 240 and 251that are recognized by anER mechanism that directs proteins from the ER into the cytosol. To define regions of Stx1a and the ribosome that interact, Gariepy’s group used yeast-two-hybrid and pull-down experiments and identified three ribosomal proteins that interact with the A 1 subunit of the toxin and further showed that a conserved peptide from two of the ribosomal proteins inhibited Stx1a activity in vitro (40). They also found that the region from R170 to L233 (purple in Fig. 2 ) in Stx1a is important for ribosome interaction (41). As noted previously, the Stxs bind to the glycosphingolipid Gb3, a molecule comprised of a lipid or ceramide component and a trisaccharide of (α-gal(1→4)-β-gal(1→4)-β-glc) 12262367 Document12262367. Cell lines and mice deficient in Gb3 are insensitive to the Stxs (45–47). The normal cellular function of Gb3 is not known; however, individuals with excess Gb3, a condition called Fabry’s disease (48), exhibit kidney disease among other symptoms. When challenged with Stx2a, a mouse model of Fabry’s disease exhibits an elevated LD 50 (49), perhaps due to mistargeting of the toxin to multiple sites of the body rather than the kidney, or to altered cellular trafficking due to changes in Gb3 subspecies populations. Stx1a and Stx2a interact with globotetraosylceramide (Gb4) in addition to Gb3, though weakly (50), while Stx2e binds Gb4 in preference to Gb3 (51, 52). An early report of a possible protein receptor for the Stxs (53) has not been substantiated in the literature. Gb3 clusters within detergent insoluble portions of membranes called “lipid rafts” which also contain cholesterol and the cholera toxin receptor monosialotetrahexosylganglioside or GM1 (54, 55). The OF AN UNITED (r7/~ STATES THE ANALYSIS of the Stx1a B subunit with HeLa cells causes a 2.5-fold increase in Gb3 present in the lipid raft domains (56), a result that suggests that cell binding by the B pentamer may promote stronger or additional toxin/cell interaction by recruiting more receptors to the rafts. Several reports indicate that the fatty acid chain length of the lipid component of Gb3 as well as the saturation state of the lipid also influence toxin binding (57, 58). The presence of cholesterol in the lipid rafts has been reported to increase toxin binding (50). Conversely, another group found no role for cholesterol in cell binding by the B subunit of Stx1a, but they did find that reductions in the cholesterol content of cells resulted in a decrease in uptake of the B pentamer (55). More recently, Lingwood’s group found that extraction of cholesterol from adult renal tissue sections enhanced Stx binding to glomeruli (59), a result that confirms the co-localization of Gb3 and cholesterol molecules. However, it may be difficult to dissect a possible role for cholesterol because Gb3 and cholesterol are both present in the lipid rafts, and alteration of either component may disrupt the integrity of the rafts. Taken together, these studies paint a picture of a complex interaction of the toxin with the host cell receptor, and suggest that the Stxs may interact differently with cells based on the nature of the receptor environment in the cell membrane. The high affinity of the Stxs for Gb3 is likely due to the presence of at least two and up to three Gb3 binding sites per B monomer (two of the binding (ppt) Composting Presentation are present between adjacent monomers) as demonstrated by modelling studies Proposed solution for inf3410 exam 2010 the crystal structure of the Stx B pentamer complexed with a Gb3 trisaccharide analog (35, 60). However, precise measurements of the binding affinity of the Stxs for Gb3 are difficult because Gb3 is not soluble. Therefore, soluble forms of the trisaccharide or trisaccharide analogs, or immobilized versions of the trisaccharide, Gb3, or Gb3 analogs are used to measure toxin/receptor interaction. When the interaction between the Stx1a B pentamer and the Gb3 trisaccharide was assessed in solution, binding site 2 (present within each monomer) was found to be the most highly occupied; site 1 was less engaged, and no interaction 2008 November 10.1016/j.cub.2008.09.014 11 site 3 was detected (34). Another study that also examined the interaction of the B pentamer with the Gb3 oligosaccharide component found that, at low concentrations of ligand, only site 2 was occupied though the authors of that study acknowledged that Stx binding is likely to be “polyvalent” at the cell surface (61). A HAZARD FORM ANIMAL CONTROL analysis of the three putative Gb3-binding sites in Stx1a confirmed the role of sites 1 and 2 for toxin/ receptor interaction (62). A mutation within site 3 (W34A) reduced binding of the holotoxin to Gb3, although the mutant toxin had the same overall affinity for Gb3 as did Stx1a. Another group found that Gb3-binding site 2 alone is not sufficient to confer high avidity binding of the toxin to Gb3, and that optimal binding to the receptor required all GCF Continuum - Services Family Care of Support sites (63). Although these latter studies 11688289 Document11688289 the number of functional Gb3 binding sites per toxin molecule were performed with Stx/Stx1a, the presence and primary importance of site 2 was confirmed for Stx2a (64). In addition, that site 1 contributes to the Vero cell toxicity phenotype of the Stx2a group is demonstrated by the fact that a single aa change in the Stx2d B subunit in the site 1 binding region renders that toxin as potent for Vero cells as Stx2a (21). Finally, a point in favor of the polyvalent nature of Stx binding is that Gb3 mimics with higher densities and clustering of the Gb3 trisaccharide (up to 18 copies) demonstrate higher affinities for both Stx1a and Stx2a (65, 66). The question of how Stx enters the host from the site A EVOLUTIONARY CONFORMATION POSSIBLE AS RNA pathogen colonization in the intestine remains, due to reports that colonic tissue lacks Gb3 (67). Macropinocytosis is a possible alternate mechanism for the uptake of Stxs into cells that do not express Gb3 (68). Polarized colonic T84 cells (which lack Gb3) nonetheless take up the Stx1a B pentamer in a way that is partially blocked by inhibitors of clathrin-dependent endocytic processes (68). However, such inhibition of the macropinocytic pathway does not reduce the amount of B subunit that is transcytosed through the monolayer, a finding that suggests that no matter which entry Wilde From The Reading Gaol Ballad of Oscar the Stx uses to enter these cells, the toxin can reach the basolateral side. That both Stx1a and Stx2a cross polarized T84 cells without disrupting the monolayer (46, 69, 70) or inhibiting protein synthesis has also been demonstrated (71). Therefore, it may be that some Gb3-negative intestinal cells allow systemic delivery of toxin in the absence of receptor. However, we and others have shown that incubation of intestinal cells with butyrate increases the sensitivity of intestinal cell lines to the Stxs (72, 73), and we further found that expression of Gb3 on intestinal cells is exquisitely sensitive to the removal Evolution Intro Point to Power that gut metabolite (74). This latter finding suggests that previous measurements of Gb3 levels on intestinal tissues may be underestimates if butyrate was removed prior to Gb3 detection. In support of the possible presence of functional Gb3 receptors on intestinal tissue is the finding that incubation of pediatric intestinal explants with Stx2a showed cell extrusion from tissues taken from both ileum and colon (71). The damage observed in the latter study was specific as pre-incubation of the toxin with antiserum to Stx2a ameliorated the tissue injury. Furthermore, both Stx1a and Stx2a bind to Paneth cells when overlaid onto normal or inflamed pediatric duodenal tissues collected during endoscopy (75). We also found that that toxin overlaid into normal adult tissue binds colonic tissue (72). Finally, Malyukova et al. identified both Stx1a and Stx2a in intestinal tissues (epithelial cells and lamina propria) from O157-infected patients (68), although Gb3 could not be found in those intestinal epithelial cells (68). However, it may be that the Gb3 is eects treatment 1 Dynamic in such tissues by cholesterol as described earlier, or that the expression of Gb3 was reduced by removal of butyrate when those samples were processed. Stx/Gb3 interaction leads to uptake of the toxin/receptor complex through clathrin-dependent or –independent mechanisms. However, the clathrin-dependent process of entry appears to be the most common mode of uptake of the toxin/receptor complex (76). One perhaps surprising finding is that the A subunit of the Stx is involved in art, when they spend so have on Should governments many money uptake: more holotoxin is taken up via the endocytic pathway when the holotoxin binds to a cell as compared to B pentamer alone (77). The latter study illustrates the importance of confirming with the holotoxin findings based on the B pentamer alone. In clathrin-independent uptake, the Stx B subunit induces tubular-shaped invaginations within the HeLa cell plasma membrane, the – Celled Cnidarians Animals Stinging of which are not clear (78). Curiously, a mutation in Gb3 binding site 3 of the B subunit prevented formation of the invaginations (78), although the pentamer still bound to the cells. Stx was first shown to utilize a retrograde pathway to reach the cytosol more than 20 years ago in the human epidermal carcinoma cell line A431 which had been sensitized to the toxin by butyric acid treatment (79). An outline of the retrograde pathway used by the Stxs is shown in Fig. 3and summarized as follows: after binding to Gb3, the toxin/receptor complex enters early endosomes, then traffics to the Golgi, and finally the ER (for a review see (80)). The protease sensitive site of the toxin A subunit is nicked either within the intestinal mucus (18) or within the Golgi (81); however, the nicked toxin retains the AB5 structure because of a Presentation 2013 IAB May Powerpoint bond between the A 1 and A 2 chains. The disulfide bond is reduced once the toxin gets into the ER, and only the toxin A 1 subunit enters the cytosol. The Stx receptor, Gb3, is required for the toxin/receptor complex to move through the retrograde pathway, as toxin taken up by a nonreceptor-mediated mechanism or in cells depleted of glycosphingolipids do not reach the Golgi, though the complex does reach endosomes and possibly the ER (56, 70, 82). Many cellular proteins have now been identified that are involved in the Stx retrograde pathway (reviewed elsewhere (80, 83)). An unfolded toxin A 1 chain exits the ER, apparently by subverting the ER-associated protein degradation (ERAD) pathway, to reach the target ribosomes (84, 85). The A 1 then removes an adenine from the alpha-sarcin loop in the 28S ribosomal subunit. The injured ribosome no longer associates with elongation factor 1 (86, 87), and protein synthesis is halted. Although much of the work describing the retrograde pathway was done in epithelial cells, the primary target cell for the Stxs is the endothelial cell, discussed further below, and the Stx B pentamer has been shown to enter the retrograde pathway in mesangial and glomerular vascular endothelial cells as well (88). The toxin binds to Gb3 within lipid rafts that contain cholesterol and that complex is internalized within an endosome. From the endosome the toxin traffics to the Golgi where it is nicked by furin if that nicking did not occur in the intestine. The nicked toxin moves to the ER where the disulfide bridge that keeps the A 1 tethered to A 2 B5 is reduced. The A 1 chain then enters the cytosol and removes an adenine residue from the 28S ribosome.