Journal articles on the topic 'Toxin A and Toxin B'
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McMillin, David E., Lycurgus L. Muldrow, and Shwanda J. Laggette. "Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction." Canadian Journal of Microbiology 38, no. 1 (January 1, 1992): 81–83. http://dx.doi.org/10.1139/m92-013.
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A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure. Key words: Clostridium difficile, polymerase chain reaction, bacterial toxins.
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Kobayashi, Kazuo. "Diverse LXG toxin and antitoxin systems specifically mediate intraspecies competition in Bacillus subtilis biofilms." PLOS Genetics 17, no. 7 (July 19, 2021): e1009682. http://dx.doi.org/10.1371/journal.pgen.1009682.
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Biofilms are multispecies communities, in which bacteria constantly compete with one another for resources and niches. Bacteria produce many antibiotics and toxins for competition. However, since biofilm cells exhibit increased tolerance to antimicrobials, their roles in biofilms remain controversial. Here, we showed that Bacillus subtilis produces multiple diverse polymorphic toxins, called LXG toxins, that contain N-terminal LXG delivery domains and diverse C-terminal toxin domains. Each B. subtilis strain possesses a distinct set of LXG toxin–antitoxin genes, the number and variation of which is sufficient to distinguish each strain. The B. subtilis strain NCIB3610 possesses six LXG toxin–antitoxin operons on its chromosome, and five of the toxins functioned as DNase. In competition assays, deletion mutants of any of the six LXG toxin–antitoxin operons were outcompeted by the wild-type strain. This phenotype was suppressed when the antitoxins were ectopically expressed in the deletion mutants. The fitness defect of the mutants was only observed in solid media that supported biofilm formation. Biofilm matrix polymers, exopolysaccharides and TasA protein polymers were required for LXG toxin function. These results indicate that LXG toxin-antitoxin systems specifically mediate intercellular competition between B. subtilis strains in biofilms. Mutual antagonism between some LXG toxin producers drove the spatial segregation of two strains in a biofilm, indicating that LXG toxins not only mediate competition in biofilms, but may also help to avoid warfare between strains in biofilms. LXG toxins from strain NCIB3610 were effective against some natural isolates, and thus LXG toxin–antitoxin systems have ecological impact. B. subtilis possesses another polymorphic toxin, WapA. WapA had toxic effects under planktonic growth conditions but not under biofilm conditions because exopolysaccharides and TasA protein polymers inhibited WapA function. These results indicate that B. subtilis uses two types of polymorphic toxins for competition, depending on the growth mode.
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Singh, Sunita, and Prachi Lad. "Assay of Bacillus cereus Emetic toxin produced in orange squash." EUREKA: Life Sciences, no. 2 (April 1, 2021): 41–55. http://dx.doi.org/10.21303/2504-5695.2021.001753.
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The contamination of squash by B. cereus, an enterotoxin producer, was found to range between 7.5×104 and 1.8×104 CFU/g in orange squash (during storage), that is hazardous. Orange squash is widely produced and consumed in India, but has a low rating of 3 on the scale of 10 (on feedback), mostly due to high sugars, not preferred these days. It can be preserved for >9 months due to added sugars and preservatives. During processing squash, if juice is not quickly cooled and/or squash is kept for long at temperatures <48 °C after processing, it can be a source of food poisoning. Reason, a large number of toxins can be produced by B. cereus. B. cereus strains, isolated from squash, produce heat stable toxin. Vacuolar assay confirmed them as emetic toxins, produced in squash. The toxin behaved like an ionophore in assay using mitochondria, extracted from liver cells of chicken with potassium ions in buffer. The toxicity of toxin by assay was 3200 IU/ng (BC IV strain) and 800 IU/ng (BC X strain). By the vacuolar expansions of mitochondria in assay, toxins of B. cereus demonstrated a toxic effect, in the range of 20.93 to 60.94 % by BC IV toxin and 43.28 to 45.02 % by BC X toxin, on the 3rd day growth of B. cereus in squash and toxin extraction for assay. It was also possible to produce antibodies against the B. cereus whole cell and toxin of BC IV, as an attempt to detect B. cereus contaminations in foods, by Ouchterlony’s immune-diffusion test
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de Melo, Janaina Viana, Gareth Wyn Jones, Colin Berry, Romero Henrique Teixeira Vasconcelos, Cláudia Maria Fontes de Oliveira, André Freire Furtado, Christina Alves Peixoto, and Maria Helena Neves Lobo Silva-Filha. "Cytopathological Effects of Bacillus sphaericus Cry48Aa/Cry49Aa Toxin on Binary Toxin-Susceptible and -Resistant Culex quinquefasciatus Larvae." Applied and Environmental Microbiology 75, no. 14 (June 5, 2009): 4782–89. http://dx.doi.org/10.1128/aem.00811-09.
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ABSTRACT The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Bacillus sphaericus strain IAB59 and is uniquely composed of a three-domain Cry protein toxin (Cry48Aa) and a binary (Bin) toxin-like protein (Cry49Aa). Its mode of action has not been elucidated, but a remarkable feature of this protein is the high toxicity against species from the Culex complex, besides its capacity to overcome Culex resistance to the Bin toxin, the major insecticidal factor in B. sphaericus-based larvicides. The goal of this work was to investigate the ultrastructural effects of Cry48Aa/Cry49Aa on midgut cells of Bin-toxin-susceptible and -resistant Culex quinquefasciatus larvae. The major cytopathological effects observed after Cry48Aa/Cry49Aa treatment were intense mitochondrial vacuolation, breakdown of endoplasmic reticulum, production of cytoplasmic vacuoles, and microvillus disruption. These effects were similar in Bin-toxin-susceptible and -resistant larvae and demonstrated that Cry48Aa/Cry49Aa toxin interacts with and displays toxic effects on cells lacking receptors for the Bin toxin, while B. sphaericus IAB59-resistant larvae did not show mortality after treatment with Cry48Aa/Cry49Aa toxin. The cytopathological alterations in Bin-toxin-resistant larvae provoked by Cry48Aa/Cry49Aa treatment were similar to those observed when larvae were exposed to a synergistic mixture of Bin/Cry11Aa toxins. Such effects seemed to result from a combined action of Cry-like and Bin-like toxins. The complex effects caused by Cry48Aa/Cry49Aa provide evidence for the potential of these toxins as active ingredients of a new generation of biolarvicides that conjugate insecticidal factors with distinct sites of action, in order to manage mosquito resistance.
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Koon, Hon Wai, David Q. Shih, Tressia C. Hing, Jun Hwan Yoo, Samantha Ho, Xinhua Chen, Ciarán P. Kelly, Stephan R. Targan, and Charalabos Pothoulakis. "Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes." Antimicrobial Agents and Chemotherapy 57, no. 7 (April 29, 2013): 3214–23. http://dx.doi.org/10.1128/aac.02633-12.
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ABSTRACTClostridium difficileinfection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality.C. difficilemediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blockingC. difficiletoxin A- and toxin B-mediated inflammatory responses and histologic damage.
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Kato, Haru, Naoki Kato, Kunitomo Watanabe, Naoichi Iwai, Haruhi Nakamura, Toshinobu Yamamoto, Kanzo Suzuki, Shin-Moo Kim, Yunsop Chong, and Eddy Bagus Wasito. "Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR." Journal of Clinical Microbiology 36, no. 8 (1998): 2178–82. http://dx.doi.org/10.1128/jcm.36.8.2178-2182.1998.
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Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.
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Jarraud, Sophie, Grégoire Cozon, François Vandenesch, Michèle Bes, Jerome Etienne, and Gerard Lina. "Involvement of Enterotoxins G and I in Staphylococcal Toxic Shock Syndrome and Staphylococcal Scarlet Fever." Journal of Clinical Microbiology 37, no. 8 (1999): 2446–49. http://dx.doi.org/10.1128/jcm.37.8.2446-2449.1999.
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We investigated the involvement of the recently described staphylococcal enterotoxins G and I in toxic shock syndrome. We reexamined Staphylococcus aureus strains isolated from patients with menstrual and nonmenstrual toxic shock syndrome (nine cases) or staphylococcal scarlet fever (three cases). These strains were selected because they produced none of the toxins known to be involved in these syndromes (toxic shock syndrome toxin 1 and enterotoxins A, B, C, and D), enterotoxin E or H, or exfoliative toxin A or B, despite the fact that superantigenic toxins were detected in a CD69-specific flow cytometry assay measuring T-cell activation. Sets of primers specific to the enterotoxin G and I genes (seg andsei, respectively) were designed and used for PCR amplification. All of the strains were positive for seg andsei. Sequence analysis confirmed that the PCR products, corresponded to the target genes. We suggest that staphylococcal enterotoxins G and I may be capable of causing human staphylococcal toxic shock syndrome and staphylococcal scarlet fever.
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Griffiths, S. L., R. A. Finkelstein, and D. R. Critchley. "Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders." Biochemical Journal 238, no. 2 (September 1, 1986): 313–22. http://dx.doi.org/10.1042/bj2380313.
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125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 × 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.
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Ma, Jie, Erich Gulbins, Michael J. Edwards, Charles C. Caldwell, Martin Fraunholz, and Katrin Anne Becker. "Staphylococcus aureus α-Toxin Induces Inflammatory Cytokines via Lysosomal Acid Sphingomyelinase and Ceramides." Cellular Physiology and Biochemistry 43, no. 6 (2017): 2170–84. http://dx.doi.org/10.1159/000484296.
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Background/Aims: Staphylococcus aureus (S. aureus) infections are a major clinical problem and range from mild skin and soft-tissue infections to severe and even lethal infections such as pneumonia, endocarditis, sepsis, osteomyelitis, and toxic shock syndrome. Toxins that are released from S. aureus mediate many of these effects. Here, we aimed to identify molecular mechanisms how α-toxin, a major S. aureus toxin, induces inflammation. Methods: Macrophages were isolated from the bone marrow of wildtype and acid sphingomyelinase-deficient mice, stimulated with S. aureus α-toxin and activation of the acid sphingomyelinase was quantified. The subcellular formation of ceramides was determined by confocal microscopy. Release of cathepsins from lysosomes, activation of inflammasome proteins and formation of Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) were analyzed by western blotting, confocal microscopy and ELISA. Results: We demonstrate that S. aureus α-toxin activates the acid sphingomyelinase in ex vivo macrophages and triggers a release of ceramides. Ceramides induced by S. aureus α-toxin localize to lysosomes and mediate a release of cathepsin B and D from lysosomes into the cytoplasm. Cytosolic cathepsin B forms a complex with Nlrc4. Treatment of macrophages with α-toxin induces the formation of IL-1β and TNF-α. These events are reduced or abrogated, respectively, in cells lacking the acid sphingomyelinase and upon treatment of macrophages with amitriptyline, a functional inhibitor of acid sphingomyelinase. Pharmacological inhibition of cathepsin B prevented activation of the inflammasome measured as release of IL-1β, while the formation of TNF-α was independent of cathepsin B. Conclusion: We demonstrate a novel mechanism how bacterial toxins activate the inflammasome and mediate the formation and release of cytokines: S. aureus α-toxin triggers an activation of the acid sphingomyelinase and a release of ceramides resulting in the release of lysosomal cathepsin B and formation of pro-inflammatory cytokines.
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Morgan, D. G., B. R. DasGupta, G. Stubbs, and J. P. Robinson. "Two dimensional crystals of botulinum toxin, serotype B." Proceedings, annual meeting, Electron Microscopy Society of America 47 (August 6, 1989): 1034–35. http://dx.doi.org/10.1017/s0424820100157152.
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Botulinum toxin is a powerful, protein neurotoxin produced by Clostridium botulinum which exerts its toxic action by inhibiting the release of acetyl choline. There are several immunologically distinguishable types of botulinum toxin and most of these have been shown to bind the ganglioside GTlb. The ganglioside binding property of these neurotoxins has allowed us to prepare two dimensional crystals of serotypes A, B, and E. We report here our preliminary observations of two dimensional crystals of serotype B.Type B botulinum toxin was purified by methods reported by DasGupta and Woody. The two dimensional crystals were prepared by slightly modified procedures used previously to prepare similar crystals of tetanus and cholera toxins. Purified toxin was dialyzed into citric acid-sodium phosphate buffer at pH 4.0 to 6.5 and ionic strength of about 0.04. Dialyzed toxin was placed into the wells of microtiter dishes in 20 μl volumes at a concentration of fifty to one hundred μgm per ml. The toxin solutions were then layered with one to two μl of a solution of one to two mg per ml of egg lecithin (Sigma Cat. No. P- 2772) in chloroform containing five to ten percent by weight of the ganglioside GT1B (Supelco Cat. No. 4-6035). The microtiter dishes were then placed in the cold and crystallization was allowed to proceed for one to four days at 4°C. The crystals were then picked up on carbon coated electron microscope grids, negatively stained with one to two percent uranium acetate and examined in the electron microscope.
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TSIOURIS (Β.Σ. ΤΣΙΟΥΡΗΣ), V. S., I. GEORGOPOULOU (ΓΕΩΡΓΟΠΟΥΛΟΥ), and E. PETRIDOU (Ε. ΠΕΤΡΙΔΟΥ). "Update on the toxins of Clostridium perfringens and their actions." Journal of the Hellenic Veterinary Medical Society 61, no. 3 (November 17, 2017): 241. http://dx.doi.org/10.12681/jhvms.14892.
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Clostridia appeared as a distinct class, approximately 2.7 billion years ago, before the initial formation of oxygen. Clostridium perfringens is widely distributed throughout the environment due to its ability to form spores. Furthermore, it is a member of intestinal microbiota in animals and human. In 2002, the complete genome of C perfringens strain 13 was published. Genomic analysis has revealed that C. perfringens lacks the genetic machinery to produce 13 essential amino acids and it obtains these in vivo via the action of its toxins. Toxins of C perfringens can be divided into major, minor and enterotoxin. C perfringens strains are classified into five toxinotypes (A, B, C, D and E), based on the production of four major toxins. Alpha toxin is the best and most studied major toxin of C perfringens and it was the first bacterial toxin established to possess enzymatic activity. It has haemolytic, necrotic and cytolytic activity, it can lyse platelets and leukocytes and it can damage fibroblasts and muscle cell membranes. Expression of epa gene, which is responsible for the production of alpha toxin by C perfringens, is down-regulated in the normal healthy gut, but it is upregulated to initiate enteric disease in response to an environmental signal. C perfringens appears to be regulated in a quorum sensing manner, using oligopeptides, AI-2 or both, to regulate expression of the epa gene, and thus the synthesis of alpha toxin. Beta toxin is recognized as an important agent in necrotic enteritis of humans and it is the second most lethal C. perfringens toxin following epsilon toxin. Beta toxin is a membrane spanning protein that oligomerizes to form channels in susceptible cells or it primarily acts as a neurotoxin. Epsilon toxin is the most potent of the C. perfringens toxins and the third most potent neurotoxin from the Clostridium spp., following botulinum and tetanus toxins. Epsilon toxin of C perfringens type D causes enterotoxaemia and pulpy kidneys disease of lambs. Iota toxin causes disruption of the actin cytoskeleton and cell barrier integrity and it is the less toxic of the major toxins of C perfringens. Although C perfringens enterotoxin is not classified as one of the major toxins of C perfringens, it is the third most common cause of food poisoning in industrialized nations. It is not secreted by the cells of growing bacteria, but it is released only with the sporulation of C perfringens. Not all strains of C perfringens carry the epe gene, which is responsible for the production of enterotoxin. Theta toxin is a pore-forming cytolysin that can lyse red blood cells. It is produced by all types of C perfringens. Together with alpha-toxin, theta-toxin modulates the host inflammatory response. ß2 toxin is a pore forming toxin which is involved in necrotic enteritis of swine and horse, in haemorragic enteritis of bovine in diarrhea cases of dogs and along with enterotoxin in diarrhea cases of humans. Recently, -NetB, a novel toxin that is associated with broiler necrotic enteritis, has been described. The mechanism of its action seems to involve the formation of small hydrophilic pores. Other toxins of C. perfringens include λ-toxin, ô-toxin, μ-toxin, v-toxin, κ-toxin, a-clostripain like protease and neuraminidase/sialidase. These toxins can act as enzymes, while many of them can act synergically or supplementally with major pore forming toxins. Potentially, C. perfringens might produce more toxins, which have not been identified. Finally, the actions of C. perfringens toxins, major or minor, in some diseases have not been figured out.
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McNichol, Beth A., Rebecca A. Bova, Kieron Torres, Lan N. Preston, and Angela R. Melton-Celsa. "Switching Shiga Toxin (Stx) Type from Stx2d to Stx2a but Not Stx2c Alters Virulence of Stx-Producing Escherichia coli (STEC) Strain B2F1 in Streptomycin (Str)-Treated Mice." Toxins 13, no. 1 (January 15, 2021): 64. http://dx.doi.org/10.3390/toxins13010064.
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Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins.
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Tse, Sirius Pui-kam, Fred Wang-fat Lee, Daniel Yun-lam Mak, Hang-kin Kong, Kenrick Kai-yuen Chan, Pak-yeung Lo, and Samuel Chun-lap Lo. "Production of Paralytic Shellfish Toxins (PSTs) in Toxic Alexandrium catenella is Intertwined with Photosynthesis and Energy Production." Toxins 12, no. 8 (July 27, 2020): 477. http://dx.doi.org/10.3390/toxins12080477.
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To investigate the mechanism for the production of paralytic shellfish toxins (PST) in toxic dinoflagellates, with a 2D-gel based approach, we had made two sets of proteomic comparisons: (a) between a toxic Alexandrium catenella (AC-T) and a phylogenetically closely related non-toxic strain (AC-N), (b) between toxic AC-T grown in a medium with 10% normal amount of phosphate (AC-T-10%P) known to induce higher toxicity and AC-T grown in normal medium. We found that photosynthesis and energy production related proteins were up-regulated in AC-T when compared to AC-N. However, the same group of proteins was down-regulated in AC-T-10%P when compared to normal AC-T. Examining the relationship of photosynthesis and toxin content of AC-T upon continuous photoperiod experiment revealed that while growth and associated toxin content increased after 8 days of continuous light, toxin content maintained constant when cells were shifted from continuous light to continuous dark for 3 days. This emphasized the cruciality of light availability on toxin biosynthesis in AC-T, while another light-independent mechanism may be responsible for higher toxicity in AC-T-10%P compared to normal AC-T. Taken all together, it is believed that the interplay between “illumination”, “photosynthesis”, “phosphate availability”, and “toxin production” is much more complicated than what we had previously anticipated.
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Jagtap, Santosh C., Chandrakant B. Jagtap, Pradeep Kumar, and R. B. Srivastava. "Detection ofBacillus sphaericusmosquitocidal toxin genes by multiplex colony PCR." Canadian Journal of Microbiology 55, no. 2 (February 2009): 207–9. http://dx.doi.org/10.1139/w08-113.
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A multiplex colony PCR assay was developed for the detection of 5 genes encoding Bacillus sphaericus mosquito larvicidal toxins, namely binA, binB, mtx1, mtx2, and mtx3. Primers designed for these 5 genes yielded specific PCR amplicons of the expected size from type cultures of B. sphaericus. This method of detecting multiple toxin genes by colony PCR in a single tube reaction is a simple, rapid, and economical technique for identification of highly toxic environmental B. sphaericus isolates.
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Lin, Qianyun, Nira R. Pollock, Alice Banz, Aude Lantz, Hua Xu, Limei Gu, Dale N. Gerding, et al. "Toxin A–Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype." Clinical Infectious Diseases 70, no. 12 (August 11, 2019): 2628–33. http://dx.doi.org/10.1093/cid/ciz727.
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Abstract Background Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A–negative, toxin B–positive (A−B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. Methods An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). Results There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23–17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). Conclusions We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.
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Kink, John A., and Jim A. Williams. "Antibodies to Recombinant Clostridium difficile Toxins A and B Are an Effective Treatment and Prevent Relapse of C. difficile-Associated Disease in a Hamster Model of Infection." Infection and Immunity 66, no. 5 (May 1, 1998): 2018–25. http://dx.doi.org/10.1128/iai.66.5.2018-2025.1998.
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ABSTRACT Clostridium difficile causes antibiotic-associated diarrhea and colitis in humans through the actions of toxin A and toxin B on the colonic mucosa. At present, broad-spectrum antibiotic drugs are used to treat this disease, and patients suffer from high relapse rates after termination of treatment. This study examined the role of both toxins in pathogenesis and the ability of orally administered avian antibodies against recombinant epitopes of toxin A and toxin B to treat C. difficile-associated disease (CDAD). DNA fragments representing the entire gene of each toxin were cloned, expressed, and affinity purified. Hens were immunized with these purified recombinant-protein fragments of toxin A and toxin B. Toxin-neutralizing antibodies fractionated from egg yolks were evaluated by a toxin neutralization assay in Syrian hamsters. The carboxy-terminal region of each toxin was most effective in generating toxin-neutralizing antibodies. With a hamster infection model, antibodies to both toxins A and B (CDAD antitoxin) were required to prevent morbidity and mortality from infection. In contrast to vancomycin, CDAD antitoxin prevented relapse and subsequent C. difficile reinfection in the hamsters. These results indicate that CDAD antitoxin may be effective in the treatment and management of CDAD in humans.
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Melconian, A. K., J. Fleurette, and Y. Brun. "Studies on staphylococci from toxic shock syndrome in France, 1981–1983." Journal of Hygiene 94, no. 1 (February 1985): 23–29. http://dx.doi.org/10.1017/s002217240006109x.
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SUMMARYStaphylococci from 22 cases of toxic shock syndrome with onsets between 1981 and March 1983 have been studied. Another four cases were detected by abstract surveillance. Three of these patients died. The case histories show that the syndrome occurs in women during menstruation as well as in males and in children, and is associated withStaphylococcus aureusinfections.The production of enterotoxins (A, B, C) and toxic shock toxin byS. aureusisolates from toxic shock syndrome was investigated. Twenty-two of the 23 isolates were found to be toxigenie: 7 produced enterotoxin A, 8 produced enterotoxin B, 3 produced enterotoxin C and 13 produced toxic shock toxin. The latter was found with enterotoxin A in five cases, and with enterotoxins A and B in only one case.Sixty-three percent of 40S. aureusstrains isolated from the vagina of patients with diseases other than toxic shock syndrome produced toxin; eight of these strains produced toxic shock toxin.
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&NA;. "Botulinum toxin B." Reactions Weekly &NA;, no. 1134 (January 2007): 9. http://dx.doi.org/10.2165/00128415-200711340-00032.
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&NA;. "Botulinum toxin B." Reactions Weekly &NA;, no. 1367 (September 2011): 11. http://dx.doi.org/10.2165/00128415-201113670-00030.
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&NA;. "Botulinum toxin B." Reactions Weekly &NA;, no. 1217 (August 2008): 9. http://dx.doi.org/10.2165/00128415-200812170-00027.
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Figgitt, David P., and Stuart Noble. "Botulinum Toxin B." Drugs 62, no. 4 (2002): 705–22. http://dx.doi.org/10.2165/00003495-200262040-00011.
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&NA;. "Botulinum toxin B." Reactions Weekly &NA;, no. 937 (February 2003): 6. http://dx.doi.org/10.2165/00128415-200309370-00021.
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&NA;. "Botulinum toxin B." Reactions Weekly &NA;, no. 950 (May 2003): 7. http://dx.doi.org/10.2165/00128415-200309500-00014.
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&NA;. "Botulinum toxin B." Reactions Weekly &NA;, no. 1071 (October 2005): 7. http://dx.doi.org/10.2165/00128415-200510710-00019.
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Song, Linan, Mingwei Zhao, David C. Duffy, Joshua Hansen, Kelsey Shields, Manida Wungjiranirun, Xinhua Chen, et al. "Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool." Journal of Clinical Microbiology 53, no. 10 (July 22, 2015): 3204–12. http://dx.doi.org/10.1128/jcm.01334-15.
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The currently available diagnostics forClostridium difficileinfection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenicC. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinicalC. difficileisolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.
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Llewellyn-Smith, I. J., C. L. Martin, L. F. Arnolda, and J. B. Minson. "Tracer-toxins: cholera toxin B-saporin as a model." Journal of Neuroscience Methods 103, no. 1 (November 2000): 83–90. http://dx.doi.org/10.1016/s0165-0270(00)00298-3.
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Janezic, Sandra, Mercedes Marín, Adoración Martín, and Maja Rupnik. "A New Type of Toxin A-Negative, Toxin B-Positive Clostridium difficile Strain Lacking a CompletetcdAGene." Journal of Clinical Microbiology 53, no. 2 (November 26, 2014): 692–95. http://dx.doi.org/10.1128/jcm.02211-14.
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Toxins A and B are the main virulence factors ofClostridium difficileand are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficiletransferase)-negative (A−B+CDT−) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) withouttcdAand with atypical organization of the PaLoc integration site.
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Drudy, Denise, Séamus Fanning, and Lorraine Kyne. "Toxin A-negative, toxin B-positive Clostridium difficile." International Journal of Infectious Diseases 11, no. 1 (January 2007): 5–10. http://dx.doi.org/10.1016/j.ijid.2006.04.003.
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Tapp, H., and G. Stotzky. "Monitoring the insecticidal toxins fromBacillus thuringiensisin soil with flow cytometry." Canadian Journal of Microbiology 43, no. 11 (November 1, 1997): 1074–78. http://dx.doi.org/10.1139/m97-153.
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The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in environmental hazards, such as toxicity to nontarget species and the selection of toxin-resistant target species. We describe the use of flow cytometry as a method for detecting and tracking the fate of these insecticidal toxins in soil that does not require their extraction and purification. The toxins from B. thuringiensis subspp. tenebrionis and kurstaki were bound on clay- or silt-sized particles separated from Kitchawan soil that was unamended (naturally contains predominantly kaolinite) or amended to 6% v/v with the clay minerals montmorillonite or kaolinite (as an internal control). The particle–toxin mixtures were suspended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powder to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, and then resuspended in a solution of anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC–Ab). Controls consisted of the particles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstaki. All particles that bound the toxin from B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with the nonspecific fluorescence from the control FITC–Ab complexes with particles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity between the toxins from B. thuringiensis subspp. tenebrionis and kurstaki and the antibodies that they induce. This method is more sensitive and rapid than the dot-blot ELISA, and processing of many samples is easily accomplished.Key words: flow cytometry, soil, insecticidal toxins, Bacillus thuringiensis, clay, silt.
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Funk, J., N. Biber, M. Schneider, E. Hauser, S. Enzenmüller, C. Förtsch, H. Barth, and H. Schmidt. "Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli." Infection and Immunity 83, no. 6 (March 30, 2015): 2338–49. http://dx.doi.org/10.1128/iai.00231-15.
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In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2of Shiga toxin-producingEscherichia coliwas determined and compared to the plasmid-encoded SubAB1and the chromosome-encoded SubAB2-1variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1exhibited cytotoxic effects in the absence of the respective B1subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1alone induced apoptosis, while the B1subunit alone did not induce cell death.
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Castagliuolo, Ignazio, Martin F. Riegler, Leyla Valenick, J. Thomas LaMont, and Charalabos Pothoulakis. "Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa." Infection and Immunity 67, no. 1 (January 1, 1999): 302–7. http://dx.doi.org/10.1128/iai.67.1.302-307.1999.
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ABSTRACT Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibitsC. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardiion toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects ofS. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii onC. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease.
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Dorca-Arévalo, Jonatan, Inmaculada Gómez de Aranda, and Juan Blasi. "New Mutants of Epsilon Toxin from Clostridium perfringens with an Altered Receptor-Binding Site and Cell-Type Specificity." Toxins 14, no. 4 (April 16, 2022): 288. http://dx.doi.org/10.3390/toxins14040288.
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Epsilon toxin (Etx) from Clostridium perfringens is the third most potent toxin after the botulinum and tetanus toxins. Etx is the main agent of enterotoxemia in ruminants and is produced by Clostridium perfringens toxinotypes B and D, causing great economic losses. Etx selectively binds to target cells, oligomerizes and inserts into the plasma membrane, and forms pores. A series of mutants have been previously generated to understand the cellular and molecular mechanisms of the toxin and to obtain valid molecular tools for effective vaccination protocols. Here, two new non-toxic Etx mutants were generated by selective deletions in the binding (Etx-ΔS188-F196) or insertion (Etx-ΔV108-F135) domains of the toxin. As expected, our results showed that Etx-ΔS188-F196 did not exhibit the usual Etx binding pattern but surprisingly recognized specifically an O-glycoprotein present in the proximal tubules of the kidneys in a wide range of animals, including ruminants. Although diminished, Etx-ΔV108-F135 maintained the capacity for binding and even oligomerization, indicating that the mutation particularly affected the pore-forming ability of the toxin.
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Cowardin, Carrie A., Brianna M. Jackman, Zannatun Noor, Stacey L. Burgess, Andrew L. Feig, and William A. Petri. "Glucosylation Drives the Innate Inflammatory Response to Clostridium difficile Toxin A." Infection and Immunity 84, no. 8 (June 6, 2016): 2317–23. http://dx.doi.org/10.1128/iai.00327-16.
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Clostridium difficileis a major, life-threatening hospital-acquired pathogen that causes mild to severe colitis in infected individuals. The tissue destruction and inflammation which characterizeC. difficileinfection (CDI) are primarily due to the Rho-glucosylating toxins A and B. These toxins cause epithelial cell death and induce robust inflammatory signaling by activating the transcription factor NF-κB, leading to chemokine and cytokine secretion. The toxins also activate the inflammasome complex, which leads to secretion of the pyrogenic cytokine IL-1β. In this study, we utilized glucosylation-deficient toxin A to show that activation of the inflammasome by this toxin is dependent on Rho glucosylation, confirming similar findings reported for toxin B. We also demonstrated that tissue destruction andin vivoinflammatory cytokine production are critically dependent on the enzymatic activity of toxin A, suggesting that inhibiting toxin glucosyltransferase activity may be effective in combating this refractory disease.
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Yi, S., A. S. D. Pang, and K. van Frankenhuyzen. "Immunocytochemical localization of Bacillus thuringiensis Cryl toxins in the midguts of three forest insects and Bombyx mori." Canadian Journal of Microbiology 42, no. 7 (July 1, 1996): 634–41. http://dx.doi.org/10.1139/m96-087.
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Light microscopy was used to investigate the relation between toxicity, cytopathological effects, and in vivo binding of Bacillus thuringiensis CrylA(b) and CrylE toxin proteins in larvae of Lymantria dispar, Choristoneura fumiferana, Actebia fennica, and Bombyx mori. These target insects were selected for their contrasting susceptibility to the two toxins. Lymantria dispar is susceptible to CrylA(b), B. mori is susceptible to CrylE, C. fumiferana is susceptible to both, and A. fennica is not susceptible to either. In the susceptible species, both toxins caused typical pathological changes in midgut epithelial cells, including disruption and shedding of the brush border membrane, vacuolization of the cytoplasm, and swelling of the cells and their nuclei, followed by disintegration and release of cytoplasmic content into the lumen. In the highly resistant A. fennica, no cell damage was observed, but the midguts of toxin-fed larvae had a shrunken appearance. Immunohistochemical staining of midgut sections from toxin-fed larvae revealed that the toxins bound to the microvilli of the midgut epithelial cells of susceptible species only, with the exception of B. mori. In this species, the CrylA(b) toxin bound to the apical microvilli without causing cell damage or larval death. In vivo binding of toxins is thus not always correlated with larval toxicity. Accumulation of the toxins at the peritrophic membrane depended on both toxin and insect species and was not correlated with larval toxicity.Key words: Bacillus thuringiensis, Cryl toxins, forest Lepidoptera, immunocytochemical localization, cytopathology.
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Perry, Michael, Lee Graham, Sweta Parida, Phoebe Katzenbach, Jose Luis Baptista Baeza, Joel Estis, Johanna Sandlund, et al. "2357. Toxin Detection Using Single Molecule Counting Technology: The Best of Both Worlds?" Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S812. http://dx.doi.org/10.1093/ofid/ofz360.2035.
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Abstract Background Accurate diagnosis of CDI remains challenging as there is no standalone laboratory test with adequate clinical sensitivity and specificity. Thus, many clinical laboratories currently employ a multistep algorithm incorporating a sensitive screening test followed by a specific toxin test. An automated ultrasensitive toxin immunoassay (Singulex Clarity® C. difficile toxins A/B assay) has demonstrated excellent performance compared with cell cytotoxicity neutralization assay (CCNA). In this study, the Clarity assay was evaluated relative to glutamate dehydrogenase (GDH), toxin EIA, toxin B gene PCR, multistep algorithms, and C. difficile culture with ribotyping. Methods Residual clinical stool samples (n = 293) were collected from patients with suspected CDI. The samples were tested on-site with GDH (C. DIFF CHEK™-60), PCR (EntericBio realtime® C. difficile assay), a membrane-type toxin EIA (Tox A/B Quik Chek®), and culture and ribotyping. In total, 188 samples were tested with GDH and 239 samples were tested by PCR. All PCR-positive samples (n = 148) and prospectively tested GDH samples (n = 97) were tested with the toxin EIA. Culture and ribotyping information were available for 205 samples. Results Three of the samples tested gave no result using the Clarity assay and were excluded from the analysis. The Singulex Clarity C. difficile toxins A/B assay had high positive percent agreement (PPA) and low negative percent agreement (NPA) compared with toxin EIA and multistep algorithms ending with toxin EIA. The Clarity assay had high NPA and low PPA compared with PCR, GDH, and the multistep algorithm ending with PCR (figure). Less than 70% of the detected C. difficile PCR positive samples had toxins present. There was no difference in toxin concentration between the ribotypes. Conclusion The Clarity assay had strong PPA compared with toxin EIA and strong NPA compared with PCR. The low NPA and PPA compared with toxin EIA and PCR, respectively, may reflect the poor sensitivity of current toxin EIAs and low specificity of PCR. The Clarity assay detected 30 different ribotype strains, and less than 70% of samples (by PCR) or strains (by ribotyping) had toxins present. The Clarity assay may be considered for use as a standalone test for CDI diagnosis. Disclosures All authors: No reported disclosures.
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Servant, Pascale, Marie-Laure Rosso, Sylviane Hamon, Sandrine Poncet, Armelle Delécluse, and Georges Rapoport. "Production of Cry11A and Cry11Ba Toxins in Bacillus sphaericus Confers Toxicity towards Aedes aegyptiand Resistant Culex Populations." Applied and Environmental Microbiology 65, no. 7 (July 1, 1999): 3021–26. http://dx.doi.org/10.1128/aem.65.7.3021-3026.1999.
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ABSTRACT Cry11A from Bacillus thuringiensis subsp.israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on theB. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegyptiand Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegyptilarvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE andCulex quinquefasciatus GeoR to B. sphaericus strain 2297.
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Kaiser, Eva, Claudia Kroll, Katharina Ernst, Carsten Schwan, Michel Popoff, Gunter Fischer, Johannes Buchner, Klaus Aktories, and Holger Barth. "Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90." Infection and Immunity 79, no. 10 (July 18, 2011): 3913–21. http://dx.doi.org/10.1128/iai.05372-11.
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ABSTRACTSome hypervirulent strains ofClostridium difficileproduce the binary actin-ADP-ribosylating toxinC. difficiletransferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity ofC. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin fromClostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin Ain vitro. In combination with our recently obtained data on the C2 toxin fromC. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.
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Davies, D. G., and R. S. Dhillon. "Botulinum toxin." BMJ 298, no. 6672 (February 25, 1989): 522. http://dx.doi.org/10.1136/bmj.298.6672.522-b.
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Hofmann, Fred, Christian Busch, and Klaus Aktories. "Chimeric Clostridial Cytotoxins: Identification of the N-Terminal Region Involved in Protein Substrate Recognition." Infection and Immunity 66, no. 3 (March 1, 1998): 1076–81. http://dx.doi.org/10.1128/iai.66.3.1076-1081.1998.
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ABSTRACT Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C. sordelliilethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C. sordellii lethal toxin and C. difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C. sordellii lethal toxin.
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Mullan, N., K. R. Hughes, and Y. R. Mahida. "Primary Human Colonic Myofibroblasts Are Resistant toClostridium difficileToxin A-Induced, but Not Toxin B-Induced, Cell Death." Infection and Immunity 79, no. 4 (January 18, 2011): 1623–30. http://dx.doi.org/10.1128/iai.00686-10.
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ABSTRACTColonic inflammation inClostridium difficileinfection is mediated by released toxins A and B. We investigated responses toC. difficiletoxins A and B by isolated primary human colonic myofibroblasts, which represent a distinct subpopulation of mucosal cells that are normally located below the intestinal epithelium. Following incubation with either purified toxin A or B, there was a change in myofibroblast morphology to stellate cells with processes that were immunoreactive for alpha-smooth muscle actin. Most of the myofibroblasts remained viable, with persistence of stellate morphology, despite exposure to high concentrations (up to 10 μg/ml) of toxin A for 72 h. In contrast, a majority of the toxin B-exposed myofibroblasts lost their processes prior to cell death over 24 to 72 h. At low concentrations, toxin A provided protection against toxin B-induced cell death. Within 4 h, myofibroblasts exposed to either toxin A or toxin B lost expression of the nonglucosylated form of Rac1, and there was also a loss of the active form of RhoA. Despite preexposure to high concentrations of toxin A for 3 h, colonic myofibroblasts were able to recover their morphology and proliferative capacity during prolonged culture in medium. However, toxin B-preexposed myofibroblasts were not able to recover. In conclusion, primary human colonic mucosal myofibroblasts are resistant to toxin A (but not toxin B)-induced cell death. Responses by colonic myofibroblasts may play an important role in mucosal protection, repair, and regeneration in colitis due toC. difficileinfection.
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Blake, J. E., F. Mitsikosta, and M. A. Metcalfe. "Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants." Journal of Medical Microbiology 53, no. 3 (March 1, 2004): 197–205. http://dx.doi.org/10.1099/jmm.0.05404-0.
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Torres, Javier F., and Ivar Lönnroth. "Production, purification and characterization of Clostridium difficile toxic proteins different from toxin A and from toxin B." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 998, no. 2 (October 1989): 151–57. http://dx.doi.org/10.1016/0167-4838(89)90267-7.
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Genth, Harald, Jörg Selzer, Christian Busch, Jürgen Dumbach, Fred Hofmann, Klaus Aktories, and Ingo Just. "New Method To Generate Enzymatically DeficientClostridium difficile Toxin B as an Antigen for Immunization." Infection and Immunity 68, no. 3 (March 1, 2000): 1094–101. http://dx.doi.org/10.1128/iai.68.3.1094-1101.2000.
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ABSTRACT The family of the large clostridial cytotoxins, encompassingClostridium difficile toxins A and B as well as the lethal and hemorrhagic toxins from Clostridium sordellii, monoglucosylate the Rho GTPases by transferring a glucose moiety from the cosubstrate UDP-glucose. Here we present a new detoxification procedure to block the enzyme activity by treatment with the reactive UDP-2′,3′-dialdehyde to result in alkylation of toxin A and B. Alkylation is likely to occur in the catalytic domain, because the native cosubstrate UDP-glucose completely protected the toxins from inactivation and the alkylated toxin competes with the native toxin at the cell receptor. Alkylated toxins are good antigens resulting in antibodies recognizing only the C-terminally located receptor binding domain, whereas formaldehyde treatment resulted in antibodies recognizing both the receptor binding domain and the catalytic domain, indicating that the catalytic domain is concealed under native conditions. Antibodies against the native catalytic domain (amino acids 1 through 546) and those holotoxin antibodies recognizing the catalytic domain inhibited enzyme activity. However, only antibodies against the receptor binding domain protected intact cells from the cytotoxic activity of toxin B, whereas antibodies against the catalytic domain were protective only when inside the cell.
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Sato, Shigeru, Yoshinobu Takata, Sunaho Kondo, Akiko Kotoda, Naoto Hongo, and Masaaki Kodama. "Quantitative ELISA Kit for Paralytic Shellfish Toxins Coupled with Sample Pretreatment." Journal of AOAC INTERNATIONAL 97, no. 2 (March 1, 2014): 339–44. http://dx.doi.org/10.5740/jaoacint.sgesato.
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Abstract A new ELISA kit to quantitate the level of paralytic shellfish poisoning (PSP) toxins in crude shellfish extracts was developed. A conjugate for preparing antigen and a novel antibody used in the ELISA wasprepared based on the unique reactions between C11-O-sulfate toxins such as gonyautoxins 2 and 3 (GTX2,3) and various thiol compounds, followed by coupling to keyhole limpet hemocyanin. The compounds necessary for competitive ELISA, labeled toxin and an artificial standard toxin to replace saxitoxin in the analysis, were also produced by the same techniques. Theresulting ELISA recognized all the toxin components tested; however, carbamoyl-N-sulfate derivatives such as B and C toxins and N1-OH toxins such as neoSTX and GTX1,4 showed low affinity to the antibody. The difference in the reactivity of the antibody observed among the toxin components prevents accurate quantification of the toxin amounts in shellfish extracts. To address this problem, the former toxin components were transformed to corresponding carbamate toxins by mild HCl treatment according to a conventional method. The reduction of N1-OH of the latter toxins to N1-H was performed by our original method using hemin as a catalyst. We report here the new ELISA kitcoupled with the pretreatment process to transform the toxin components favorable for the quantitative analysis of PSP toxins.
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Wolf, Anne A., Michael G. Jobling, David E. Saslowsky, Eli Kern, Kimberly R. Drake, Anne K. Kenworthy, Randall K. Holmes, and Wayne I. Lencer. "Attenuated Endocytosis and Toxicity of a Mutant Cholera Toxin with Decreased Ability To Cluster Ganglioside GM1 Molecules." Infection and Immunity 76, no. 4 (January 22, 2008): 1476–84. http://dx.doi.org/10.1128/iai.01286-07.
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ABSTRACT Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM1. The homopentomeric B-subunit of the toxin can bind up to five GM1 molecules at once. Here, we examined the role of polyvalent binding of GM1 in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM1. The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM1 when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM1 molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM1 molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.
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Kovbasnjuk, Olga, Michael Edidin, and Mark Donowitz. "Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells." Journal of Cell Science 114, no. 22 (November 15, 2001): 4025–31. http://dx.doi.org/10.1242/jcs.114.22.4025.
Full textAbstract:
Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens. The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb3, on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome. Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization. Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb3 in lipid rafts with another glycosphingolipid GM1 on the apical surface of highly polarized intestinal epithelial Caco-2 cells. Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit. Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells.
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See, Raymond H., Gerald Krystal, and Anthony W. Chow. "Receptors for toxic shock syndrome toxin-1 and staphylococcal enterotoxin A on human blood monocytes." Canadian Journal of Microbiology 38, no. 9 (September 1, 1992): 937–44. http://dx.doi.org/10.1139/m92-151.
Full textAbstract:
Staphylococcal toxic shock syndrome toxin-1 (TSST-1) as well as staphylococcal enterotoxin A (SEA) and B (SEB) have recently been shown to bind directly to the class II major histocompatibility antigen, HLA-DR. Whereas others have characterized TSST-1 and SEA binding to HLA-DR on transfected L cells or B lymphoma cell lines, we sought evidence for direct binding of TSST-1 and SEA to HLA-DR on purified human monocytes. A single class of high-affinity receptors was found for both TSST-1 (dissociation constant (Kd) 40 nM, 3.4 × 104 receptors per cell) and SEA (Kd 12 nM, 3.2 × 104 receptors per cell) on normal human monocytes. Affinity cross-linking of 125I-labeled toxins to monocytes revealed the presence of two membrane protein subunits with molecular masses consistent with the α and β chains of human HLA-DR (35 and 28 kDa, respectively). The anti-HLA-DR monoclonal antibody L243, but not L203 or 2.06, inhibited radiolabeled toxin binding to human monocytes and neutralized the mitogenic response of human T lymphocytes to both toxins. However, L243 was consistently more effective in blocking radiolabeled TSST-1 than SEA binding to human monocytes from the same donors, suggesting that TSST-1 and SEA may be binding to overlapping epitopes rather than to the same epitope on HLA-DR. Because TSST-1 and SEB bind to distinct epitopes on HLA-DR and because SEA cross competes with both TSST-1 and SEB on the HLA-DR receptor, we postulate that SEA occupies a binding site within HLA-DR that overlaps both TSST-1 and SEB. Future studies focused on receptor-mediated binding of these toxins to human monocytes and T lymphocytes from normal donors and toxic shock syndrome patients may reveal the underlying anomalies that predispose particular individuals to toxic shock syndrome. Key words: monocytes, staphylococcal toxic shock syndrome toxin-1, receptors, HLA-DR, staphylococcal enterotoxin A.
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Li, Lu, Leah E. Cole, Utsav Jetley, Jinrong Zhang, Kristl Pacheco, Fuqin Ma, Jianxin Zhang, et al. "Increasing Domain Coverage Improves Neutralizing Potency of C. difficile Toxin-specific Antibodies." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 117.38. http://dx.doi.org/10.4049/jimmunol.200.supp.117.38.
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Abstract Clostridiumdifficile (C.difficile)is a significant human pathogen. C.difficile infection (CDI) causesclinical symptoms ranging from diarrhea to life-threatening fulminant pseudo membranous colitis. The pathogenesis of C. difficile is mediated by two large exotoxins, toxins A and B. These two toxins are highly homologous, single chain proteins consisting of four functional domains: N-terminal glucosyl transferase domain (GTD), cysteine protease domain (CPD), translocation domain (TLD) and a C-terminal receptor-binding domain (RBD). The important role played by anti-toxin sera and antibodies in the prevention of primary and recurrent CDI has been described and demonstrated in both clinical and pre-clinical studies; however, work focused on the impact of the toxin domain specificity of these antibodies is limited. To address this deficit, sera from a C. difficile vaccine immunized hamsters and toxin-specific human monoclonal antibodies (mAbs) were used to assess the impact of toxin domain specificity on antibody mediated inhibition of cytotoxicity in a Vero cell-based functional assay. Results from toxin domain immunoabsorption assays using hamster anti-toxinsera indicated that no single domain fragment from either toxin A or toxin B could inhibit neutralizing activities. Anti-toxin A activity was prevented with a combination of GTD and CTD fragments while anti-toxin B activity required the GTD, CTD and CPD fragments to block activity. Assays with human mAbs demonstrated that combining mAbs that target multiple toxin domains greatly improves neutralizing potency when compared to equivalent concentrations of either a single mAb or a combination of mAbs against a single domain.
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From, Cecilie, Rudiger Pukall, Peter Schumann, Víctor Hormazábal, and Per Einar Granum. "Toxin-Producing Ability among Bacillus spp. Outside the Bacillus cereus Group." Applied and Environmental Microbiology 71, no. 3 (March 2005): 1178–83. http://dx.doi.org/10.1128/aem.71.3.1178-1183.2005.
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ABSTRACT A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42°C, and no reduction in activity was observed even after 24 h of growth at 42°C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22°C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.
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Klyueva, S. N., T. N. Shchukovskaya, T. P. Shmel’Kova, A. L. Kravtsov, S. A. Bugorkova, N. I. Smirnova, O. A. Volokh, et al. "Immunobiological Characteristics of Cholera Toxin B-Subunit." Problems of Particularly Dangerous Infections, no. 3(113) (June 20, 2012): 67–70. http://dx.doi.org/10.21055/0370-1069-2012-3-67-70.
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Carried out was immunobiological evaluation of cholera toxin B subunit preparations obtained using different experimental and production methods. It was demonstrated that B subunit preparations were non-toxic for biomodels and did not cause significant pathological alterations in their organs and tissues. They also did not alter the condition of immunocompetent cells at different stages of their cycle, and promote anti-toxic antibodies production. Thus, B subunit preparations under study can be considered as promising components of cholera vaccine.