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1
Javid-Khojasteh, Vahideh. "Toxic Shock Syndrome Toxin-1 : detection of the toxin, anti-toxin antibodies and producer organisms in a paediatric burns unit." Thesis, University of Salford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365993.
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Guttenberg, Gregor [Verfasser], and Manfred [Akademischer Betreuer] Jung. "Clostridiale Glukosylierende Toxine: Untersuchungen zur Autoprozessierung von Clostridium sordellii Letalem Toxin und Clostridium novyi alpha-Toxin sowie funktionelle Charakterisierung von Clostridium perfringens TpeL-Toxin." Freiburg : Universität, 2012. http://d-nb.info/1123467994/34.
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Maldonado-Arocho, Francisco J. "Characterization of host-pathogen interaction of two bacterial toxins anthrax edema toxin and Escherichia coli cytolethal distending toxin /." Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1973060671&sid=4&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Fernandes, da Costa Sérgio Paulo. "Molecular and structural characterisation of epsilon toxin and necrotic enteritis toxin B : two pore-forming toxins from Clostridium perfringens." Thesis, University of Exeter, 2013. http://hdl.handle.net/10871/14608.
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Epsilon toxin (Etx) and necrotic enteritis toxin B (NetB) are two pore-forming toxins produced by C. perfringens. While Etx has been shown to be the key virulence factor for enterotoxemia in goats and sheep, NetB has been associated with the pathogenesis of avian necrotic enteritis (NE), a gastro-intestinal disease causing economic damage to the poultry industry worldwide. The crystal structure of Etx H149A (an Etx variant with 6x reduced toxicity relative to wild type toxin) was solved to 2.4 Å and showed that the H149A mutation in domain III does not affect organization of the receptor binding region in domain I. The Etx H149A structure also revealed a second putative glycan binding site in domain III. In addition, site-directed mutagenesis in domain I of Etx H149A affirmed the important role of tyrosine residues for toxin binding and demonstrated the capability of Etx H149A to be used as a platform for further receptor binding studies in the future. The crystal structure of the pore-form of NetB was solved to 3.9 Å and revealed high similarities to the Staphylococcus aureus α-hemolysin heptameric structure. However, in particular the region thought to interact with the target cell membrane showed some interesting divergence in amino acid composition. Site-directed mutagenesis within this domain significantly affected binding and toxicity of NetB to target cells. Mutagenesis within the β-sandwich domain of NetB revealed important amino acid residues for toxin oligomerisation and pore-formation. In order to test NetB toxoids as candidate vaccines, a NetB genetic toxoid and a formaldehyde NetB toxoid were used to immunise poultry in an in vivo NE disease model. Vaccination with any of the two antigens resulted in the induction of specific antibody responses against NetB and provided significant protection against disease.
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Hovey, Bianca T. "Cholera toxin and heat-labile enterotoxin : structural studies of assembly and design of active A-subunit constructs /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/9263.
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Edwards-Jones, Valerie. "Toxic shock syndrome toxin production in relation to burned patients." Thesis, University of Salford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244871.
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Kuk, Chiu Ying. "Anthrax Lethal Toxin Is a Tumor Hemorragic Toxin." Thesis, Van Andel Research Institute, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10973827.
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Blood supply is crucial for tumor growth and metastasis. However, current anti-angiogenic therapy is not as effective as predicted, thus a better understanding of the tumor angiogenic process and new anti-angiogenic agent are urgently required. Anthrax lethal toxin (LeTx) has an anti-angiogenic effect on tumors. Tumors treated with LeTx are smaller, paler, and have lower mean vessel density compared to control treated tumors. Most interestingly, compared to current anti-angiogenic treatment, LeTx does not cause normalization of tumor vessels. Instead, tumors treated with LeTx have massive hemorrhages, pointing to a potential alternative mechanism to inhibit tumor angiogenesis. I hypothesize that instead of causing “normalization” of tumor vasculature, LeTx’s anti-angiogenic effects works in a manner similar to a hemorrhagic toxins. To test this hypothesis, I compared the effect of LeTx to snake venom metalloproteinase, a known hemorrhagic toxin, in tumor vasculature. Quantified by Nuance multispectral imaging system, both LeTx and SVMP caused an increase in tumor hemorrhage. Futher analysis of vasculature integrity using continued vessel length showed disruption of vessels by LeTx and SVMP. With these results, I conclude that the anti-angiogenic effects of LeTx are due to its hemorrhagic nature, and not due to normalization of tumor vasculature. Further understanding of LeTx mechanism can help design novel anti-angiogenic agent that compliments current therapy.
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Pellino, Christine A. "Characterization of Shiga Toxin Potency and Assembly." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1418909563.
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Penha, Marcelo De Luca. "Detecção dos genes das toxinas alfa, beta e épsilon de Clostridium perfringens isolados a partir de amostras clínicas de bovinos pela reação em cadeia da polimerase." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-06072005-101119/.
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O Clostridium perfringens é um microrganismo anaeróbio que está presente no solo e no trato intestinal dos mamíferos. Provoca intoxicação alimentar nos seres humanos, doenças enterotoxêmicas nos animais domésticos e gangrena gasosa em ambos os grupos. O C. perfringens é classificado em cinco tipos (A, B, C, D e E) mediante a produção de quatro toxinas principais (alfa, beta, épsilon e iota). Neste trabalho foi possível padronizar a técnica de PCR para detectar a presença dos genes cpa, cpb e etx a partir de culturas de C. perfringens. A sensibilidade analítica da técnica de PCR a partir de culturas de C. perfringens foi de 2,27 ng/µL para o gene cpa, 22,7 pg/µL para o gene cpb e 22,7 pg/µL para o gene etx. A pesquisa dos genes cpa, cpb e etx partir de 35 amostras de C. perfringens isoladas de bovinos revelou que 16 (45,7%) eram do tipo A; 18 (51,4%) eram do tipo C e 1 (2,9%) era do tipo B. Não foi observada nenhuma amostra do tipo D. A metodologia de PCR revelou-se útil na tipificação de amostras de C. perfringens isoladas de bovinos, contribuindo para o diagnóstico dessa bacteriose neste país, eliminando as dificuldades de tipificação oriundas do alto custo e da indisponibilidade de anti-soros para a tipificação pela reação de soroneutralização e evitando a utilização de animais de laboratório.Clostridium perfringens is an anaerobic micro-organism that is present in the soil and gastrointestinal tract of mammals. It causes food poisoning in humans, enterotoxemic diseases in domestic animals and gas gangrene in both. C. perfringens is classified into five types (A, B, C, D and E) according to the production of four major toxins (alpha, beta, epsilon and iota). In this trial was possible to standardize the PCR?s technique to detect cpa, cpb and etx genes from cultures of C. perfringens. PCR?s analythical sensibility was 2.27 ng/µL for cpa gene, 22.7 pg/µL for cpb gene and 22.7 pg/µL for etx gene. The research of cpa, cpb and etx genes from 35 samples of C. perfringens isolated from cattle reveals that 16 (45.7%) were classified as type A, 18 (51.4%) as type C and 1 (2.9%) as type B. No sample of type D was observed. PCR?s technique reveals to be usefull to typify samples of C. perfringens isolated from cattle, contributing to diagnose of this bacterial disease in this country and solving typifing problems represented by the high costs of the process and by the lack of antiserum that is required to typify the micro-organism by seroneutralization. PCR?s technique avoid the use of laboratory animals, too.
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Rosten, Patricia Melanie. "The role of toxic shock syndrome toxin-1 in the pathogenesis of toxic shock syndrome." Thesis, University of British Columbia, 1986. http://hdl.handle.net/2429/26527.
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Toxic shock syndrome toxin-1 (TSST-1), an exoprotein produced by some strains of Staphylococcus aureus, is implicated in the pathogenesis of menstrual TSS. However, its role in nonmenstrual TSS is less certain. In order to study the pathogenetic role of TSST-1 in TSS, three approaches were taken: a) to develop an ELISA for detection of TSST-1 in biologic fluids in order to verify TSST-1 production in vivo in TSS patients, b) to quantitate TSST-1 specific antibodies in the serum of TSS patients and controls to determine whether such antibodies are protective, and c) to attempt to identify other staphylococcal products which may be implicated in some forms of TSS.
A sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) capable of detecting TSST-1 at concentrations from 0.5 to 16 ng/ml was developed. This assay did not detect other staphylococcal enterotoxins including A, B, C₁, C₂, C₃, D and E. Possible interference by protein A was readily eliminated by pretreatment of test samples with 10% nonimmune rabbit serum. The assay was adapted for rapid screening of TSST-1 production by S. aureus isolates in culture supernatants in vitro, and for the detection of TSST-1 in vaginal washings and urine of TSS patients and healthy controls in vivo. All 35 S. aureus isolates confirmed to be TSST-1 positive by Ouchterlony immunodiffusion, and 59 of 60 isolates confirmed to be TSST-1 negative, gave concordant results by ELISA. Interestingly, toxigenic S. aureus strains isolated from TSS patients quantitatively produced significantly more toxin in vitro compared to toxigenic control strains (p<0.05, Mann-Whitney rank sum test). TSST-1 could be detected by ELISA in 3 of 4 vaginal washings collected within 3 days of hospitalization from 3 women with acute menstrual TSS, compared to 0 of 17 washings from 9 TSS women collected greater than 3 days after hospitalization (p=0.003, Fisher's exact test) and 1 of 15 washings from 14 healthy control women (p=0.016). TSST-1 was not detected in the urine of 4 acute TSS patients, 2 convalescent TSS patients or in 3 control urine tested.
A sensitive and reproducible ELISA was also developed for the quantitation of TSST-1 specific IgG in serum. Anti-TSST-1 was assessed in acute and convalescent sera from 16 nonmenstrual (9 female, 7 male) and 14 menstrual TSS patients, and from 87 healthy women and 66 healthy men as controls. Quantitative levels of anti-TSST-1 in the study groups were calculated as the percent of standard activity (POSA) relative to a medium titre reference serum standard. ELISA titers in acute sera from menstrual TSS (26.2 ± 5.2, mean POSA ± S.E.M.), but not nonmenstrual TSS women (71.8 ± 18.6), were significantly lower than in healthy controls (78.9 ± 7.3) (p<0.01, Mam-Whitney test). Titers from menstrual TSS patients remained low (25.2 ± 10.7) even during late convalescence (mean duration 20 months after illness onset), compared to healthy female controls (p<0.05). Acute titers in males with TSS (37.0 ± 15.6) were also significantly lower than those in control men (114.6 + 11.0) (p<0.05). An inverse relationship of recovery of toxigenic S. aureus and anti-TSST-1 titers in acute sera of TSS patients was observed. Interestingly, antibody titers in control men were significantly higher than in control women (p<0.001). No age-dependent effects or interactive effects of age and sex on ELISA titers were observed.
To enable immunoblot analyses, TSST-1 was produced and partially purified using column chromatography techniques. Percent recovery of TSST-1 from culture supernatant through to the final procedure was approximately 15.5%. The relative purity of TSST-1 (TSST-l/total protein, w/w) was increased from 0.21% in culture supernatants to 94.4% in the final product. Ouchterlony immunoprecipitation against reference rabbit antitoxin demonstrated identity with reference TSST-1 as well as with TSST-1 prepared in other laboratories. Physical characterization demonstrated a molecular weight of 24 kd and a pi of 7.0. Using pooled normal human serum as a first antibody probe, several bands in addition to the 24 kd TSST-1 band were visualized by immunoblot against our partially purified toxin as well as similar preparations obtained from other investigators. To determine whether any of the additional bands might be implicated in TSS, acute and convalescent sera from TSS patients were used to probe for immunoreactive bands in our partially purified TSST-1 as well as a commercially obtained preparation. Seroconversion was demonstrated to the 24 kd TSST-1 protein in 7 of 10 TSS patients from whom toxigenic S. aureus was isolated. In addition, seroconversion was noted to a 49 kd band in 4 patients, to a 21 kd band in 3 patients, to a 28 kd band in 1 patient and to a 32 kd band in 2 patients.
In conclusion: 1) the ability to measure TSST-1 in biologic fluids lends stronger support for the role of TSST-1 in menstrual TSS patients; 2) the serologic data support the etiologic role of TSST-1 in menstrual TSS and in nonmenstrual TSS patients from whom toxigenic S. aureus could be cultured, but not for nonmenstrual TSS women from whom toxigenic S. aureus was not isolated; 3) immunoblotting results with acute and convalescent sera from TSS and control patients, not only add further support to the role of TSST-1 in patients from whom toxigenic S. aureus could be isolated, but also indicate that there may be several other staphylococcal products implicated in TSS, particularly in whom antibody to TSST-1 pre-existed in acute sera. The nonresponsiveness or lack of seroconversion to TSST-1 in some patients could suggest either: a) TSST-1 was not the etiologic agent for such patients; b) TSST-1 was the etiologic agent, but the exposure was sufficient for an immune response (similar to tetanus), or; c) some immunologic defect may be present. Future studies are required to clarify these possibilities.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Massey, Christopher. "Cellular and Molecular Mechanisms of Toxin Resistance For Endoplasmic Reticulum Translocating Toxins." Master's thesis, University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2687.
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The endoplasmic reticulum (ER) is the site of co- and post-translational modification for secretory proteins. In order to prevent vesicular transport and secretion of misfolded or misassembled proteins, a highly regulated mechanism called ER-associated degradation (ERAD) is employed. This pathway recognizes misfolded proteins in the ER lumen and targets them to the cytosol for ubiquitination and subsequent degradation via the 26S proteasome. Sec61 and Derlin-1 are ER pores through which export occurs. AB-type protein toxins such as cholera toxin (CT), Shiga toxin (ST), exotoxin A (ETA), and ricin have evolved means of exploiting the ERAD pathway in order to reach their cytosolic targets. AB-type protein toxins consist of a catalytic A-subunit and a cell-binding B-subunit. The B-subunit recognizes cell surface receptors for the toxin. This begins a series of vesicle trafficking events, collectively termed retrograde trafficking, that lead to the ER. Dissociation of the A and B subunits occurs in the ER, and only the A subunit enters the cytosol. The exact mechanism of A subunit translocation from the ER to the cytosol is unknown. Toxin translocation occurs through a pore in the ER membrane. Exit through the pore requires the toxin to be in an unfolded conformation. The current model for toxin translocation proposes that ER chaperones actively unfold the toxin A chain for translocation. After the translocation event, the toxin spontaneously refolds to an active conformation. Our model suggests that unfolding in the ER is spontaneous and refolding in the cytosol is dependent upon cytosolic chaperones. Based on our model, we hypothesize that blockage of the A subunit unfolding and/or the ERAD translocation step will confer a phenotype of non-harmful multi-toxin resistance to cells. In support of this model, we have shown that, at 37[degrees]C, the isolated catalytic subunit of cholera toxin (CTA1) is in an unfolded and protease sensitive confirmation that identifies the toxin as misfolded by the ERAD pathway. Stabilization of CTA1 via glycerol inhibits the loss of its tertiary structure. This stabilization results in decreased translocation from the ER to the cytosol and increased secretion of CTA1 to the extracellular medium. Treatment with glycerol also prevents CTA1 degradation by the 20S proteasome in vitro. These data indicate that the thermal stability of CTA1 plays an important role in intoxication. These data also suggest that stabilization of CTA1 tertiary structure is a potential target for therapeutic agents. Our model asserts that CTA1 behaves as a normal ERAD substrate upon dissociation from the holotoxin. In support of this model, we have shown that the ER luminal protein HEDJ, known to be involved in ERAD, interacts with CTA1. The interactions between HEDJ and CTA1 occur only at temperatures in which the toxin is in an unfolded conformation. We have also shown that HEDJ does not affect the thermally stability of CTA1 since there is no alteration in its pattern of temperature-dependent protease sensitivity. Alteration of the normal HEDJ-CTA1 interaction via a dominant-negative HEDJ construct resulted in decreased translocation from the ER to the cytosol and, as a result, decreased intoxication. Our work demonstrated toxin resistance can result through effects on toxin structure or ERAD chaperones. To identify other potential inhibitors, we developed a novel assay to detect the activity of other AB toxins and compared it with an established toxicity assay. We generated a Vero cell line that expressed a destabilized variant of enhanced green fluorescent protein (EGFP). These cells were used to monitor the Stx-induced inhibition of protein synthesis by monitoring the loss of EGFP fluorescence from cells. We screened a panel of 13 plant compounds, and indentified grape seed extract and grape pomace extract as inhibitors of Stx activity. Grape seed extract and grape pomace extract were also shown to block the toxic activities of ETA and ricin, providing the basis for a future high-throughput screen for multi-toxin inhibitors.Ph.D.
Department of Biomolecular Science
Burnett College of Biomedical Sciences
Biomedical Sciences PhD
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Wilczek, Claudia. "Identifizierung des zellulären Rezeptors für das binäre Toxin von Clostridium spiroforme." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-166768.
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Erst kürzlich wurde der Lipolyse-stimulierte Lipoproteinrezeptor (LSR, engl.
lipolysis-stimulated lipoprotein receptor) als der zelluläre Oberflächenrezeptor
von CDT und Iota-Toxin, zweier Vertreter der Iota-Toxin-Familie der
clostridialen Aktin-ADP-ribosylierenden Toxine, identifiziert. In dieser Arbeit
sollte geprüft werden, ob CST, ein weiterer Vertreter der Iota-Toxin-Familie,
ebenfalls LSR für den Zelleintritt nutzt.
Zunächst wurden die Toxinkomponenten CSTa und CSTb erstmals
rekombinant hergestellt. Dazu wurden die für CSTa und CSTb codierenden
Genabschnitte mittels PCR amplifiziert und anschließend in einen
Expressionsvektor kloniert. Als Expressionsvektor wurde in dieser Arbeit der
pHis1522-Vektor verwendet. Zur Amplifizierung wurden die Plasmide in E. coli transformiert und anschließend aufgereinigt. Die Proteinexpression erfolgte in B. megaterium, weil dieses Bakterium sich bereits zur Expression anderer clostridialer Toxine bewährt hatte. Zur Aufreinigung der 6xHis-getaggten Proteine wurde die Nickel-Affinitätschromatographie eingesetzt.
Als nächstes wurde gezeigt, dass die rekombinant hergestellten
Toxinkomponenten CSTa und CSTb biologisch aktiv waren. Dazu wurden
CaCo2-Zellen mit CST behandelt und anschließend die Morphologie der Zellen untersucht. CaCo2-Zellen, die mit CSTa und CSTb behandelt wurden, wiesen Vergiftungserscheinungen wie eine typische Zellabrundung auf. Mit dem „Aktin-Nach-ADP-Ribosylierungs-Assay“ und der fluoreszenzmikroskopischen Untersuchung von TRITC-Phalloidin-gefärbtem Aktin wurde gezeigt, dass das rekombinant hergestellte CST Aktin-ADP-ribosylierende Eigenschaften besaß.
Nachdem gezeigt war, dass rekombinant hergestelltes CST sich wie ein
biologisch aktives, binäres Aktin-ADP-ribosylierendes Toxin verhält, konnte
mithilfe der Vergiftung von H1-HeLa(+LSR)-Zellen und nativen H1-HeLa-Zellen, die kein LSR exprimierten, nachgewiesen werden, dass die Wirkung des Toxins LSR-abhängig ist. FACS-Analysen und Kolokalisationsstudien mit Alexa488-gefärbtem CSTb und Antikörper-gefärbtem LSR erbrachten zusätzlich den Beweis, dass CSTb auf der Zelloberfläche an LSR bindet und bei der Aufnahme in die Zellen mit LSR in endozytischen Vesikeln kolokalisiert. Die Ergebnisse dieser Arbeit zeigen, dass das C. spiroforme Toxin (CST) ebenfalls LSR als Rezeptor für den Zelleintritt verwendet.
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Pigatto, Caroline Peters [UNESP]. "Carcaterização denotípica e genotípica de Escerichia coli produtora de toxina shiga (STEC) isoladas de bovinos de corte no Estado do Paraná." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/103826.
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Escherichia coli produtoras de toxina Shiga (STEC) são reconhecidas como agentes causadores de infecções em humanos em todo o mundo. O principal reservatório é o bovino. Neste trabalho, cepas de STEC previamente isoladas de fezes bovinas foram caracterizadas usando PCR multiplex para determinar os genes de virulência (stx1, stx2, ehxA, eaeA e saa), soroaglutinação passiva reversa em látex (RPLA-VTEC screen) para avaliar a expressão da toxina Shiga, PCR-RFLP e sequenciamento para obter os subtipos e a variabilidade dos genes stx2, respectivamente. Foram determinados também os sorotipos, o perfil de sensibilidade e a viabilidade das cepas de STEC em queijo minas frescal. A freqüência de STEC nas amostras de fezes bovinas foi de 37%. Foram encontrados trinta e quatro sorotipos de STEC sendo os mais freqüentes o ONT:H7 (10%), O22:H8, O22:H16 e ONT:H21 (7% cada). Onze sorotipos encontrados não tinham sido associados com STEC até o momento. A maioria das STEC (96%) foi susceptível a todos os antimicrobianos testados. A produção de toxina Shiga determinada pelo ensaio RPLA foi de 89%. Os marcadores de virulência foram encontrados em 11 diferentes combinações, a mais freqüente foi stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% cada). Foram detectados 8 subtipos de stx2: stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc e stx2O48. Os genes que apresentaram maior freqüência foram: stx2 e stx2c. As seqüências parciais obtidas sugerem a presença de elevada variabilidade nos genes do tipo stx2 nas STEC analisadas. A viabilidade de STEC não-O157 em queijo minas revelou que diferentes cepas de STEC podem ser detectadas nos queijos após 10 dias de armazenamento sob refrigeração. Os dados encontrados neste trabalho sugerem isolados com alto potencial de patogenicidade oferecendo risco de desencadear graves infecções à população.
Shiga toxin-producing Escherichia coli (STEC) is recognize worldwide as an organism capable to cause human diseases. Cattle are the main source of STEC. In this research, STEC strains previously isolated were analyzed using multiplex-PCR for virulence genes, the RPLA assay to detect the Shiga toxin production and serotyping. PCR-RFLP and nucleotide sequence were analyzed to detect stx2 genes subtypes and their variability. Moreover tests for antimicrobial susceptibility and the vialbility of STEC in Minas Frescal cheese were done. The frequency of cattle shedding STEC was 37%. Thirty-four serotypes of STEC were found, the most frequent being ONT:H7 (10%), O22:H8, O22:H16 and ONT:H21 (7% each). Eleven serotypes had not been associate with STEC until the moment. Most of the strains (96%) were susceptible to all antimicrobial agents tested. Production of Shiga toxin by the RPLA assay was detected in most (89%) of the STEC strains. The frequency of virulence markers were found in 11 diferent combinations: stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% each). Eigth stx2 subtypes were detect (stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc; stx2O48) and the most frequent were: stx2; stx2c. The partial sequences of stx2 genes suggested a high variability of stx2 types in the STEC analyzed. The STEC viability in cheese could be detected after 10 days of storage under refrigeration. The results found in this work suggest strains with high potential of pathogenicity offering risk to lead serious infections to the population.
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Passalacqua, Edward F. "X-ray crystallographic studies of toxic shock syndrome toxin-1 and related superantigens." Thesis, University of Bath, 1995. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295442.
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Leka, Oneda. "Structural and functional characterization of A-B toxins: diphtheria toxin and clostridial neurotoxins." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3421803.
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I performed my doctorate research activity studying three important human pathogens that are A-B toxins: Diphtheria Toxin (DT), Tetanus Neurotoxin (TeNT) and Botulinum neurotoxins (BoNTs), the etiologic agents of diphtheria, tetanus and botulism respectively. In terms of structural organization these toxins consist of three domains, which are termed L chain (the N-terminal catalytic domain), HN (the transmembrane domain), and HC (the C-terminal binding domain). These domains are closely related to the common four step mechanism of action: membrane binding mediated by HC, endocytosis, membrane translocation mediated by HN and L-chain mediated substrate modification.
I studied the conformational change of diphtheria toxin at acidic pH. DT includes a T domain which is known to mediate the pH-dependent membrane translocation, by forming a channel through which the catalytic domain crosses the endocytic vesicle membrane. To date no structural data are available about the pore/channel formed by the T domain, nor is known if it is monomeric or oligomeric. I have performed biochemical and structural studies to characterize the T domain of DT. The T domain is also considered a prospective anti-cancer agent for the targeted delivery of cytotoxic therapy to cancer cells. I obtained the crystal structure of DT in the presence of lipid bicelles (which simulate the endocytic vesicle membrane) and grown at pH 5.5, pH that mimics the acidic environment where translocation takes place. The reported structure throws lights on the initial event of this process, the destabilization of the three α-helices present at the bottom of the toxin (Leka et al., 2014).
I then worked on a project which aimed to unravel the three dimensional structure of tetanus neurotoxin by crystallization studies. Because TeNT is considered “uncrystallizable” I focused on the use of antibody fragments (Fabs) as crystallization chaperons to aid the structural determination. Native gel analysis and size exclusion chromatography showed the formation of a stable complex in vitro between TeNT and the relative Fabs. Several crystallization experiments were carried out by high throughput crystallization screens.
Further, I performed functional analysis on the trafficking of botulinum neurotoxin at the neuromuscular junction (NMJ). I expressed the binding domains of different BoNT serotypes, which are both necessary and sufficient for binding to the neuronal surface and internalization. The two step purifications, chromatography and gel filtration, were sufficient to yield purifications of each binding domain to >90% purity. Using cerebellum granular neurons (CGNs), I tested their functionality and specificity. I performed also in vivo assays in order to analyze their distribution along the NMJ. The data from fluorescence analysis show high specificity of these binding domains at the NMJ, and a different staining between different BoNT serotypes, reflecting their different time of intoxication, and perhaps a different pathway of vesicular trafficking.Ho effettuato la mia attività di ricerca studiando tre importanti patogeni umani, che sono tossine di tipo A-B: la tossina difterica (DT), la neurotossina tetanica (TeNT) e le neurotossine botuliniche (BoNTs), gli agenti eziologici di difterite, tetano e botulismo, rispettivamente. In termini di organizzazione strutturale queste tossine sono costituite da tre domini: il dominio catalitico (LH), il dominio di translocazione (HN) e il dominio di legame (HC). Questa organizzazione dei domini è strettamente correlata al loro comune meccanismo d’azione che comprende: il legame alla membrane cellulare mediato dal HC, la traslocazione del dominio catalitico nel citoplasma mediata dal canale di permeazione formato dal HN. Ho studiato il cambiamento conformazionale della tossina difterica a pH acido. DT include un dominio di translocazione (dominio T), che forma il canale attraverso il quale il dominio catalitico attraversa la membrana della vescicola endosomica. Fino ad oggi non ci sono dati strutturali che riguardano il canale formato dal dominio T, non si sa neanche se è un monomero o oligomero. Ho eseguito studi biochimici e strutturali per caratterizzare il dominio T di DT. Il dominio T è anche considerato un agente anti-cancro nelle terapie mirate contro le cellule tumorali. Ho ottenuto la struttura tridimensionale della tossina difterica in presenza di doppi strati lipidici (che simulano la membrana della vescicola endosomica) ed in condizioni di pH 5,5 (pH corrispondente all'ambiente acido in cui avviene la il processo di traslocazione). La struttura riportata getta luci sull'evento iniziale di questo processo, la destabilizzazione di tre alfa-eliche presenti nella parte inferiore della tossina (Leka et al., 2014). Ho poi lavorato su un progetto che mirava a caratterizzare la struttura tridimensionale della tosssina tetanica. Poiché la cristallizzazione di questa tossina risulta d’essere molto difficile, mi sono concentrata sull'utilizzo di frammenti di anticorpi (Fab) come tools per aiutare la determinazione strutturale. Analisi da gel nativo e da cromatografia ad esclusione mostrano la formazione di un complesso stabile in vitro tra la tossina ed i relativi Fab. Diversi esperimenti di cristallizzazione sono stati eseguiti, e per il momento non abbiamo ancora informazioni strutturali sulla tossina. Inoltre, ho studiato anche la localizzazione ed il processo di internalizzazione delle tossine botuliniche a livello della giunzione neuromuscolare (NMJ). Ho espresso i domini di legame di diversi sierotipi di tossine botuliniche, domini che sono necessari e sufficienti per il legame alla superficie dei neuroni. I domini di legame sono stati purificati utilizzando cromatografia di affinità e per esclusione, ottendo alla fine una purezza > 90% . Utilizzando i neuroni granulari di cervelletto (CGN), ho testato la loro funzionalità e specificità. Questi domini sono stati iniettati in vivo al fine di analizzare la loro localizzazione a livello della giunzione neuromuscolare. I dati ottenuti con analisi di microscopia confocale ed a fluorescenza mostrano che questi domini si localizzano proprio a livello della giunzione muscolare. Nelle marcature si osserva anche una colorazione diversa tra i diversi sierotipi BoNT, e questo risultato riflette il diverso tempo di intossicazione tra i vari serotipi di tossine botuliniche, e forse anche una diversa localizzazione in diverse vescicole endosomiche.
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Bader, Carly. "The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin." Master's thesis, University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5762.
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Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gs? target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals. The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs?, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of post-intoxication treatment options.M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biomedical Sciences; Biomedical Sciences
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Gao, Haifei. "Chemical biology approaches to study toxin clustering and lipids reorganization in Shiga toxin endocytosis." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB147.
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La toxine bactérienne de Shiga se lie au glycosphingolipide (GSL) globotriaosylcéramide (Gb3) afin d’entrer par endocytose dans les cellules en utilisant une voie dépendante et indépendante de la clathrine. Dans la voie indépendante de la clathrine, la toxine de Shiga réorganise les lipides de la membrane de façon à imposer une contrainte mécanique sur la bicouche, conduisant ainsi à la formation de pic d’invagination d'endocytose profonds et étroits. Mécaniquement ce phénomène n’est pas encore compris, notamment il reste énigmatique, comment se traduisent les propriétés géométriques de l’agrégation des glycosphingolipides GSLS et de la toxine. Dans mon travail de thèse, via l’utilisation de la sous-unité B de la toxine de Shiga (STxB) comme un modèle, différentes espèces moléculaires de son récepteur Gb3 ont été synthétisés avec des structures délibérément choisis. Les études réalisées par imagerie de haute résolution et par la modélisation informatique ont permis d’élucider les contraintes mécano-chimique sous-jacente conduisant à une réorganisation efficace qui a pour résultat l’agrégation de la toxine et la réorganisation des lipides. En combinant des expériences de simulation sur ordinateur de dynamique des particules dissipatives (DPD) et des expériences sur des modèles de membranes cellulaires, nous avons fourni la preuve de l’induction d’une force de fluctuation-membrane, de type « force de Casimir », conduisant à l'agrégation des molécules de toxines associées à la membrane à des échelles de longueur mésoscoiques. Nous avons observé et mesuré, en outre la condensation lipidique induite par la toxine, quantitativement sur des monocouches de Langmuir en utilisant la réflectivité des rayons X (XR) et par la mesure de la diffraction des rayons X par incidence rasante (GIXD), fournissant ainsi une preuve directe de l'hypothèse que la toxine a le potentiel de réduire de façon asymétrique la surface moléculaire sur la partie membranaire exoplasmique, ce qui conduit à une déformation locale de la membrane. Durant ma thèse, nos efforts ont été consacrés à la réalisation de nouveaux glycosphinolipides (GSL) comme outils chimiques à visée biologique. Par ailleurs, une nouvelle stratégie de reconstitution de GSL fonctionnels sur la membrane cellulaire, basée sur une réaction de ligation de type « click » entre un glycosyl-cyclooctyne et un azido-sphingosine a été étudiée. Les résultats obtenus sur les cellules se sont avérés beaucoup moins efficace que ceux in vitro. Une poursuite de l'optimisation de cette méthodologie est actuellement en cours. Une sonde fluorescente du glycosphinolipide Gb3, marquée à l’Alexa Fluor 568 lui-même lié par l'intermédiaire d'un bras PEG-α à la position de la chaîne acyle, a été synthétisée. Cette sonde se lie à la STxB sur couche mince de TLC, mais pas sur des membranes modèles. D'autres améliorations sont discutéesBacterial Shiga toxins bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3) to enter cells by clathrin-dependent and independent endocytosis. In the clathrin-independent pathway, Shiga toxin reorganizes membrane lipids in a way such as to impose mechanical strain onto the bilayer, thus leading to the formation of deep and narrow endocytic pits. Mechanistically how this occurs is not yet understood, and notably how the geometric properties of toxin-GSLs complexes translate into function has remained enigmatic. In my thesis work, using the B-subunit of Shiga toxin (STxB) as a model, different molecular species of its receptor Gb3 have been synthesized with deliberately chosen structures, coupled with high resolution imaging and computational modeling, to understand the underlying mechano-chemical constraints leading to efficient toxin clustering and lipids reorganization. By combining dissipative particle dynamics (DPD) computer simulation and experiments on cell and model membranes, we provided evidence that a membrane fluctuation-induced force, termed Casimir-like force, drives the aggregation of tightly membrane-associated toxin molecules at mesoscopic length scales. Furthermore, toxin-induced lipid condensation was observed and measured quantitatively on Langmuir monolayers using X-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), thereby providing direct evidence for the hypothesis that the toxin has the potential to asymmetrically reduce the molecular area of the exoplasmic membrane leaflet, leading to local membrane deformation. During my PhD, effort was also invested to develop new GSL tools applied to the biological setting. A novel strategy based on the Cu-free click reaction between glycosyl-cyclooctyne and azido-sphingosine was designed with the goal to functionally incorporate GSLs into cellular membranes. Following the synthesis work, click reactions have been performed in solution and on cells. Compared to the former, results on cells were far less efficient. Further optimization is currently ongoing. A fluorescently labeled Gb3 probe with Alexa Fluor 568 coupled via a PEG linker to the α-position of the acyl chain, was synthesized, to which STxB bound on TLCs, but not on model membranes. Further improvements are discussed
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Karlsson, Sture. "Toxin production in Clostridium difficile /." Stockholm : Karolinska institutet, 2004. http://diss.kib.ki.se/2004/91-77349-812-2/.
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Strack, Julia [Verfasser], and Andreas [Akademischer Betreuer] Bechthold. "Osteoklastendifferenzierung durch Pasteurella multocida-Toxin." Freiburg : Universität, 2014. http://d-nb.info/1123480834/34.
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Sharma, Davinder Kumar. "Toxin production by Clostridium botulinum." Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301991.
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The endopeptidase activity assay developed for measurement of purified botulinum neurotoxin type A (BoNT/A) in clinical therapeutic preparations has been adopted to provide a specific measure of BoNT/A activity in culture supernatants of proteolytic C. botulinum type A. Electrophoretic studies and inhibition of BoNT/A activity by anti-A antibody confirmed the specificity of the assay. The minimum detection limit was 0.2 MLD50/ml indicating the assay as more sensitive than the standard mouse bioassay or any other in vitro assay available to date. Whilst the assay did not exhibit any cross reactions with non-proteolytic (saccharolytic) clostridia, proteolytic C. botulinum types B and F and C. sporogenes showed some cross reactions. The endopeptidase assay was used to investigate physiological aspects of BoNT/A production by proteolytic C. botulinum type A strain NCTC 7272. Growth studies at 15°C, 25°C and 37°C with strain NCTC 7272 demonstrated that the first appearance of BoNT/A (0.1-1.0 MLD50 ml) occurred during mid-late exponential or early stationary phase of growth. Extracellular BoNT/A formation was not proportional to viable count. Slightly more BoNT/A was detected at 25°C than 37° or 15°C. The results of BoNT/A formation by one of the growth curves at 25°C measured by the endopeptidase assay and mouse bioassays were very similar confirming the specificity of the assay. A simple method was developed to lyre the cells so that BoNT/A formation could be subsequently measured in the endopeptidase assay. The data obtained following lysis of cells and measurement of intracellular BoNT/A showed that both intracellular BoNT/A and total BoNT/A formation is not constitutive but are more closely proportional to viable count than extracellular BoNT/A. Release of BoNT/A from cells was not associated with autolysis. The conversion of BoNT/A from the single-chain to dichain form during growth has been measured. The use of the endopeptidase assay has been also exploited to study BoNT/A formation by this strain within the population of cells. There was only a four-fold difference in BoNT/A production by cells of strain NCTC 7272, and further work in this area is warranted. Attempts were made to use MAPs for the production of monoclonal antibodies to SNAP-25 following cleavage by BoNT/E. Whilst the outcome was unsuccessful, the soundness of the principle was demonstrated
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Díaz, Ocaña Raquel. "Recombinant self-assembling nanoparticles for cancer therapy based on toxin and venom compounds." Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/670483.
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La plataforma desenvolupada d’enginyeria de proteïnes auto-acoblables permet dissenyar nanopartícules únicament proteiques (NPs) capaces d’atacar i actuar selectivament sobre les cèl.lules canceroses mitjançant la interacció amb receptors que es sobreexpressen. Les estructures esfèriques estables de les NPs desenvolupades i la seva mida adequada, en combinació amb els pèptids d’orientació , milloren la seva especificitat. A més, la incorporació de segments de toxina i verí ha millorat els efectes terapèutics d’aquestes estructures que són totalment biocompatibles i que no tenen cap portador extern o material agregat, complint d’aquesta manera amb el nou concepte per a medicaments de precisió, que involucra un fàrmac recombinant lliure de vehicle, auto-acoblat, auto-dirigit i eficient.
Una versió modificada de la cadena catalítica ricina A, amb la capacitat de disminuir els efectes secundaris no desitjats de la síndrome de vessament vascular però conservant la seva citotoxicitat natural, es va adaptar a la plataforma de proteïnes. El disseny es va desenvolupar amb el pèptid T22, que s’uneix a CXCR4, en l’extrem N-terminal, i una cua d’histidines a la terminal-C, en combinació amb un fragment del lloc escindible de furina per alliberar la proteïna intracel.lularment, i una seqüència KDEL per evitar la secreció del reticle endoplàsmic. Les NPs de cadena de ricina A solubles purificades dirigides a CXCR4 amb un diàmetre mitjà de 11 nm, van assolir un increment de 100 vegades en la seva citotoxicitat amb un IC50 de 13 ± 0,5 x 10 -9 M en cèl.lules HeLa. També es van produir per mètodes recombinants i es van purificar cossos d’inclusió insolubles de 400-600 nm, amb resultats citotòxics parcials. El mecanisme d’entrada dependent del receptor d’T22-MRTA-H6 es va verificar i avaluar en un model de ratolí amb leucèmia mieloide aguda (AML) mitjançant la injecció sistèmica a la vena de la cua on es va verificar un bloqueig important de les cèl.lules leucèmiques sense toxicitat sistèmica o histològica lateral en els òrgans sans.
De manera similar, la clorotoxina (CTX) també es va incorporar a la plataforma de proteïnes per tal d’aprofitar la seva d’orientació i efecte terapèutic en glioblastoma (GBM), ambdues funcions en un sol pèptid. Es van dissenyar dues versions que s’uneixen a la proteïna anexina-2 i la metaloproteinasa de matriu MMP-2; CTX-GFP-H6 i CTX-KRKRK-GFP-H6. Les NPs solubles d’un diàmetre mitjà de 12 nm es van incubar en cèl·lules HeLa, sobreexpressant annexina-2, i en cèl.lules U87MG, sobreexpressant MMP2. Les dues versions eren completament fluorescents, però CTX-GFP-H6 va presentar efectes citotòxics lleus, mentre que CTX-KRKRK-GFP-H6 va mostrar ser més citotòxic en les cèl.lules U87MG que en les cèl.lules HeLa. L’afinitat selectiva de CTX es va confirmar mitjançant l’avaluació de la seva selectivitat utilitzant anticossos monoclonals i un sèrum policlonal contra la proteïna de la superfície cel.lular, actuant com un receptor de la CTX.La plataforma desarrollada de ingeniería de proteínas autoensamblables permite diseñar nanopartículas únicamente proteicas (NPs) capaces de atacar y actuar selectivamente sobre las células cancerosas mediante la interacción con receptores que se sobreexpresan. Las estructuras esféricas estables de las NPs desarrolladas y su tamaño adecuado, en combinación con los péptidos de direccionamiento involucrados, mejoran su especificidad. Además, la novedosa incorporación de segmentos de toxina y veneno ha mejorado los efectos terapéuticos de estas estructuras que son totalmente biocompatibles y que no tienen ningún portador externo o material agregado, cumpliendo de esta manera con el concepto emergente para medicamentos de precisión que involucra un fármaco recombinante libre de vehículo, autoensamblado, auto-dirigido y eficiente. Una versión modificada de la cadena catalítica de ricina A, con la capacidad de disminuir los efectos secundarios no deseados del síndrome de derrame vascular, pero conservando su citotoxicidad natural, se adaptó a la plataforma de proteínas. El diseño se desarrolló con el péptido T22, que se une a CXCR4, en el extremo N-terminal, y una cola de histidinas en el extremo C-terminal, en combinación con un fragmento del sitio escindible de furina para liberar la proteína intracelularmente, y una secuencia KDEL para evitar secreción del retículo endoplásmico. Las NPs de cadena de ricina A solubles purificadas dirigidas a CXCR4, con un diámetro promedio de 11 nm, alcanzaron un incremento de 100 veces en su citotoxicidad con un IC50 de 13 ± 0,5 x 10 -9 M en células HeLa. Pero también se produjeron por métodos recombinantes y se purificaron cuerpos de inclusión insolubles de 400-600 nm, con resultados citotóxicos parciales. El mecanismo de entrada dependiente del receptor de T22-mRTA-H6 se verificó y evaluó en un modelo de ratón con leucemia mieloide aguda (AML) mediante la inyección sistémica en la vena de la cola, donde se verificó un bloqueo importante de las células leucémicas sin toxicidad sistémica o histológica lateral en los órganos sanos. De manera similar, la clorotoxina (CTX) también se incorporó a la plataforma de proteínas con el fin de aprovechar su direccionamiento y efecto terapéutico en glioblastoma (GBM), ambas funciones en un solo péptido. Se diseñaron dos versiones que se unen a la proteína anexina-2 y la metaloproteinasa de matriz MMP-2; CTX-GFP-H6 y CTX-KRKRK-GFP-H6. Lss NPs solubles, de un diámetro promedio de 12 nm, se incubaron en células HeLa sobreexpresando anexina-2, y en células U87MG, sobreexpresando MMP2. Ambas versiones eran completamente fluorescentes, pero CTX-GFP-H6 presentó efectos citotóxicos leves, mientras que CTX-KRKRK-GFP-H6 mostró ser más citotóxico en las células U87MG que en las células HeLa. La afinidad selectiva de CTX se confirmó mediante la evaluación de su direccionamiento utilizando anticuerpos monoclonales y un suero policlonal contra la proteína de la superficie celular, actuando como un receptor de la CTX.
The developed self-assembling platform allows the engineering of protein-only nanoparticles (NPs) capable to target and act selectively over cancer cells by means of the interaction with overexpressed receptors. The stability of the spherical NP structures and their adequate size, in combination with the involved targeting peptides, enhance their specificity. Also, the novel incorporation of toxin and venom segments have improved the therapeutic effects of these fully biocompatible materials, without the need of any external carrier or added material, thus fulfilling the newfangled concept for precision medicines that involve self-assembled, self-targeted and efficient vehicle-free recombinant drugs. A modified version of the catalytic ricin A chain, with the ability to diminish the undesired vascular leak syndrome side effects but retaining its natural cytotoxicity, was adapted to the protein platform. The design was developed with the peptide T22 in the N-terminal, which binds CXCR4, and a his-tag in the C-terminal. This was combined with a furin cleavable site fragment in order to release the protein intracellularly, and a KDEL sequence to avoid endoplasmic reticulum secretion. Purified soluble CXCR4-targeted ricin A chain NPs with an average diameter of 11 nm, reached a 100-fold cytotoxic improvement with an IC50 of 13 ± 0.5 x 10 -9 M in HeLa cells. Also, insoluble 400-600 nm inclusion bodies were produced by recombinant methods and purified, with partial cytotoxic results. The receptor-dependent mechanism of T22-mRTA-H6 was verified and evaluated in an acute myeloid leukemia (AML) mouse model by systemic administration through a vein tail injection where an important blockage of the leukemic cells was verified without side systemic or histological toxicity in healthy organs. In a similar way, chlorotoxin (CTX) was also incorporated to the protein platform in order to take advantage of its targeting and therapeutic effect in glioblastoma (GBM), both functions in one peptide. Two versions that target protein Annexin-2 and the matrix metalloproteinase MMP-2 were engineered, namely CTX-GFP-H6 and CTX-KRKRK-GFP-H6. The soluble NPs of an average dimeter of 12 nm were incubated with HeLa cells, overexpressing annexin-2, and in U87MG cells, overexpressing MMP2. Both versions were fully fluorescent but CTX-GFP-H6 presented mild cytotoxic effects, whereas CTX-KRKRK-GFP-H6 showed to be more cytotoxic in U87MG cells than in HeLa cells. The selective affinity of CTX was confirmed by means of evaluating its targeting using a monoclonal antibody and a polyclonal serum against the cell surface protein, acting as a CTX receptor.
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Zimmermann, Leigh A. "Environmental regulation of toxin production : comparison of hemolytic activity of Amphidinium carterae and Amphidinium klebsii /." Electronic version (PDF), 2006. http://dl.uncw.edu/etd/2006/zimmermannl/leighzimmermann.pdf.
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Trescos, Yannick. "Effets des toxines de Bacillus anthracis sur le cytosquelette des cellules immunitaires : implication sur la phagocytose et les fonctions immunitaires." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV026/document.
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Bacillus anthracis, agent de la maladie du charbon, est aussi un agent majeur de la menace biologique. Sa virulence est liée à deux principaux facteurs : une capsule et deux toxines, la toxine oedémateuse (ET = EF + PA) et la toxine létale (LT = LF + PA). EF est une adénylate cyclase, calcium et calmoduline dépendante, produisant une élévation de la concentration en AMPc intracellulaire tandis que LF est une métalloprotéase à zinc clivant la majorité des Mitogen Activated Protein Kinase Kinases. Les toxines jouent un rôle central dans la pathogénie de la maladie du charbon et dans la dérégulation des fonctions des cellules du système immunitaire. Le cytosquelette d'actine participe activement aux fonctions de phagocytose et de migration des macrophages et des cellules dendritiques.Cependant, peu d'études analysent l'implication du cytosquelette d'actine des cellules immunitaires dans la physiopathologie des toxines. ET induit une rétraction temps-dépendant des cellules dendritiques et des macrophages normalisés sur des micropatterns de fibronectine, s'accompagnant d'une dépolymérisation de l'actine et d'une perte des points d'ancrage des cellules dendritiques. Précocement, ET active la cofiline par l'activation de la voie de signalisation AMPc – PKA – Protéines phosphatases. Malgré ces altérations du cytosquelette d'actine, ET n'induit pas de modification des capacités phagocytaires des cellules dendritiques, à l'exception d'une dérégulation de la maturation des phagosomes. ET conduit également à une augmentation de la migration des cellules dendritiques in vitro par activation et expression de CCR7 et CXCR4 à la surface des cellules dendritiques.A l'inverse, LT conduit à un étalement temps-dépendant des cellules dendritiques normalisées, accompagné d'une dérégulation de la dynamique de l'actine provoquant des regroupements anormaux d'actine filament. LT active les myosines phosphatases via la voie RhoA-ROCK pour déphosphoryler les myosines II. A la différence de ET, LT inhibe les capacités phagocytaires des cellules dendritiques mais ne conduit pas à une modification de la migration des cellules dendritiques in vitroBacillus anthracis, the agent of anthrax, is also a major agent of biological warfare threat. Its virulence is caused by two main factors : the capsule and two toxins, edema toxin (ET = PA + EF) and lethal toxin (LT = LF + PA). EF is a calcium and calmodulin-dependent adenylate cyclase, producing a rise in intracellular cAMP concentration, while LF is a zinc metalloprotease cleaving the majority of Mitogen Activated Protein Kinase Kinases. The toxins play a central role in the pathogenesis of the disease and the deregulation of the functions of immune cells. The actin cytoskeleton is actively participating in the phagocytosis and the migration of macrophages and dendritic cells.However, few studies analyze the involvement of the actin cytoskeleton of immune cells in the pathogenesis of toxins. ET induces a time-dependent retraction of dendritic cells and macrophages on fibronectin micropatterns, accompanied by actin depolymerization and a loss of the anchor points of dendritic cells. ET early activates cofilin by activating the cAMP - PKA - Protein phosphatases signaling pathway. Despite these alterations of the actin cytoskeleton, ET does not induce any change in the phagocytic capacity of dendritic cells, except for a deregulation of the phagosomes maturation. ET also leads to an increase in the migration of dendritic cells in vitro by activation and expression of CCR7 and CXCR4 on the surface of dendritic cells.In contrast, LT results in a time-dependent spreading of micropatterned dendritic cells, accompanied by a dysregulation of actin dynamics causing abnormal combinations of actin filament. LT activates myosin phosphatase via the RhoA-ROCK pathway to dephosphorylate myosin II. Unlike ET, LT inhibits the dendritic cells phagocytosis but does not lead to a change in dendritic cells migration in vitro
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Rystedt, Alma. "Botulinum Toxin : Formulation, Concentration and Treatment." Doctoral thesis, Uppsala universitet, Neurologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-181667.
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Botulinum toxin (BTX) is used in various fields of medicine, including the treatment of hyperhidrosis and cervical dystonia. Botox®, Dysport®, Xeomin® and NeuroBloc® are commercially available BTX products, which are formulated differently and their dosing units are unique. Dosage and concentration of the prepared solution for injection varies considerably among studies comparing the products. Improved guidelines on concentration and dosing when changing from one product to another are warranted. This would ensure the use of the lowest effective doses for good effect, minimal risk of antibody formation and side-effects as well as reduced costs. The aim of the present work was to find the most appropriate BTX concentration for each of the four products to achieve the highest sweat reducing effect and to investigate dose conversion ratios between Botox and Dysport in the treatment of cervical dystonia when the products are diluted to the same concentration, 100 U/ml. Paper I and II clearly confirm that it is crucial to consider the BTX concentration in a treatment regimen, especially when changing between different products. The optimal concentration to reduce sweating varies among the products and was found to be 25 U/ml for Botox and Xeomin, approximately 100 U/ml for Dysport and 50 U/ml for NeuroBloc. However, for NeuroBloc the optimal concentration might be even lower. In Paper III, which is a retrospective study using casebook notes from 75 patients with cervical dystonia, it was found that the most appropriate dose conversion ratio to use when switching from Botox to Dysport was 1:1.7. In Paper IV, Botox and Dysport were prospectively compared in a double-blind, randomized clinical trial in two different dose conversion ratios (1:3 and 1:1.7) when diluted to the same concentration (100 U/ml). No statistically significant difference was seen between Botox (1:3) and Dysport nor between Botox (1:1.7) and Dysport four weeks after treatment. Some of the secondary outcome observations, however, did indicate that the ratio 1:3 resulted in suboptimal efficacy of Botox but this must be further validated in a larger patient material.
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Taft, Sarah C. "Anthrax toxin immunity and receptor activity /." Cincinnati, Ohio : University of Cincinnati, 2007. http://www.ohiolink.edu/etd/view.cgi?ucin1195584188.
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Thesis (Ph.D.)--University of Cincinnati, 2007.Advisor: Alison A. Weiss. Title from electronic thesis title page (viewed Feb. 5, 2008). Keywords: Bacillus anthracis, anthrax toxin, AVA. Includes abstract. Includes bibliographical references.
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Chatwell, Nicola. "Nucleic acid approaches to toxin detection." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606582.
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PCR is commonly used for detecting contamination of foods by toxigenic bacteria. However, it is unknown whether it is suitable for detecting toxins in samples which are unlikely to contain bacterial cells, such as purified biological weapons. Quantitative real-time PCR assays were developed for amplification of the genes encoding Clostridium botulinum neurotoxins A to F, Staphylococcal enteroxin B (SEB), ricin, and C. perfringens alpha toxin. Botulinum neurotoxins, alpha toxin, ricin and V antigen from Yersinia pestis were purified at Dstl using methods including precipitation, ion exchange, FPLC, affinity chromatography and gel filtration. Additionally, toxin samples of unknown purity were purchased from a commercial supplier. Q-PCR analysis showed that DNA was present in crudely prepared toxin samples. However, the majority of purified or commercially produced toxins were not detectable by PCR. Therefore, it is unlikely that PCR will serve as a primary toxin detection method in future. Immuno-PCR was investigated as an alternative, more direct method of toxin detection. Several iterations of the method were investigated, each using a different way of labelling the secondary antibody with DNA. It was discovered that the way in which antibodies are labelled with DNA is crucial to the success of the method, as the DNA concentration must be optimised in order to fully take advantage of signal amplification without causing excessive background noise. In general terms immuno-PCR was demonstrated to offer increased sensitivity over conventional ELISA, once fully optimised, making it particularly useful for biological weapons analysis.
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Promdonkoy, Boonhiang. "Molecular biology of a microbial toxin." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621541.
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Martínez-García, Juan Carlos. "Expression cloning of insecticidal toxin receptors." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621840.
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Bergmann, Stefan [Verfasser], and Klaus [Akademischer Betreuer] Aktories. "Charakterisierung zytotoxischer Pasteurella multocida-Toxin–Chimären." Freiburg : Universität, 2015. http://d-nb.info/1114996289/34.
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TAFT, SARAH C. "ANTHRAX TOXIN: IMMUNITY AND RECEPTOR ACTIVITY." University of Cincinnati / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1195584188.
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Tallett, April. "Structure and function of pertussis toxin." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/20238.
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Tan, Yian Kim. "Novel functions of anthrax lethal toxin." Fairfax, VA : George Mason University, 2009. http://hdl.handle.net/1920/3451.
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Thesis (Ph.D.)--George Mason University, 2009.Vita: p. 141. Thesis director: Charles Bailey. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biodefense. Title from PDF t.p. (viewed June 10, 2009). Includes bibliographical references (p. 110-140). Also issued in print.
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MacMaster, Kayleigh A. "Characterization of Cellular Pathways and Potency of Shiga Toxin on Endothelial Cells." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1439304438.
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Michaelides, Alecos. "Chemical and enzymatic fragmentation of tetanus toxin and immunological studies on anti-tetanus toxin and toxoid sera." Thesis, University of Ottawa (Canada), 1996. http://hdl.handle.net/10393/9661.
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This thesis describes the immunization protocols for the production of antibodies against tetanus toxin and toxoid in guinea pigs and mice. Antibodies were successfully raised against the toxin without mortalities in either species. The murine sera obtained, were isotyped by ELISA and the toxin was proven to be a superior antigen in eliciting production of IgG$\rm\sb{2a}$ and IgG$\sb3$. The two isotypes which have demonstrated antitumor activity. The anti-toxoid sera exhibited a lower reactivity towards the toxin and toxoid when compared with anti-toxin sera. The reactivity of recombinant tetanus toxin fragment C was studied and the results indicated that in the murine serum, 72% of anti-toxin or anti-toxoid antibodies were directed against epitopes on fragment C. The study of the guinea pig sera suggested that similar to mouse serum, it can develop in response to toxin as an antigen, antibodies against toxin which are mostly directed against the fragment C portion. On the other hand. guinea pigs seem to respond to the toxoid as an antigen by producing antibodies to more than fragment C. (Abstract shortened by UMI.)
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Hostetter, Shannon Jones. "Role of Shiga toxin dissemination and inflammation in the pathogenesis of Shiga toxin-producing Escherichia coli infection." [Ames, Iowa : Iowa State University], 2009.
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SANTOS, Danilo Mamede da Silva. "Detecção de Microcystis potencialmente tóxicas em reservatórios de Pernambuco, através de marcadores moleculares para o operon da sintetase da microcistina - mcyB e mcyA." Universidade Federal Rural de Pernambuco, 2008. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4757.
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Cyanobacterias or cyanoficeas, even they have many similarities with eukariotics seaweed and occupy the same ambient niches, they belong to the Eubactéria domain. In Brazil, the occurrence of blooms of cyanobacterias was evidenced, mainly in the state of Pernambuco, where some patients died in 1996. Amongst the cyanobacterias capable of producing toxins, the Genus Microcystis is distinguished; being required some genes (mcyA the J) for toxin production. This work has the objective to verify the presence of operon for the genes, mcyB and mcyA, in populations of cyanobacterias in the reservoirs of the state of Pernambuco. The taxonomic survey of the reservoirs made possible the identification of 16 taxon represented by three orders: Chroococcales; Nostocales and Oscilatoriales, which M. aeruginosa was more widely distributed species in the studied reservoirs. The reservoir of the Agreste was the one where could be found the biggest number of organisms for L-1 with the value of 51,423,078 L-1, followed by reservoirs of Sertão and Zona da Mata. Operon of the ficocianine, proved the presence of cyanobacterias for all the studied reservoirs. The gene of mcyB was presented for all reservoirs. The mcyA only for the Tapacurá reservoir in the rainy period, representing for 11,11% of the samples. Amongst the primers employed for mcyB, mcyB-FR demonstrated to be more specific than mcyB-FRA, not showing unexpected bands. The assays in HPLC had performed so far only for two reservoirs, Arcoverde and Jazigo in rainy season, being positive only for the Jazigo reservoir. The results demonstrate the necessity for a periodic monitoring in the Pernambuco’s reservoirs state emphasized for the potentially toxic cyanobacterias, as PCR an appropriate method with respect to the detention of potentials microcystins producer in environment samples.
Cianobactérias ou algas cianofíceas, embora tenham muitas semelhanças com algas eucarióticas e ocupem os mesmos nichos ambientais, pertence ao domínio Eubacteria. No Brasil, foi evidenciada a ocorrência de florações de cianobactérias, principalmente no Estado de Pernambuco, ocasionando a morte de vários pacientes em 1996. Dentre as cianobactérias capazes de produzir toxinas, destaca-se o gênero Microcystis, sendo requeridos alguns genes (mcyA a J) para produção de toxina. Este trabalho tem como objetivo verificar a presença do operon para os genes, mcyB e mcyA, em populações de cianobactérias ocorrentes em reservatórios do Estado de Pernambuco. O levantamento taxonômico dos reservatórios possibilitou a identificação de 16 táxons representados por três ordens: Chroococcales; Nostocales e Oscilatoriales, onde M. aeruginosa foi à espécie mais amplamente distribuída nos reservatórios estudados. O reservatório do Agreste foi o que apresentou o maior número de organismos por L-1, com o valor de 51.423.078 org. L-1, seguido dos reservatórios do Sertão e Zona da Mata. O operon da ficocianina comprovou a presença de cianobactérias para todos os reservatórios estudados. O gene do mcyB esteve presente para todos os reservatórios e mcyA apenas para o reservatório de Tapacurá, período chuvoso, representando por 11,11% das amostras. Dentre os primers utilizados para o mcyB, o mcyB-FR demonstrou ser mais específico que o mcyB-FRA, por não apresentar bandas inespecíficas. Os ensaios em HPLC foram efetuados até o presente momento apenas para dois reservatórios, Arcoverde chuvoso e Jazigo chuvoso, sendo positivo apenas para o reservatório de Jazigo. Os resultados apontam uma necessidade periódica de monitoramento dos reservatórios do Estado de Pernambuco frente a cianobactérias potencialmente tóxicas, sendo o método de PCR apropriado para a detecção de potenciais produtores de microcistinas em amostras ambientais.
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Herrera, Alfa. "Staphylococcus aureus TSST-1 and Beta-toxin contribute to infective endocarditis via multiple mechanisms." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/5775.
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Staphylococcus aureus is a gram positive bacterium asymptomatically colonizing 30-40% of the human population. S. aureus causes a variety of infections including superficial skin lesions, toxic shock syndrome, and infective endocarditis (IE). There are 100,000 cases of IE each year in the United States. IE is a life threatening infection of native/prosthetic valves and the lining of the heart. It is characterized by the formation of vegetations, “cauliflower-like” structures composed of bacteria and host factors. S. aureus is the most commonly identified pathogen (up to 40%) in patients with IE. USA200 (Clonal Complex 30) strains of S. aureus are significantly associated with IE, all of which produce toxic shock syndrome toxin-1 (TSST-1) and β-toxin.
TSST-1 characterizes the staphylococcal Group I superantigens (SAgs). The major mechanism of activity of TSST-1 and other SAgs is the ability to activate T-cells and APCs by non-specifically cross-bridging Vβ-chains of T-cell receptors (TCRs) with α and/or β-chains of major histocompatibility complex II (MHCII) molecules on antigen presenting cells (APCs). In a rabbit model of IE and sepsis, TSST-1 is critical for the development of vegetations and the associated colony forming units (CFUs). β-toxin has a molecular mass of 35 kDa, a basic pI (>10.0), and is a member of the DNase I superfamily. This cytotoxin has two distinct mechanisms of action: sphingomyelinase (SMase) activity and DNA biofilm ligase activity. β-toxin is critical for causing IE in a rabbit model that strongly resembles human disease. This toxin association had been observed, but studies have not been completed to determine what role TSST-1 and β-toxin play independently and in cooperation with one another, and more specifically which mechanism each uses, during IE infections.
While TSST-1 and β-toxin are both important for IE, they are very different toxins. My studies determined that the presence of TSST-1 and β-toxin in combination results in the highest levels of lethality in a rabbit model of IE. A strain expressing TSST-1 lacking superantigenic activity has decreased lethality compared to the same strain expressing wild type TSST-1. My study is the first to begin characterization of the DNA biofilm ligase active site by identifying important residues via a DNA binding and biofilm formation assays. Furthermore, my research shows that a β-toxin mutant lacking SMase activity is decreased in lethality and vegetation formation compared to wild type. β-toxin mutants disrupted in biofilm ligase activity do not decrease lethality but are deficient in vegetation formation compared to wild type. Utilizing in vitro assays to assess cellular events during IE, I established that β-toxin causes changes to morphology and is cytotoxic to human aortic endothelial cells (HAECs), inhibits production of IL-8, and modulates the expression levels of cluster of differentiation 40 (CD40) and vascular cell adhesion molecule 1 (VCAM-1).
My work shows these two virulence factors (TSST-1 and β-toxin) produced by USA200 strains and other clonal groups play important roles in causing IE.
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Taylor, J. M. Walsh. "Identification and isolation of emetic toxin producing Bacillus Cereus and heat-stable toxins from other Bacillus species." Thesis, Glasgow Caledonian University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415442.
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Daimon, Yasushi. "Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σE in Escherichia coli." Kyoto University, 2016. http://hdl.handle.net/2433/215457.
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Leeper, Molly Maitland. "Trends in Toxin Profiles of Human Shiga Toxin-Producing Escherichia Coli (STEC) O157 Strains, United States, 1996-2008." Atlanta, Ga. : Georgia State University, 2009. http://digitalarchive.gsu.edu/iph_theses/57/.
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Thesis (M.P.H.)--Georgia State University, 2009.Title from file title page (Digital Archive@GSU, viewed June 16, 2010) Karen Giseker, committee chair; Peter Gerner-Smidt, committee member. Includes bibliographical references (p. 101-105).
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Blower, Timothy Robert. "Functional and structural studies of the toxIN abortive infection and toxin-antitoxin locus from Erwinia carotovora subspecies atroseptica." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608655.
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Kerzmann, Amy N. "Mechanistic analysis of Clostridium difficile toxin A." [Bloomington, Ind.] : Indiana University, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378359.
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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2009.Title from home page (viewed on Jul 12, 2010). Source: Dissertation Abstracts International, Volume: 70-10, Section: B, page: 6182. Advisers: Andrew L. Feig; James T. Drummond.
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Li, Feng. "Studies on inhibition against anthrax lethal toxin." Oklahoma City : [s.n.], 2010.
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Adams, Toni Elizabeth. "Bordetella bronchiseptica dermonecrotic toxin, purification and characterisation." Thesis, University of Leicester, 1997. http://hdl.handle.net/2381/29723.
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Dermonecrotic toxin (DNT) is produced by all the Bordetella species, and DNT from B. bronchiseptica is considered to be an important virulence factor in turbinate atrophy of pigs.;Recombinant DNT (rDNT) was purified by sonication, ion-exchange and hydroxylapatite chromatography. Other methods for the purification of wild-type DNT and rDNT, including preparative isoelectric focusing and hydrophobic chromatography, were investigated in detail.;Partially pure preparations of rDNT contained a 145 kDa protein band and were cytotoxic to embryonic bovine lung (EBL) cells. Partially pure rDNT induced the formation of actin stress fibres and focal adhesions in Swiss 3T3 cells. In addition, rDNT stimulated DNA synthesis in quiescent Swiss 3T3 cells but prevented cell proliferation, resulting in binucleated cells. Recombinant DNT has been shown to directly modify the small GTP-binding protein, Rho, (Pullinger, unpublished), which regulates the cell cytoskeleton. Results from this thesis indicate that rDNT causes the assembly of actin stress fibres and focal adhesion possibly by direct activation of the Rho protein.;Partially purified rDNT with a site-directed mutation in a putative nucleotide-binding motif did not induce cytoskeletal rearrangements and did not stimulate DNA synthesis in Swiss 3T3 cells. This suggests that the nucleotide-binding motif is essential for activity.;Two lines of evidence indicate that the toxin is internalised in the endosomal/lysosomal compartment: i) stimulation of DNA synthesis by transient exposure of Swiss 3T3 cells to rDNT, and ii) blocking of rDNT-induced DNA synthesis with methylamine.;Three monoclonal antibodies (mAbs) were produced against B. bronchiseptica DNT. These mAbs recognised rDNT and B. pertussis DNT, but none neutralised the cytotoxic activity of DNT on EBL cells.;The partial purification of rDNT and characterisation of its biological effects provide valuable information for further studies of the toxin, including analysis of its enzymatic mode of action and its role in infection. Also, DNT may prove to be a useful tool for analysis of cell responses involving the important signalling molecule, Rho.
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Rizzolo, Flavio. "ToXin, an indexing scheme for XML data." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ58778.pdf.
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Hayes, Edward. "Fragment based design of cholera toxin inhibitors." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534749.
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Rooks, David John. "Molecular ecology of shiga-toxin encoding bacteriophage." Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548790.
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Schmidt, Elke. "Structure-function characterisation of zonula occludens toxin." Thesis, University of Strathclyde, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501858.
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Eames, Niall W. A. "Botulinim toxin A and gastrocnemius muscle length." Thesis, Queen's University Belfast, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387872.
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Smyth, Martin Gerard. "Structural characterization of the pasteurella multocida toxin." Thesis, Birkbeck (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338390.
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