Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 156-168.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Notre système immunitaire détecte les microorganismes via des molécules absentes de l’hôte appelées MAMPs. Mais étant donné que les MAMPs sont exprimés par tous les microorganismes indépendamment de leur potentiel pathogène, ce mécanisme n’explique pas comment le système immunitaire distingue les microorganismes pathogènes des non-pathogènes. De récents travaux ont mis en évidence un mécanisme de détection de l’activité des facteurs de virulence bactériens. En utilisant la drosophile, notre laboratoire a précédemment démontré que l’activation de la Rho GTPase Rac2 par la toxine CNF1 d’Escherichia coli induisait une réponse immunitaire innée conservée au cours de l’évolution chez le mammifère et similaire à l’immunité induite par les effecteurs chez la plante. Par la suite, nous avons évalué l’importance de cette réponse immunitaire au cours de la bactériémie chez la souris et démontré le rôle central de la cytokine IL-1β dans l’élimination des bactéries en réponse à la détection de CNF1. Des expériences in vitro nous ont permis d’identifier les mécanismes moléculaires mis en jeu et l’inflammasome responsable de l’activation de la caspase-1 et du clivage de l’IL-1β. De manière intéressante, CNF1 est toujours co-exprimée avec la toxine hémolysine-α (HlyA) dans les souches pathogènes d’E. coli. En outre, nous avons découvert que l’HlyA bloquait l’élimination des bactéries induite par CNF1 au cours de la bactériémie et inhibait la sécrétion de l’IL-1β. Ici, nous avons rapporté le premier exemple d’immunité induite par une toxine (CNF1) et contrecarrée par une autre (HlyA). / Our immune system detects microorganisms via molecules absent from the host called MAMPs. Since MAMPs are shared by all microorganisms regardless of their pathogenic potential, this mechanism does not explain how the immune system distinguishes between pathogenic and non pathogenic microorganisms. The detection of the activities of pathogen-encoded virulence factors has emerged as a new paradigm of pathogen recognition. Using Drosophila we previously demonstrated that the Escherichia coli CNF1 toxin-induced activation of the Rho GTPase Rac2 is sufficient to initiate a defense signal evolutionarily conserved from flies to mammals and similar to Effector-Triggered Immunity in plants. We further addressed the importance of this innate immune mechanism during bacteremia in mice and demonstrated the central role of the IL-1β cytokine in the clearance of bacteria in response to the detection of CNF1. In vitro experiments allowed us to identify the involved molecular mechanisms and the inflammasome responsible of caspase-1 activation and IL-1β maturation. Interestingly, CNF1 is always co-expressed with α-hemolysin toxin in pathogenic E. coli. In addition, we found that HlyA blocked the elimination of bacteria induced by CNF1 during bacteremia and inhibited the secretion of IL-1β. Here, we have reported the first example of immunity induced by a toxin (CNF1) and counteracted by another (HlyA).

No description available.

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Les systèmes toxine-antitoxine (TA) sont composés d’une toxine intracellulaire qui cible un processus cellulaire essentiel et qui est neutralisée par une antitoxine. Ces systèmes sont très abondant chez les bactéries et sont impliqués dans la réponse aux stress, la formation de biofilm, le phénomène de persistance, etc.<p>Mon projet de thèse a porté sur l’étude de la diversité des systèmes TA à deux niveaux. Dans un premier temps, plusieurs toxines de la famille RelE provenant de différentes espèces bactériennes et associées à des antitoxines non-canoniques ont été étudiées. Dans la seconde partie de ma thèse, nous avons caractérisé l’activation spécifique de deux systèmes TA d’Escherichia coli au niveau de la régulation transcriptionnelle du système et de l’activation de la toxine. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

246 samples of bulk and packaged spices from retail stores in the western, southeastern, southern, midwestern, and northeastern areas of the U.S. were examined for the presence of Clostridium -perfringens. Isolates were checked for the presence of the lecithinase gene (cpa) and enterotoxin genes (cpe) by PCR. Enterotoxin formation during sporulation was investigated using the Oxoid Toxin Detection Kit. Forty-three confirmed isolates (from 17% of total samples) were cpa-positive. Of those, 27 were cpe-positive. Together, levels of C. perfringens spores ranged from 3.6-2400/gm. The amount of enterotoxin in cell extracts ranged from 2-16 ng/ml. Some of the SEM images of isolated spore (# 78) and one plasmid-borne ent control (FD-153) showed an organized surface structure termed “candy-wrapper”. This extracellular structure remained after treatment with 0.1 % SDS for 1 hr, suggesting it was not composed of membrane debris from the mother cell. The D values of spores ranged from 1.19- 3.31 min. The addition of lysozyme in the plating medium elevated the recovery rate of heat-treated spores. The growth rate of a cocktail of spores from spices (# 31, # 32, # 45) between 4 to 5 hr after inoculation was determined with a doubling time of 6.82 min in hamburger. A cocktail of spores of plasmid-borne ent control showed an optimum growth rate between 5 to 8 hr after inoculation with doubling time of 15.98 min. However; spice isolate cocktail, plasmid-borne ent control cocktail (FD-5603 and FD-153), and a chromosome-borne ent control (NTCT 8239) were unable to germinate and outgrowth at 20oC. Inoculation in laboratory medium FTG indicated the same result as hamburger at 20oC. The ability of C. perfringens spores in spices to potentially survive cooking procedures can be followed by germination and growth of vegetative cells during improper cooling to levels associated with foodborne illness caused by this organism. Our results suggest that retail spices are potential vehicles of transmission of enterotoxin-positive C. perfringens.

Este trabalho realizou importantes avanços na elucidação do potencial das toxinas termo-lábeis do tipo II (LT-IIs) como adjuvantes por via intradérmica e transcutânea. Os dados gerados indicam que LT-IIb e LT-IIc nativas atuam como potentes adjuvantes vacinais por via intradérmica induzindo respostas imunológicas antígeno específicas, como medido pela produção de anticorpos sistêmicos (IgG) e ativação de linfócitos T CD8+ citotóxicos. Soma-se ao potencial adjuvante demonstrado para as LT-IIs por essa via a baixa reatogenicidade das moléculas, com menores níveis de edema e reduzida migração leucocitária para o sítio de inoculação em comparação a LT-I. Os resultados indicam também que o efeito adjuvante das LTs aplicadas por via transcutânea pode estar relacionado à capacidade de ligação ao gangliosídeo GM1 já que a toxina LT-IIb, incapaz de interagir com este receptor, não apresenta efeito adjuvante por essa via. O conjunto de dados apresentados abre perspectivas para o emprego das LT-IIs nativas como adjuvantes parenterais em vacinas para animais e humanos. / This work has made significant advances in the understanding of the potential of type II heat-labile toxins (LT-IIs) as vaccine adjuvants by intradermal and transcutaneous route. The generated data indicate that native forms of LT-IIb and LT-IIc act as potent vaccine adjuvants when intradermally injected inducing antigen-specific immune responses, as measured by the generation of systemic serum antibody (IgG) and activation of cytotoxic CD8+ T lymphocytes. Besides the adjuvant effects, LT-IIs show reduced side effects, measured by the lesser edema formation and reduced leukocytes migration to the site of injection, in comparison to LT-I. The results also indicate that the adjuvant activity of LTs applied transcutaneously may be related to the the ganglioside GM1 binding property since the LT-IIb toxin, unable to interact with this receptor, has no adjuvant effect by this route. The presented data set opens prospects for the employment of native LT-IIs as parenteral adjuvants in vaccines for animals and humans.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.