Programmed Cellular Necrosis Mediated by the Pore-Forming α-Toxin from Clostridium septicum

Programmed necrosis is a mechanism of cell death that has been described for neuronal excitotoxicity and ischemia/reperfusion injury, but has not been extensively studied in the context of exposure to bacterial exotoxins. The α-toxin of Clostridium septicum is a β-barrel pore-forming toxin and a potent cytotoxin; however, the mechanism by which it induces cell death has not been elucidated in detail. We report that α-toxin formed Ca2+-permeable pores in murine myoblast cells, leading to an increase in intracellular Ca2+ levels. This Ca2+ influx did not induce apoptosis, as has been described for other small pore-forming toxins, but a cascade of events consistent with programmed necrosis. Ca2+ influx was associated with calpain activation and release of cathepsins from lysosomes. We also observed deregulation of mitochondrial activity, leading to increased ROS levels, and dramatically reduced levels of ATP. Finally, the immunostimulatory histone binding protein HMGB1 was found to be released from the nuclei of α-toxin-treated cells. Collectively, these data show that α-toxin initiates a multifaceted necrotic cell death response that is consistent with its essential role in C. septicum-mediated myonecrosis and sepsis. We postulate that cellular intoxication with pore-forming toxins may be a major mechanism by which programmed necrosis is induced

Meningitic Escherichia coli K1 Penetration and Neutrophil Transmigration Across the Blood–Brain Barrier are Modulated by Alpha7 Nicotinic Receptor

Alpha7 nicotinic acetylcholine receptor (nAChR), an essential regulator of inflammation, is abundantly expressed in hippocampal neurons, which are vulnerable to bacterial meningitis. However, it is unknown whether α7 nAChR contributes to the regulation of these events. In this report, an aggravating role of α7 nAChR in host defense against meningitic E. coli infection was demonstrated by using α7-deficient (α7-/-) mouse brain microvascular endothelial cells (BMEC) and animal model systems. As shown in our in vitro and in vivo studies, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the blood-brain barrier (BBB) were significantly reduced in α7-/- BMEC and α7-/- mice. Stimulation by nicotine was abolished in the α7-/- cells and animals. The same blocking effect was achieved by methyllycaconitine (α7 antagonist). The tight junction molecules occludin and ZO-1 were significantly reduced in the brain cortex of wildtype mice infected with E. coli and treated with nicotine, compared to α7-/- cells and animals. Decreased neuronal injury in the hippocampal dentate gyrus was observed in α7-/- mice with meningitis. Proinflammatory cytokines (IL-1β, IL-6, TNFα, MCP-1, MIP-1alpha, and RANTES) and adhesion molecules (CD44 and ICAM-1) were significantly reduced in the cerebrospinal fluids of the α7-/- mice with E. coli meningitis. Furthermore, α7 nAChR is the major calcium channel for nicotine- and E. coli K1-increased intracellular calcium concentrations of mouse BMEC. Taken together, our data suggest that α7 nAChR plays a detrimental role in the host defense against meningitic infection by modulation of pathogen invasion, PMN recruitment, calcium signaling and neuronal inflammation

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development

Background Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host’s signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. Results In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatisinfection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-β (PDGFRβ) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRβ that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatisdevelopment. Conclusion Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases

Micropropagation and rejuvenation of Sequoia sempervirens (Lamb) Endl: a review

This article describes the botanical, biological and forest-tree characteristics of Sequoia sempervirens, the reasons for interest in its in vitro vegetative multiplication, the difficulty in achieving this from old and remarkable trees, and reviews means of overcoming this limitation. Among such means are the repeated culture of stem fragments on media containing appropriate hormonal combinations, the micrografting of buds originating from old trees onto juvenile rootstocks, and regeneration of buds from previously rejuvenated material. The value and limitations of these protocols and of morphological, physiological and biochemical markers of rejuvenation are discussed. The experimental conditions required for the formation of somatic embryos are described. Increased knowledge of in vitro micropropagation will be essential to enhance the use of clonal selection and offer practical outlets to studies concerning somatic hybridization and somatic embryogenesis.Micropropagation et rajeunissement du Sequoia sempervirens (Lamb) Endl : revue. Cet article présente les principales caractéristiques botaniques, biologiques et forestières du Sequoia sempervirens. Il analyse l'intérêt de la multiplication végétative réalisée in vitro soit par micropropagation sensu stricto (figs 1, 2 et 3), soit par régénération (figs 6, 7 et 9); il discute les raisons de la difficulté à la réaliser à partir d'arbres âgés et remarquables ainsi que les moyens de la contourner. Parmi ces moyens figurent la réitération des cultures de fragments de tige sur des milieux contenant un équilibre hormonal adéquat (tableaux I et II), le microgreffage de bourgeons appartenant à des plantes âgées sur des porte-greffes juvéniles (figs 4 et 5), la régénération de bourgeons à partir de matériel préalablement rajeuni selon l'un des protocoles précédents (figs 10 et 11). L'intérêt et les limites de ces protocoles sont discutés, en considérant les marqueurs morphologiques (figs 12 et 13), physiologiques et biochimiques du rajeunissement. Les conditions d'obtention de l'embryogenèse somatique chez cet arbre sont décrites (fig 8). Finalement, l'accroissement de nos connaissances en micropropagation in vitro apparaît essentiel pour augmenter la qualité de la sélection clonale et offrir des débouchés pratiques aux travaux concernant l'hybridation somatique et l'embryogenèse somatique. L'acquisition de telles connaissances de base devrait permettre une meilleure utilisation de cet arbre

Réflexion sur les modalités d'appréciation du rajeunissement in vitro chez le Sequoia sempervirens

International audienc

Auxin metabolism and rooting in young and mature clones of Sequoia sempervirens

International audienc