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

Actin-based confinement of calcium responses during Shigella invasion.

Shigella, the agent of bacillary dysentery, invades epithelial cells by locally inducing actin reorganization. Upon cell invasion, Shigella induces calcium (Ca(2+)) signalling, but its role in invasion has remained unclear. Here we show that components involved in inositol 1, 4, 5- trisphosphate (InsP3) signalling are implicated in Shigella invasion. Although global Ca(2+) responses are dispensable for bacterial invasion, local Ca(2+) responses of unprecedented long duration are associated with invasion sites. Fluorescence recovery after photo-bleaching experiments indicate that diffusion of small solutes is hindered at Shigella-invasion sites and that diffusion hindrance is dependent on bacterially induced actin reorganization. Computational simulations and experimental challenge of the model support the notion that local accumulation of InsP3 permitted by restricted diffusion and enrichment of InsP3 receptors account for sustained local Ca(2+) increases at entry sites. Thus, cytoskeletal reorganization through diffusion hindrance shapes the duration of local Ca(2+) signals.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death

BACKGROUND: Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca(2+) concentration ([Ca(2+)]i) elevation induced by infection can cause cell death, but [Ca(2+)]i changes and high [Ca(2+)]i-induced death of macrophages due to infection of Leptospira have not been previously reported. METHODOLOGY/PRINCIPAL FINDINGS: We first used a Ca(2+)-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca(2+)]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca(2+)]i elevation was caused by both extracellular Ca(2+) influx through the purinergic receptor, P(2)X(7), and Ca(2+) release from the endoplasmic reticulum, as seen by suppression of [Ca(2+)]i elevation when receptor-gated calcium channels were blocked or P(2)X(7) was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP(2)) into inositol-1,4,5-trisphosphate (IP(3)), which in turn induces intracellular Ca(2+) release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S(-1). Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca(2+)]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca(2+)]i. Moreover, PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca(2+)]i increases induced apoptosis and necrosis of macrophages, while mild [Ca(2+)]i elevation only caused apoptosis. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that L. interrogans infection induced [Ca(2+)]i elevation through extracellular Ca(2+) influx and intracellular Ca(2+) release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca(2+)]i elevation