Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH-and temperature-dependent pore-forming activity of LLO, which is unique among cholesteroldependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca 2 þ oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca 2 þ uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

Chlorine (Cl(2)) is a highly reactive oxidant gas used extensively in a number of industrial processes. Exposure to high concentrations of Cl(2) results in acute lung injury that may either resolve spontaneously or progress to acute respiratory failure. Presently, the pathophysiological sequelae associated with Cl(2)-induced acute lung injury in conscious animals, as well as the cellular and biochemical mechanisms involved, have not been elucidated. We exposed conscious Sprague-Dawley rats to Cl(2) gas (184 or 400 ppm) for 30 min in environmental chambers and then returned them to room air. At 1 h after exposure, rats showed evidence of arterial hypoxemia, respiratory acidosis, increased levels of albumin, IgG, and IgM in bronchoalveolar lavage fluid (BALF), increased BALF surfactant surface tension, and significant histological injury to airway and alveolar epithelia. These changes were more pronounced in the 400-ppm-exposed rats. Concomitant decreases of ascorbate (AA) and reduced glutathione (GSH) were also detected in both BALF and lung tissues. In contrast, heart tissue AA and GSH content remained unchanged. These abnormalities persisted 24 h after exposure in rats exposed to 400 ppm Cl(2). Rats injected systemically with a mixture of AA, deferoxamine, and N-acetyl-L-cysteine before exposure to 184 ppm Cl(2) had normal levels of AA, lower levels of BALF albumin and normal arterial Po(2) and Pco(2) values. These findings suggest that Cl(2) inhalation damages both airway and alveolar epithelial tissues and that resulting effects were ameliorated by prophylactic administration of low-molecular-weight antioxidants.

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-a activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-a inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-a activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI) 1/2 /arginase II (AII) 2/2 C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI 1/1 /AII 2/2 counterparts, indicating an important role for arginase I in PLYinduced endothelial hyperpermeability. These results identify PKC-a and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.Keywords: PKC; arginase; pneumococcus; pneumolysin; TNF Although severe pneumonia remains the leading cause of mortality worldwide in children aged less than 5 years (1), community-acquired pneumonia represents a major cause of morbidity and mortality mainly in elderly patients (2). Despite the use of potent antibiotics and aggressive intensive care support, the fatality rate associated with Streptococcus pneumoniae, accounting for 45% of all cases of community-acquired pneumonia, is approximately 20% (1, 2). Pulmonary permeability edema, a major complication of severe pneumonia characterized by endothelial hyperpermeability, can occur days after initiation of antibiotics therapy when tissues are sterile and the pneumonia is clearing and correlates with the presence of the bacterial virulence factor pneumolysin (PLY) (3, 4). This cytoplasmic hemolytic protein is released during bacterial lysis, as occurs after treatment with b-lactam antibiotics (5). PLYinduced lung injury was suggested to result from direct pneumotoxic effects on the alveolar-capillary barrier rather than from resident or recruited phagocytic cells (6).Upon binding of PLY to cholesterol in cell membranes, oligo...

Chlorine (Cl(2)) is a reactive oxidant gas used extensively in industrial processes. Exposure of both humans and animals to high concentrations of Cl(2) results in acute lung injury, which may resolve spontaneously or progress to acute respiratory failure. Injury to airway and alveolar epithelium may result from chemical reactions of Cl(2), from HOCl (the hydrolysis product of Cl(2)), and/or from the various reaction products, such as chloramines, that are formed from the reactions of these chlorinating species with biological molecules. Subsequent reactions may initiate self-propagating reactions and induce the production of inflammatory mediators compounding injury to pulmonary surfactant, ion channels, and components of lung epithelial and airway cells. Low-molecular-weight antioxidants, such as ascorbate, glutathione, and urate, present in the lung epithelial lining fluid and tissue, remove Cl(2) and HOCl and thus decrease injury to critical target biological targets. However, levels of lung antioxidants of animals exposed to Cl(2) in concentrations likely to be encountered in the vicinity of industrial accidents decrease rapidly and irreversibly. Our measurements show that prophylactic administration of a mixture containing ascorbate and desferal N-acetyl-cysteine, a precursor of reduced glutathione, prevents Cl(2)-induced injury to the alveolar epithelium of rats exposed to Cl(2). The clinical challenge is to deliver sufficient quantities of antioxidants noninvasively, after Cl(2) exposure, to decrease morbidity and mortality.

Listeriosis can lead to potentially lethal pulmonary complications in newborns and immune compromised patients, characterized by extensive permeability edema. Listeriolysin (LLO), the main virulence factor of Listeria monocytogenes, induces a dose-dependent hyperpermeability in monolayers of human lung microvascular endothelial cells in vitro. The permeability increasing activity of LLO, which is accompanied by an increased reactive oxygen species (ROS) generation, RhoA activation and myosin light chain (MLC) phosphorylation, can be completely inhibited by the protein kinase C (PKC) α/β inhibitor GÖ6976, indicating a crucial role for PKC in the induction of barrier dysfunction. The TNF-derived TIP peptide, which mimics the lectin-like domain of the cytokine, blunts LLO-induced hyperpermeability in vitro, upon inhibiting LLO-induced protein kinase C-α activation, ROS generation and MLC phosphorylation and upon restoring the RhoA/Rac 1 balance. These results indicate that the lectin-like domain of TNF has a potential therapeutic value in protecting from LLO-induced pulmonary endothelial hyperpermeability.