The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell

ZnO nanoparticle tracking from uptake to genotoxic damage in human colon carcinoma cells

Zinc Oxide (ZnO) nanoparticles are widely used both in the industry and in biomedical applications for their chemical and physical nanomaterial properties. It is therefore essential to go in depth into the cytotoxicity mechanisms and interactions between nanomaterials and cells. The aim of this work was to evaluate the dissolution of ZnO nanoparticles and their uptake, from a few minutes after treatments up to 24 h. ZnO nanoparticles routes of entry into the human colon carcinoma cells (LoVo) were followed at different times by a thorough ultrastructural investigation and semiquantitative analysis. The intracellular release of Zn2 + ions by Zinquin fluorescent dye, and phosphorylated histone H2AX (γ-H2AX) expression were evaluated. The genotoxic potential of ZnO nanoparticles was also investigated by determining the levels of 8-hydroxyl-2′-deoxyguanosine (8-oxodG). The experimental data show that ZnO nanoparticles entered LoVo cells by either passive diffusion or endocytosis or both, depending on the agglomeration state of the nanomaterial. ZnO nanoparticles coming into contact with acid pH of lysosomes altered organelles structure, resulting in the release of Zn2 + ions. The simultaneous presence of ZnO nanoparticles and Zn2 + ions in the LoVo cells determined the formation of reactive oxygen species at the mitochondrial and nuclear level, inducing severe DNA damage

Iron availability influences aggregation, biofilm, adhesion and invasion of Pseudomonas aeruginosa and Burkholderia cenocepacia

Pseudomonas aeruginosa and Burkholderia cenocepacia are predominant opportunistic pathogens in cystic fibrosis (CF) patients. In healthy humans the lower respiratory tract as well as all mucosa, contains a very low free iron concentration (10(-18) M), while in CF patients' sputum iron concentration is very high, showing a median value of 63x10(-6) M. Accumulation of catalytic reactive iron heavily contributes to subsequent clinical complications in the lung disorders by the production of reactive oxygen species and increases bacterial growth and virulence. The data reported in this study indicate that low iron concentration (Fe3+ 1 mu M) induced free-living forms and motility both in P. aeruginosa and B. cenocepacia, while high iron concentrations (Fe3+ 10 and 100 mu M) stimulated aggregation and biofilm formation already in the fluid phases, so demonstrating that aggregation and biofilm formation are positively iron-modulated in these bacteria. Moreover, the different morphological forms (free-living, aggregates and biofilm) showed different capabilities of adhering and invading the bronchial cell line A549. P. aeruginosa PAO1 aggregates, and mostly biofilm, exerted the highest adhesion efficiency, while B. cenocepacia PV1 aggregates or biofilm the lowest. A significant reduction in invasion efficiency by P. aeruginosa biofilm and a significant increase in cell internalization by B. cenocepacia biofilm has been reported. Therefore, the iron availability is an important signal to which P. aeruginosa and B. cenocepacia counteract by leaving the motile free-living forms and entering into a new lifestyle, i.e. biofilm. These data could contribute to explain that the iron-overload of the sputum of CF patients, inducing nonmotile forms, aggregates and biofilm, may facilitate penetration of host epithelial barriers contributing to the establishment of infection, colonization, persistence and systemic spread of these opportunistic pathogens

Bovine lactoferrin inhibits the efficiency of invasion of respiratory A549 cells of different iron-regulated morphological forms of Pseudomonas aeruginosa and Burkholderia cenocepacia

Pseudomonas aeruginosa and Burkholderia cenocepacia are two important opportunistic respiratory pathogens of cystic fibrosis (CF) patients. Infections caused by these microorganisms are particularly difficult to eradicate because they are usually highly resistant to several currently available broad-spectrum antibiotics. Lactoferrin (Lf), a glycoprotein found in physiological fluids of mammals and present at high concentrations in infected and inflamed tissues, plays an important role in the natural defence mechanism against pathogens and in immune regulation. In the present study, we evaluate the ability of bovine lactoferrin (W) to influence P aeruginosa PAO1 and B. cenocepacia PV1 adhesiveness and invasiveness, using the A549 human bronchial cell line. Three different iron-induced morphological forms of bacteria (free-living, aggregates and biofilm) were assayed. The addition of bLf to cells just before infection had little influence on adhesion efficiency for all three of the morphological forms of B. cenocepacia PV1, while a slight increase in adhesion efficiency by R aeruginosa PAO1 was noticed. Conversely, invasion of all three morphological forms of both P aeruginosa and B. cenocepacia was strongly inhibited by the presence of bLf, independently of its degree of iron-binding activity. This is the first report demonstrating an anti-invasive property of bLf for strains of R aeruginosa and B. cenocepacia