Preliminary Study On The Interaction Between RTX Toxins Of Actinobacillus Pleuropneumoniae And Host Cells

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a contagious pulmonary disease of pigs which causes great economic losses in industrialized pig production worldwide. Apx toxins are Ca2+-dependent haemolytic and/or cytotoxic toxins, belonging to the pore-forming RTX (repeats in toxin) toxin family. To date, four different Apx toxins, Apxâ… , Apxâ…¡, Apxâ…¢and Apxâ…£, have been described, and each of the 15 serotypes of A. pleuropneumoniae secretes various combinations of the Apx toxins. Generally, Apx toxin is encoded by an operon that is consist of four contiguous genes, C, A, B and D. The structural toxin encoded by the A gene is activated by the C gene-encoded protein. The proteins encoded by the B and D genes are involved in the secretion of the RTX toxins. Apoptosis and necrosis, two forms of cell death with distinct morphological and biochemical features, have both been detected in tissue cultures exposed to an RTX toxin.In previouse research, it was revealed that high concentrations of RTX toxin induces very rapid target cell death, with features reminiscent of necrosis. By contrast, cells exposed to low concentrations of toxin exhibited the morphological, biochemical and molecular changes associated with apoptosis. Little is known about the interactions between individual species of Apx toxins with their target cells because of the complex nature of Apx. This study aimed to characterize the interaction between the Apx toxins (Apxâ… and Apxâ…¡) and host cells (porcine peripheral blood leucocyte and lung epithelial cell).The apoptosis of porcine peripheral blood leucocyte (PPBL) induced by Apx toxins: Native Apxâ… and Apxâ…¡toxins were extracted from the cultural supernatants of Actinobacillus pleuropneumoniae reference strains of serovars 10 and 7 respectively. Recombinant Apxâ… toxins (rApxâ… ) were prepared by in vitro expression. The distinction establishement natural toxin Apxâ… group, the natural toxin ApxII group, the recombinant protein rApxIA group, caspase-3,-9 and general caspase inhibitor groups of three protein, then established the time gradient and the concentration gradient separately, affected on PPBL. DNA LADDER was used to analyze their apoptosis, further more, to delineat the underling signaling pathways. The experimental result showed that PPBL apoptosis activated the way together through caspase-3,-9 by Apxâ…¡, which could not work by Apxâ… and rApxIA at low concentrations.The apoptosis of Porcine Lung Epithelial Cells (SJPL) induced by Apx toxins:We set terms as Apxâ… , recombinant protein rApxâ… A, caspase general inhibitor, caspase-3 inhibitor and caspase-9 inhibitor. Concentration-and time-dependent manners were set in order to observe apoptosis of SJPL cells by obove terms, further more, delineate the underlying signaling pathways. The study revealed that Apxâ… could cause the apoptosis of SJPL cells at low concentrations, but rApxIA could not. Caspase-3 was activated after Apxâ… treated SJPL cells.The effect on apoptosis of Porcine Lung Epithelial Cells(SJPL) by native toxin Apxâ… under the condition of antibiotical culture:In previous study, it revealed that under the long-term antibiotical cultured condition, slim changes of physiological and biochemistry often appeare on cells. In order to research the phenomenon above, we set two terms of SJPL cells(two-antibiotics condition and none antibiotic) induced by native Apxâ… toxin. DNA ladder assay was used to detect weather antibiotics have effects on apoptosis of target cells. In this research, we found the phenomenon of delay on apoptosis of target cells in condition of antibiotical cultured, what's more,the level of density has effects to delay the time that cells apoptosis.