The Research On The Pyolysin Of Arcanobacterium Pyogenes Induced Macrophage Apoptosis

Arcanobacterium pyogenes, a gram-positive, normally commensal bacterium,resides on themucous membranes of cattle, sheep, swine, and other ecomomically important animals. It can, throughan as yet unknown mechanism, disseminate to cause a wide variety of suppurative infection. Herdsinfected with A.pyogenes can incur serious economic losses. Pyolysin(PLO), the hemolytic exotoxinexpressed by Arcanobacterium pyogenes, is a major virulence factor in infections by A.pyogenes. PLOhas directly cytotoxic for polymorphonuclear leukocytes and macrophages with unknown mechanism.In this study, we cloned gene encoding PLO, expressed in Escherichia coli and then analysed itshemolytic activity. Recombinant PLO has hemolytic activity. Then prepared the polyclonal antibodyagainst recombinant PLO protein used purified His-PLO in rabbits and can completely neutralizedhemolytic activity of recombinant PLO.To study the mechanism of PLO of A.pyogenes, recombinant protein PLO was added into culturesupernatant of murine marcrophage-like cell line RAW264.7cells and cocultured with the cells. Thetypical morphological featuers of apoptosis were observed after PLO treatment at0.5μg/mL for24h and48h, such as the appearance of apoptotic bodies, nuclear pyknosis, chromatin aggregation. DNA Laddertest showed that: the chromosome DNA appeared the typical "ladder" strip in agarose gelelectrophoresis. The apoptosis rate of RAW264.7cells was examined by Annexin V-FITC/PI-stain andthe apoptosis rate was50.1%after His-PLO treatment at0.5μg/mL for48h. PLO presented the apparentproliferation inhibition on RAW264.7cells in a time–and-dose dependent manner measured by MTT.The activity of caspase3was increased compared with control after treated with His-PLO. And secretionof TNF-α, IL-1β, IL-6, IL-8, IL-10was detected after His-PLO treatment, and cytokines secretion wasimproved in different degree, significant difference except IL-1β was not change with His-PLOtreatment. All of these showed that Pyolysin could induce macrophage cells apoptosis and secrete avrious of cytokines.In summary,in this study we expressed PLO of the A.pyogenes in Escherichia coli andidentification of its biological function and then studied the effect on RAW264.7cells in order to studythe effect of PLO inducing apoptosis.All of these provide necessary basic materials for the mechanismof PLO and pathogenesis of A.pyogenes.