The Mechanism For The Activation Of Nf-?b System In Macrophage By Pyolysin

Trueperella pyogenes(T.pyogenes)is a kind of symbiotic bacteria commonly found in animal respiratory tract,digestive tract mucosa and breast.T.pyogenes is also a conditional pathogen.When animals are in stress state,T.pyogenes can cause diseases Pyolysin(PLO)is the only haemolysin secreted by T.pyogenes.It is a cholesterol-dependent cytolysins(CDCs)and a pore-forming toxin(PFT).Previous studies have shown that PLO can induce inflammation in mice.But,the mechanism of PLO-induced inflammation is still unclear,so,elucidating the mechanism of PLO-induced inflammation has a positive significance for revealing the pathogenesis of T.pyogenes.In this study,recombinant PLO(r PLO),mutant proteins r PLO W497 F and r PLO D238 R,truncated proteins r PLO D123,r PLO D4 and r PLO D4 W497 F were expressed and purified,and their hemolytic activities were analyzed.The results showed that r PLO had the strongest hemolytic activity,followed by r PLO D238 R.r PLO W497 F and truncated proteins r PLO D123,r PLO D4 and r PLO D4 W497 F had almost no hemolytic activity.J774 A.1 cells were treated with recombinant proteins of different concentrations in different time.The results showed that r PLO protein showed almost lower cytotoxicity in the range of 30-60 ng/m L concentration,and with the increase of concentration,the cytotoxicity gradually increased.The mutant proteins r PLO W497 F and r PLO D238 R showed little cytotoxicity.J774 A.1 cells incubated with calcium ion(Ca2+)fluorescent probe were treated with recombinant proteins of different concentrations.The results showed that the intracellular Ca2+ concentration decreased in sublysis dose r PLO group,and the Ca2+concentration in r PLO D238 R and r PLO W497 F treated cells was significantly higher than that in r PLO treated showed that when the extracellulacells.Co-incubation of r PLO with extracellular fluid containing different concentrations of Ca2+ fluid contained 1 mmol/L Ca2+,r PLO could decrease the concentration of Ca2+ in cells.When the concentration of Ca2+ in extracellular fluid is lower than normal concentration,r PLO could increase the concentration of Ca2 + in cells.JI74 A.1cells were treated with different concentrations of PLO for 0.5 h.The results showed that 30-60ng/m L of had no significant effect on the activity of Calpain(CAPN)in cells.The activity of CAPN in cells treated with 120 ng/m L r PLO was significantly inhibited.When 30-60 ng/m L of r PLO was used to treat cells for 2 h,the activity of CAPN in cells was significantly inhibited.J774 A.1 cells were treated with the sublysed dose of r PLO,and western-blot results showed that r PLO could significantly inhibit the phosphorylation of NF-?B p65 and the expression of NFATc1,and the cells treated for 0.5 h showed significant inhibitory effects.Thephosphorylation level of NF-?B p65 and the expression of NFATc1 in cells treated with r PLO D238 R and r PLO W497 F were up-regulated.J774 A.1 cells were incubated with sublysis dose of r PLO and different concentration of Ca2+ The results showed that in RPMI 1640 medium containing 1 mmol/L Ca Cl2,the decrease of phosphorylation of NF-?B p65 induced by r PLO was alleviated,and the expression of NFATc1 was up-regulated.J774 A.1 cells were treated with sublysis dose of r PLO.Several key proteins in the non-classical NF-?B pathway were detected.The results showed that the contents of NIK and p52 increased and the phosphorylation levels of p100 and IKK ?/? increased at 0.5 h.The BMDMs were tested with different recombinant proteins to detect the activity of the Calcineurin(CN).The results showed that all the recombinant protein cells did not significantly affect the CN activity of the cells.Bone marrow derived macrophages(BMDMs)were treated with these proteins.The results showed that r PLO could stimulate cells to produce large amounts of IL-1?,r PLO W497 F and r PLO D123 could stimulate cells to produce large amounts of IL-10.Then,the tlr4 gene-deleted BMDMstlr4-was stimulated with the recombinant protein and the results showed that r PLO could still stimulate the cells to produce a large amount of IL-1?,while IL-10 produced by r PLO W497 F and r PLOD123 stimulated cells was significantly inhibited.BMDMs were stimulated by D123 domain truncated polypeptide fragments r PLO 1-110,r PLO 95-156,r PLO 141-296 and r PLO 81-393.The results showed that r PLO 95-156 could up-regulate the expression of IL-10 in BMDMs.In conclusion,our study demonstrated that pyolysin acted on macrophage(M?)by activating NF-?B signaling with two ways.When PLO maintains its intact structure and function,PLO activates non-classical NF-?B signaling pathway to play its role;However,if the cell membrane binding function is impaired,PLO activates the classical NF-?B signal pathway by binding to TLR4.This study provides some theoretical foundation for comprehensive understanding the mechanism of T.pyogenes-induced infectious inflammation.