The effect of intermedilysin on human bile duct cells

中间链球菌是人类口腔、胃肠道和泌尿菌群的共栖成员，但也可能与深层化脓性感染有关，特别是在肝脓肿中。最近有报道称，细菌成孔毒素的功能是双相的。当毒素浓度较高时，它们不仅对有核靶细胞起细胞溶解作用，而且当毒素浓度较低时，它们也可以影响细胞中的信号转导途径。中间溶素（Intermedilysin， ILY）是一种人类特有的成孔细胞溶素，被认为是中间葡萄球菌潜在的重要毒力因子，而Ca^<2+>是细胞存活的关键调节因子，由于Ca^<2+>持续升高而导致细胞内稳态的破坏能够引发细胞死亡。本研究的目的是研究低亚溶浓度的ILY是否通过诱导[Ca^<2+>]I振荡和[Ca^<2+>]I相关的影响人胆管细胞Ca^<2+>稳态。更多其他细胞反应（钙调磷酸酶/NFAT通路、PI3/AKT/MTOR通路和内质网应激的激活）。从钙成像来看，5次降雨ILY处理使[Ca^<2+>]i水平从50 nM的平均基线上升到400 nM的平均峰值，每分钟出现两次重复振荡。在ily处理的细胞中，最初在细胞核中检测到[Ca^<2+>]i升高，随后ER面积明显增加。免疫细胞化学和western blotting分析显示，ILY处理可激活钙调磷酸酶/NFAT1信号通路，增加AKT（p-S473）、BiP和PTEN的表达。有趣的是，在ILY治疗后1小时，BiP和AKT（p-S473）的表达下降。钙调磷酸酶抑制剂环孢素A可阻止ili诱导的钙调磷酸酶/NFAT1活化、细胞死亡和钙振荡。ILY在浓度为10-40 ng/ml时能够诱导钙振荡，激活钙调磷酸酶/NFAT1信号通路；PI3K/AKT/MTOR通路失活，引起内质网应激障碍，导致HuCCT1细胞非凋亡性细胞死亡。少

Streptococcus intermedius is a commensal member of the human oral, gastrointestinal and urinary floras, but may also be associated with deep-seated purulent infections, particularly in the liver abscess.It was recently reported that the function of bacterial pore-forming toxins is biphasic. Not only do they act cytolytically on nucleated target cells when present in higher concentrations, but they can also affect signal-transduction pathways in cells when the toxin is present in low, sublytic concentrations. Intermedilysin (ILY), a human-specific pore-forming cytolysin, has been suggested as a potentially important virulence factor of S.intermedius.Whereas Ca^<2+> is a key regulator of cell survival, the breakdown of Ca^<2+> homeostasis due to its sustained elevation was able to trigger cell death. The goals here were to examine whether a low, sublytic concentration of ILY affects Ca^<2+> homeostasis in human bile duct cells by induction of [Ca^<2+>]I oscillation and [Ca^<2+>]I related … More other cell responses (the activation of calcineurin/NFAT pathway, PI3/AKT/MTOR pathway, and endoplasmic reticulum stress).Fron the calcium imaging, five rain ILY treatment generated an increase in[Ca^<2+>]i level from the average base line of 50 nM up to the average peak of 400 nM marked by two repeated oscillations every minute. Increased [Ca^<2+>]i was initially detected in the nuclei of ILY-treated cells subsequently followed with markedly increase in ER area. Immunocytochemistry and Westernbloting analysis, ILY treatment caused activation of calcineurin/NFAT1 signalling pathway and increased expression of AKT(p-S473),BiP, and PTEN. Interestingly the expression of BiP and AKT(p-S473)decreased in an hour post ILY treatment. Calcineurin inhibitor cyclosporine A prevented ILY-induced calcineurin/NFAT1 activation, cell death, and calcium oscillations.ILY in concentration of 10-40 ng/ml capable of inducing calcium oscillations, activation of calcineurin/NFAT1 signalling pathway ; inactivation of PI3K/AKT/MTOR pathway, and caused ER stress disorder resulted non-apoptotic cell death in HuCCT1 cells. Less