表达Panton-Valentine杀白细胞素(PVL)的金黄色葡萄球菌诱导人THP-1单核细胞自噬和凋亡

Staphylococcus aureus expressing Panton-Valentine leucocidin(PVL)induces autophagy and apoptosis of human THP-1 monocytes

目的研究表达Panton-Valentine杀白细胞素(PVL)的金黄色葡萄球菌诱导人THP-1细胞自噬和凋亡机制。方法在大肠杆菌中表达重组杀白细胞素(rPVL),并免疫家兔制备多克隆抗血清。采用反转录PCR和Western blot法鉴定表达PVL的耐甲氧西林金黄色葡萄球菌(MRSA)。分别使用表达PVL的MRSA(MRSA^(PVL+))和不表达PVL的MRSA(MRSA^(PVL-))感染THP-1细胞,于感染后1、3、6 h收集细胞,采用实时定量PCR检测自噬相关基因3(ATG3)、ATG4B、ATG5、ATG7、ATG12;Western blot法检测beclin-1、微管相关蛋白1轻链3(LC3)、磷脂酰肌醇3激酶(PI3K)、磷酸化的PI3K(p-PI3K)、蛋白激酶B(AKT)、磷酸化的AKT(p-AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化的mTOR(p-mTOR)、裂解型胱天蛋白酶3(c-caspase-3)、caspase-3的蛋白水平;二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针负载法检测细胞活性氧(ROS)水平;异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测细胞凋亡。结果成功制备PVL多克隆抗体,鉴定出1株MRSA^(PVL-)菌和1株MRSA^(PVL+)菌。分别以两株菌与THP-1细胞共培养1 h,MRSA^(PVL+)组仅ATG7表达量显著高于MRSA^(PVL-)组和正常对照组;共培养3 h,MRSA^(PVL+)组ATG7和ATG12表达量高于MRSA^(PVL-)组和正常对照组,其他基因表达量变化不明显。感染1、3、6 h,MRSA^(PVL+)感染组细胞PI3K、AKT和mTOR磷酸化水平显著低于MRSA^(PVL-)感染组和正常对照组;感染1 h和3 h,MRSA^(PVL-)感染组细胞PI3K、AKT和mTOR磷酸化水平显著低于正常对照组;感染6 h,MRSA^(PVL-)菌感染组细胞mTOR磷酸化水平显著低于正常对照组。感染1、3、6 h,MRSA^(PVL+)组细胞ROS水平和凋亡率均高于MRSA^(PVL-)组和正常对照组,且随着感染时间的延长细胞ROS水平和凋亡率持续增加;MRSA^(PVL-)组细胞ROS水平和凋亡率也高于正常对照组。结论MRSA^(PVL+)对THP-1细胞PI3K/AKT/mTOR信号通路抑制作用更强,可促进ATG12/ATG7/ATG5自噬通路活化增加THP-1细胞自噬;同时,MRSA^(PVL+)诱导THP-1细胞ROS产生增加,促进细胞凋亡。

Objective To study the mechanism by which Staphylococcus aureus expressing Panton-Valentine leucocidin(PVL)induces the autophagy and apoptosis of human THP-1 cells.Methods Recombinant PVL was expressed in Escherichia coli BL21 strain,and rabbits were immunized to prepare poly-antiserum.Real-time fluorescent quantitative PCR and Western blotting were used to identify methicillin-resistant Staphylococcus aureus(MRSA)expressing PVL.MRSA strains with or without PVL expression were used to infect THP-1 cells.The cells were collected 1 hour,3 hours and 6 hoursafter the infection,and the autophagy-related genes 3(ATG3),ATG4B,ATG5,ATG7 and ATG12 were detected by real-time fluorescent quantitative PCR.Western blotting was performed to determine the expression of beclin-1,LC3,phosphatidylinositol 3 kinase(PI3K),phosphorilated PI3K(p-PI3K),AKT,phosphorilated AKT(p-AKT),mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),cleaved-capase-3(c-casepase-3)and casepase-3.In addition,2,7-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe method was used to detect the expression of reactive oxygen species(ROS).Annexin V-FITC/PI double labeling combined with flow cytometry was used to detect cell apoptosis.Results PVL polyclonal antibody was successfully prepared.Western blot and PCR identified one MRSA strain that did not express PVL(MRSA^(PVL-))and one MRSA strain that expressed PVL(MRSA^(PVL+)).Two strains were co-cultured with THP-1 cells.At 1 hour,only ATG7 expression in the MRSA^(PVL+)group was significantly higher than that in the MRSA^(PVL-) group and normal control group.At 3 hours,ATG7 and ATG12 expression in the MRSA^(PVL+) group were higher than those in the MRSA^(PVL-) group and normal control group,and there was no statistically significant change in the expression of other genes.At 1 hour,3 hours,and 6 hours after the infection,the phosphorylation levels of PI3K,AKT and mTOR in the MRSA^(PVL+) strain infection group were significantly lower than those in the MRSA^(PVL-) strain infection group and normal control group;at 1 hour and 3 hours,the phosphorylation levels of PI3K,AKT and mTOR in the MRSA^(PVL-) strain infection group were significantly lower than those in the normal control group;at 6 hours after the infection,the mTOR phosphorylation level in the MRSA^(PVL-) strain infection group was significantly lower than that in the normal control group.At 1 hour,3 hours,and 6 hours after the infection,the expression of ROS and the apoptosis rate in the MRSA^(PVL+) group were higher than those in the MRSA^(PVL-) group and normal control group,and the expression of ROS in the cells increased along with the infection time and the apoptosis rate continued to increase;ROS expression and apoptosis rate in the MRSA^(PVL-) group were also higher than those in the normal control group.Conclusion MRSA^(PVL+)strain are more capable of inhibiting the activation of the PI3K/AKT/mTOR signaling pathway in THP-1 cells than MRSA^(PVL-) strain.It can increase the THP-1 cell autophagy via ATG12-ATG7-ATG5 autophagy pathway.At the same time,MRSA^(PVL+)strain can induce THP-1 cells to express more ROS than MRSA^(PVL-) strain,and induce more THP-1 cell apoptosis.