伤寒沙门菌质粒对巨噬细胞自噬过程的影响

The influence of Salmonella enterica serovar Typhi plasmid on macrophage autophagy

为研究伤寒沙门菌质粒pRST98对人巨噬细胞THP-1自噬过程的影响,以携带伤寒沙门菌质粒pRST98的野生株ST6、消除pRST98的突变株ST6-ΔpRST98和将pRST98经接合转移的回补株ST6-c-pRST98为受试菌,与THP-1共培养建立感染模型,并加入自噬作用阻断剂氯喹(CQ)进行干预。首先测定CQ单独作用对细胞及细菌的影响,确定CQ最适浓度;在细菌和细胞共作用后的不同时间点收集细胞,通过蛋白免疫印迹法(WB)和mRFP-GFP-LC3质粒转染法检测细胞LC3Ⅱ蛋白、p62蛋白、自噬体及自噬溶酶体的变化。结果显示,30μmol/LCQ对细胞自噬的阻断效果明显,且细胞存活率超过50%,对细菌也无明显影响。WB结果显示,用该浓度CQ干预后,ST6-ΔpRST98感染组细胞的LC3Ⅱ及p62表达量上升程度显著高于野生株ST6组及回补株ST6-c-pRST98组;CQ干预组3株受试菌感染细胞LC3点状结构数量均高于未加CQ组,且ST6-ΔpRST98感染组细胞的点状结构数量明显增加。以上结果提示,伤寒沙门菌质粒pRST98在自噬前期发挥作用,早于溶酶体降解的过程。

In the present study, the influence of Salmonella enterica serovar Typhi （S. typhi） plasmid pR_ST98 on autophagy of human-derived macrophage-like cell line THP-1 was investigated. THP-1 cells were infected with wild type strain （ST6）, plasmid-free mutant strain （ST6-△pR_ST98） and ST6-△pR_ST98 with a complemented plasmid encoding pR_ST98 （ST6-c-pR_ST98） respectively, and challenged with lysosomotropic agent chloroquine （CQ）. Cells were collected at different time points after cocultivation with bacteria, and the levels of protein LC3 Ⅱ and p62 were measured by Western blotting, and the impacts on autophagosomes and autolysosomes were detected by detecting monomeric red fluorescent protein-green fluorescent protein （mRFP-GFP）-LC3 introduced by plasmid transfection. The results revealed that 30μmol/L CQ could significantly block the autophage process, and had no obvious influence on the cells and bacteria. The Western blotting analysis showed that the augment of autophagy protein LC3 Ⅱ and 1362 in ST6-△pR_ST98 group was higher than those in ST6 and ST6-c-pR_ST98 groups after CQ treatment. Similarly, the LC3 dots in the groups treated with CQ were higher than those in the groups without CQ treatment. Specifically, more LC3 dots were detected in ST6-ApR_ST98 group than that in the wild type group. All these evidences suggest that S. typhi plasmid pR_ST98 works in the early stage of autophagy, prior to the degradation of autolysosomes.