布鲁氏菌Ⅳ型分泌系统对树突状细胞自噬、胞内生存和炎症反应的影响

doi:10.16431/j.cnki.1671-7236.2021.01.028

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (1): 257-264.doi: 10.16431/j.cnki.1671-7236.2021.01.028

布鲁氏菌Ⅳ型分泌系统对树突状细胞自噬、胞内生存和炎症反应的影响

杨琴^1,2, 邓肖玉^1,2, 赵晓丽^1,2, 张欢^1,2, 席静^1,2, 易继海^1,2, 王勇^1,2, 张倩^1,3, 王震^1,4, 陈创夫^1,2

1. 石河子大学动物科技学院, 石河子 832000;
2. 人兽共患传染性疾病防治协同创新中心, 石河子 832000;
3. 新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室, 石河子 832000;
4. 动物疫病防控兵团重点实验室, 石河子 832000

收稿日期:2020-07-29 出版日期:2021-01-20 发布日期:2021-01-15
通讯作者: 王震, 陈创夫 E-mail:wzhen2018@shzu.edu.cn;ccf-xb@163.com
作者简介:杨琴(1995-),女,新疆昌吉人,硕士生,研究方向:预防兽医学,E-mail:1377972771@qq.com
基金资助:
国家自然科学基金项目（U1803236、31760020）；兵团重大科技项目（2017AA003）

Effects of Brucella Type Ⅳ Secretion System on Autophagy,Intracellular Survival and Inflammatory Response of Dendritic Cells

YANG Qin^1,2, DENG Xiaoyu^1,2, ZHAO Xiaoli^1,2, ZHANG Huan^1,2, XI Jing^1,2, YI Jihai^1,2, WANG Yong^1,2, ZHANG Qian^1,3, WANG Zhen^1,4, CHEN Chuangfu^1,2

1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
2. Collaborative Innovation Center for the Prevention and Control of Zoonotic Infectious Diseases, Shihezi 832000, China;
3. State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding Jointly Built by Xinjiang Academy of Agricultural Reclamation Sciences, Shihezi 832000, China;
4. Key Laboratory of Animal Disease Prevention and Control of Corps, Shihezi 832000, China

Received:2020-07-29 Online:2021-01-20 Published:2021-01-15

摘要/Abstract

摘要： 为探究Ⅳ型分泌系统在布鲁氏菌（Brucella）感染过程中的作用，深入了解布鲁氏菌Ⅳ型分泌系统在疫苗开发中的潜力，本研究以牛种布鲁氏菌A19疫苗株为研究对象，使用A19 VirB启动子缺失株感染小鼠树突状细胞（DCs），通过菌落计数（CFU）评估Ⅳ型分泌系统对布鲁氏菌黏附侵袭及胞内生存的影响，同时对感染的细胞进行RNA和总蛋白的提取，分别通过实时荧光定量PCR和Western blotting检测自噬基因Beclin-1的转录和表达情况；收集感染后的细胞上清液，利用ELISA检测炎症因子白细胞介素-6（IL-6）和IL-10的分泌水平。黏附侵袭结果显示，布鲁氏菌VirB启动子缺失株与亲本株A19的黏附侵袭水平无显著差异（P>0.05）；胞内生存试验发现，感染的4 h，布鲁氏菌VirB启动子缺失株的胞内存活能力显著低于亲本株A19（P<0.05），感染后0、24和48 h极显著低于亲本株A19（P<0.01）；实时荧光定量PCR和Western blotting结果显示，感染后4、8和12 h，布鲁氏菌VirB启动子缺失株刺激细胞产生Beclin-1的水平极显著高于亲本株A19（P<0.01）；ELISA结果显示，感染后8和12 h，布鲁氏菌VirB启动子缺失株刺激细胞产生IL-6的水平显著高于亲本株A19（P<0.05），而在感染后8和12 h，布鲁氏菌VirB启动子缺失株刺激细胞分泌IL-10的水平显著低于亲本株A19（P<0.05），感染24 h时极显著低于亲本株A19（P<0.01）。综上所述，当VirB启动子缺失后，布鲁氏菌对DCs的黏附侵袭能力并未明显改变，但显著降低了布鲁氏菌在DCs内的存活能力，提升了DCs的自噬水平，促进了DCs IL-6的分泌，抑制了IL-10的分泌。本研究初步探究了布鲁氏菌Ⅳ型分泌系统在感染DCs过程中的生物学作用，为后续布鲁氏菌疫苗改造研究奠定了理论基础。

关键词: 布鲁氏菌VirB启动子缺失株; Ⅳ型分泌系统; 树突状细胞; 胞内生存; 炎症细胞因子; 自噬

Abstract: In order to explore the role of the type Ⅳ secretion system in the process of Brucella infection,in-depth understanding of the potential of the type Ⅳ secretion system of Brucella in vaccine development,in this study,the A19 vaccine strain of Brucella abortus was used as the research object,and the A19 VirB promoter deletion strain was used to infect mouse dendritic cells (DCs),evaluate the effect of type Ⅳ secretion system on the adhesion,invasion and intracellular survival of Brucella by colony count (CFU).At the same time,RNA and total protein were extracted from the infected cells,and the transcription and expression of the autophagy gene Beclin-1 were detected by Real-time PCR and Western blotting respectively.The cell supernatant after infection was collected,and ELISA was used to detect the secretion levels of inflammatory factors IL-6 and IL-10.The adhesion and invasion results showed that the adhesion and invasion levels of Brucella VirB promoter deletion strain and parent strain A19 were not significantly different (P>0.05).The intracellular survival experiment found that the intracellular survival ability of the Brucella VirB promoter deletion strain was significantly lower than that of the parent strain A19 at 4 h after infection (P<0.05),and it was extremely significantly lower at 0,24 and 48 h after infection (P<0.01).The results of Real-time PCR and Western blotting showed that the level of Beclin-1 production was extremely significantly higher than that of the parent strain A19 (P<0.01).ELISA results showed that at 8 and 12 h after infection,the level of IL-6 stimulated by VirB promoter deletion strain was significantly higher than that of parental strain A19 (P<0.05).However,at 8 and 12 h after infection,the level of IL-10 stimulated by VirB promoter deletion strain was significantly lower than that of parental strain A19 (P<0.05),and was extremely significantly lower than that of parental strain A19 at 24 h after infection (P<0.01).This study preliminarily explored the biological role of Brucella type Ⅳ secretion system in the process of infection of DCs,and laid a theoretical foundation for the follow-up Brucella vaccine transformation research.

Key words: Brucella VirB promoter deletion strain; type Ⅳ secretion system; DCs; intracellular survival; cytokines; autophagy

中图分类号:

S852.61

杨琴, 邓肖玉, 赵晓丽, 张欢, 席静, 易继海, 王勇, 张倩, 王震, 陈创夫. 布鲁氏菌Ⅳ型分泌系统对树突状细胞自噬、胞内生存和炎症反应的影响[J]. 中国畜牧兽医, 2021, 48(1): 257-264.

YANG Qin, DENG Xiaoyu, ZHAO Xiaoli, ZHANG Huan, XI Jing, YI Jihai, WANG Yong, ZHANG Qian, WANG Zhen, CHEN Chuangfu. Effects of Brucella Type Ⅳ Secretion System on Autophagy,Intracellular Survival and Inflammatory Response of Dendritic Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 257-264.

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布鲁氏菌Ⅳ型分泌系统对树突状细胞自噬、胞内生存和炎症反应的影响

Effects of Brucella Type Ⅳ Secretion System on Autophagy,Intracellular Survival and Inflammatory Response of Dendritic Cells

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