牛种布鲁氏菌Ⅳ型分泌系统对巨噬细胞内质网应激和细胞凋亡的影响

doi:10.11843/j.issn.0366-6964.2022.04.019

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (4): 1192-1200.doi: 10.11843/j.issn.0366-6964.2022.04.019

牛种布鲁氏菌Ⅳ型分泌系统对巨噬细胞内质网应激和细胞凋亡的影响

杨琴^1,2, 邓肖玉^1,2, 谢珊珊^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:2021-06-15 出版日期:2022-04-23 发布日期:2022-04-25
通讯作者: 王震,主要从事动物疫病防控研究,E-mail:wzhen2018@shzu.edu.cn;陈创夫,主要从事病原微生物致病机制研究,E-mail:ccf-xb@163.com
作者简介:杨琴(1995-),女,新疆昌吉人,硕士生,主要从事病原分子生物学研究
基金资助:
国家自然基金项目(U1803236；32002245)；兵团重大科技项目(2017AA003)

Effects of Brucella bovis Type IV Secretion System on Endoplasmic Reticulum Stress and Apoptosis of Macrophages

YANG Qin^1,2, DENG Xiaoyu^1,2, XIE Shanshan^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 Infectious Diseases, Shihezi 832000, China;
3. Institute of Animal Husbandry and Veterinary of Xinjiang Agricultural Reclamation Science Academy, Shihezi 832000, China;
4. Key Laboratory of Animal Disease Prevention and Control of Corps, Shihezi 832000, China

Received:2021-06-15 Online:2022-04-23 Published:2022-04-25

摘要/Abstract

摘要： 旨在研究牛种布鲁氏菌Ⅳ型分泌系统(T4SS)对巨噬细胞内质网应激和细胞凋亡的影响，深入探究布鲁氏菌的分子致病机制。本研究以牛种布鲁氏菌疫苗株A19及其T4SS启动子缺失株A19ΔVirB侵染小鼠巨噬细胞，分别在侵染的6、12、24 h收集细胞RNA和总蛋白，通过qRT-PCR以及Western blot检测内质网应激标志性分子(GRP78和CHOP)、凋亡相关分子(BAX和BCL-2)的转录水平和蛋白表达水平；通过流式细胞术检测不同时间点的细胞凋亡情况。结果显示，在侵染的6、12和24 h，缺失株A19ΔVirB组GRP78的转录水平及蛋白表达水平均显著低于亲本株A19组(P<0.05)；在侵染后的6 h，缺失株组CHOP的转录水平及蛋白表达水平也显著低于亲本株(P<0.05)，但到了24 h，出现了显著高于亲本株(P<0.05)的现象；在侵染的24 h，缺失株A19ΔVirB组BAX的转录水平及蛋白表达水平显著高于亲本株(P<0.05)；而在侵染的24 h，缺失株组BCL-2的转录水平及蛋白表达水平显著低于亲本株(P<0.05)；流式结果进一步显示，在侵染的24 h，缺失株组的细胞凋亡率显著高于亲本株(P<0.05)；本试验的结果表明，T4SS缺失后，会导致牛种布鲁氏菌引起内质网应激的能力减弱，诱导细胞凋亡的能力增强。本研究为进一步解析布鲁氏菌的致病机制奠定了基础，也为今后布鲁氏菌T4SS的功能研究提供了科研依据。

关键词: 布鲁氏菌, 缺失株, T4SS, 内质网应激, 细胞凋亡

Abstract: In order to study the effect of Brucella bovis type IV secretion system (T4SS) on the endoplasmic reticulum stress and apoptosis of macrophages, the molecular pathogenic mechanism of Brucella was deeply explored. In this study, bovine Brucella vaccine strain A19 and its T4SS promoter deletion strain A19ΔVirB were used to infect mouse macrophages. RNA and total protein were collected at 6, 12 and 24 hours after infection, transcription level and protein expression level of endoplasmic reticulum stress marker molecules GRP78 and CHOP, apoptosis related molecules BAX and BCL-2 were detected by qRT-PCR and Western blot, apoptosis at different time points was detected by flow cytometry. The results showed that at 6, 12 and 24 h after infection, the transcription level and protein expression level of GRP78 in deletion strain A19ΔVirB group were significantly lower than those in parent A19 group (P<0.05); at 6 h after infection, the transcription level and protein expression level of CHOP in the deletion strain group were also significantly lower than those in the parent strain (P<0.05), but at 24 h, it was significantly higher than that of the parent strain (P<0.05); at 24 h after infection,transcription level and protein expression level of BAX in deletion strain A19ΔVirB group were significantly higher than those in parental strains (P<0.05); at 24 h after infection,transcription level and protein expression level of BCL-2 in deletion strain A19ΔVirB group were significantly lower than those in parental strain (P<0.05); the results of flow cytometry further showed that the apoptosis rate of the deletion strain group was significantly higher than that of the parent strain (P<0.05). The results showed that deletion of T4SS would weaken the ability of Brucella bovis to induce endoplasmic reticulum stress and enhance its ability to induce apoptosis.This study laid a foundation for further understanding the pathogenic mechanism of Brucella, and also provided a scientific basis for the functional research of Brucella T4SS in the future.

Key words: Brucella, deletion strain, T4SS, endoplasmic reticulum stress, apoptosis

中图分类号:

杨琴, 邓肖玉, 谢珊珊, 易继海, 王勇, 张倩, 王震, 陈创夫. 牛种布鲁氏菌Ⅳ型分泌系统对巨噬细胞内质网应激和细胞凋亡的影响[J]. 畜牧兽医学报, 2022, 53(4): 1192-1200.

YANG Qin, DENG Xiaoyu, XIE Shanshan, YI Jihai, WANG Yong, ZHANG Qian, WANG Zhen, CHEN Chuangfu. Effects of Brucella bovis Type IV Secretion System on Endoplasmic Reticulum Stress and Apoptosis of Macrophages[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1192-1200.

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牛种布鲁氏菌Ⅳ型分泌系统对巨噬细胞内质网应激和细胞凋亡的影响

Effects of Brucella bovis Type IV Secretion System on Endoplasmic Reticulum Stress and Apoptosis of Macrophages

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