摘要
目的研究宿主细胞自噬相关基因4B(ATG4B)对贝氏柯克斯体胞内生长繁殖的影响。方法用ATG4B特异小干扰RNA(siRNA)处理宿主细胞(THP-1和Vero),以非特异siRNA和无siRNA处理的细胞为对照;逆转录实时荧光定量PCR(qRT-PCR)和Western印迹检测抑制效果;Western印迹分析细胞自噬标识蛋白(p62和LC3)的量变;qRT-PCR检测贝氏柯克斯体感染细胞中细菌增殖水平;共聚焦显微镜观察贝氏柯克斯体感染细胞中细菌寄生囊泡发育状况。结果与对照组比较,ATG4B特异siRNA处理组的细胞自噬标识蛋白p62和LC3-Ⅱ明显增多,细菌繁殖量更低,细菌寄生囊泡为散在分布小囊泡而不是单一成熟大囊泡。结论宿主细胞ATG4B表达下调可部分阻断自噬,自噬的部分阻断使贝氏柯克斯体寄生囊泡发育受阻,进而抑制贝氏柯克斯体胞内生长繁殖,为阐明宿主细胞的自噬促进贝氏柯克斯体繁殖的致病机制提供了新的依据。
Objective To investigate the effects of autophagy-related gene 4B(ATG4B)on the intracellular replication of Coxiella burnetii(C.burnetii)and the development of Coxiella-containing vacuoles(CCVs).Methods Host cells(THP-1cells and Vero cells)treated with ATG4B-targeting siRNA served as the experimental group,those treated with non-targeting siRNA served as the negative control group(NC),and untreated ones served as the mock control.The expression levels of autophagy molecular markers(p62,LC3)in the three groups were detected by Western blotting.The bacterial loads in these groups were detected using qRT-PCR.The development of CCV in these groups was observed using confocal microscopy.Results Compared with the control groups,the autophagy molecular markers(p62,LC3)in the experimental group significantly increased while bacterial loads became much lower,and reduced CCV sizes and a multi-vacuolar CCV phenotype in ATG4B-silenced cells were observed.Conclusion The results suggest that ATG4B play an important role in modulating autophagy,which could be required for the development of CCV and the replication of C.burnetii in host cells.This study can shed light on the relationship between autophagy machinery and the pathogenic mechanism of C.burnetii.
作者
张家宁
付梦姣
焦俊
欧阳譞
于永慧
赵明亮
张珊
温博海
熊小路
孙岩松
ZHANG Jia-ning;FU Meng-jiao;JIAO Jun;OUYANG Xuan;YU Yong-hui;ZHAO Ming-liang;ZHANG Shan;WEN Bo-hai;XIONG Xiao-lu;SUN Yan-song(State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)
出处
《军事医学》
CAS
2022年第8期569-575,共7页
Military Medical Sciences
基金
国家自然科学基金(31970178,32000140)。
