Mycobacterium tuberculosis gene SapM inhibits autophagy in human embryonic kidney cell line 293A
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- 英文论文题名：Mycobacterium tuberculosis gene SapM inhibits autophagy in human embryonic kidney cell line 293A
- 论文作者：Zhang Wei ; Dong Yuanshu ; Xiong Sidong
- 英文论文作者：Zhang Wei ; Dong Yuanshu ; Xiong Sidong ; Institutes of Biology and Medical Sciences ; Soochow University
- 年：2016
- 作者机构：Institutes of Biology and Medical Sciences,Soochow University;
- 英文论文关键词：Tuberculosis ; SapM ; Autophagy
- 会议召开时间：2016-11-04
- 会议录名称：第十一届全国免疫学学术大会壁报交流集
- 英文会议录名称：Proceedings of 2016 CSI Congress on Immunology
- 语种：英文
- 分类号：R378.911
- 学会代码：IGMD
- 会议名称：第十一届全国免疫学学术大会
- 会议地点：中国安徽合肥
- 主办单位：中国免疫学会
- 学会名称：中国免疫学会
- 页数：1
- 文件大小：313k
- 原文格式：D
- 会议级别：全国
摘要
Background:Tuberculosis(TB) is a permanently respiratory disease which has afflicted human life and health.Autophagy is a cytoplasmic maintenance process which plays a pivot role against mycobacterium tuberculosis(Mtb).It has been reported that Sap M(also named Rv3310),a secreted acid phosphatase of Mtb,inhibits host cell autophagy by blocking autophagosome-lysosome fusion,but its role in the initiation of autophagy is unknown.Objective:Investigate the role of Mtb gene Sap M in regulating the initiation of autophagy in HEK293 A cells.Methods:We constructed the plasmid p FLAG-CMV2-Sap M to express Mtb gene Sap M in eukaryotic cells,and established 293 A cell line that stably expressing Sap M and GFP-LC3(Sap M-GFP-LC3 293A) by co-transfection.Sap M-GFPLC3 293 A cells and Vector-GFP-LC3 293 A cells were treated with autophagy inducer rapamycin or starvation,with or without chloroquine,and LC3 Ⅱ protein levels were detected by western blot,flow cytometry,or Fluorescence microscopy.Results:With Rapamycin treatment,LC3 II level was obviously lower in cells overexpressing Sap M compared to that in vector control cells.Moreover,after blocking autophagosome-lysosome fusion by chloroquine,the number of rapamycin induced GFP-LC3 punta in Sap M-GFP-LC3 group were dramatically lesser than that in vector group.Similar results were observed in starvation-induced autophagy.Conclusions:Our results indicated that Sap M inhibited a complete autophagic flux in HEK293 A cells and it suppressed the initiation of autophagy.These findings facilitated our understanding of the mechanisms how Sap M promoting the infection of Mtb,and may provide a new target for potential vaccines or drugs against tuberculosis.
Background:Tuberculosis(TB) is a permanently respiratory disease which has afflicted human life and health.Autophagy is a cytoplasmic maintenance process which plays a pivot role against mycobacterium tuberculosis(Mtb).It has been reported that Sap M(also named Rv3310),a secreted acid phosphatase of Mtb,inhibits host cell autophagy by blocking autophagosome-lysosome fusion,but its role in the initiation of autophagy is unknown.Objective:Investigate the role of Mtb gene Sap M in regulating the initiation of autophagy in HEK293 A cells.Methods:We constructed the plasmid p FLAG-CMV2-Sap M to express Mtb gene Sap M in eukaryotic cells,and established 293 A cell line that stably expressing Sap M and GFP-LC3(Sap M-GFP-LC3 293A) by co-transfection.Sap M-GFPLC3 293 A cells and Vector-GFP-LC3 293 A cells were treated with autophagy inducer rapamycin or starvation,with or without chloroquine,and LC3 Ⅱ protein levels were detected by western blot,flow cytometry,or Fluorescence microscopy.Results:With Rapamycin treatment,LC3 II level was obviously lower in cells overexpressing Sap M compared to that in vector control cells.Moreover,after blocking autophagosome-lysosome fusion by chloroquine,the number of rapamycin induced GFP-LC3 punta in Sap M-GFP-LC3 group were dramatically lesser than that in vector group.Similar results were observed in starvation-induced autophagy.Conclusions:Our results indicated that Sap M inhibited a complete autophagic flux in HEK293 A cells and it suppressed the initiation of autophagy.These findings facilitated our understanding of the mechanisms how Sap M promoting the infection of Mtb,and may provide a new target for potential vaccines or drugs against tuberculosis.
Background:Tuberculosis(TB) is a permanently respiratory disease which has afflicted human life and health.Autophagy is a cytoplasmic maintenance process which plays a pivot role against mycobacterium tuberculosis(Mtb).It has been reported that Sap M(also named Rv3310),a secreted acid phosphatase of Mtb,inhibits host cell autophagy by blocking autophagosome-lysosome fusion,but its role in the initiation of autophagy is unknown.Objective:Investigate the role of Mtb gene Sap M in regulating the initiation of autophagy in HEK293 A cells.Methods:We constructed the plasmid p FLAG-CMV2-Sap M to express Mtb gene Sap M in eukaryotic cells,and established 293 A cell line that stably expressing Sap M and GFP-LC3(Sap M-GFP-LC3 293A) by co-transfection.Sap M-GFPLC3 293 A cells and Vector-GFP-LC3 293 A cells were treated with autophagy inducer rapamycin or starvation,with or without chloroquine,and LC3 Ⅱ protein levels were detected by western blot,flow cytometry,or Fluorescence microscopy.Results:With Rapamycin treatment,LC3 II level was obviously lower in cells overexpressing Sap M compared to that in vector control cells.Moreover,after blocking autophagosome-lysosome fusion by chloroquine,the number of rapamycin induced GFP-LC3 punta in Sap M-GFP-LC3 group were dramatically lesser than that in vector group.Similar results were observed in starvation-induced autophagy.Conclusions:Our results indicated that Sap M inhibited a complete autophagic flux in HEK293 A cells and it suppressed the initiation of autophagy.These findings facilitated our understanding of the mechanisms how Sap M promoting the infection of Mtb,and may provide a new target for potential vaccines or drugs against tuberculosis.
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