Post-translational knockdown and post-secretional modification of EsxA determine contribution of EsxA membrane permeabilizing activity for mycobacterial intracellular survival.
Post-translational knockdown and post-secretional modification of EsxA determine contribution of EsxA membrane permeabilizing activity for mycobacterial intracellular survival.
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DOI：
10.1080/21505594.2020.1867438
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发表时间：
2021-12
期刊：
影响因子：
5.2
通讯作者：
Sun J
中科院分区：
文献类型：
--
作者：
Bao Y;Wang L;Sun J
目前的遗传学研究（例如基因敲除）表明，EsxA和EsxB作为分泌的毒力因子发挥作用，这些毒力因子对于结核分枝杆菌（Mtb）的细胞内存活至关重要，特别是介导吞噬体破裂和Mtb易位到宿主细胞的胞质溶胶中，这进一步促进了Mtb的细胞内复制和细胞间扩散。EsxA介导的细胞内存活可能是通过其pH依赖性膜透化活性（MPA）实现的。然而，来自其他研究的数据产生了关于EsxA MPA在分枝杆菌细胞内存活中的作用的差异，这引起了人们的担忧，即基因操作，如esxB-esxA操纵子或RD-1位点的缺失，可能会影响其他共同依赖分泌的因子，这些因子也可能直接参与胞质易位，或刺激其他基因表达的扩展干扰。为了避免基因敲除的缺点，我们首先工程化了海分枝杆菌（Mm）菌株，其中DAS 4+标签融合到EsxB的C末端以允许在翻译后水平诱导EsxB（也是EsxA）的敲减。我们还通过将SpyTag（ST）融合到EsxA的C末端来工程化Mm菌株，这允许通过与SpyCatcher-GFP的共价连接在分泌后水平抑制EsxA-ST MPA。EsxA的翻译后敲除和功能抑制均导致肺上皮细胞或巨噬细胞中Mm细胞内存活的减弱，这明确证实了EsxA MPA在分枝杆菌细胞内存活中的直接作用。
Current genetic studies (e.g. gene knockout) have suggested that EsxA and EsxB function as secreted virulence factors that are essential for Mycobaterium tuberculosis (Mtb) intracellular survival, specifically in mediating phagosome rupture and translocation of Mtb to the cytosol of host cells, which further facilitates Mtb intracellular replicating and cell-to-cell spreading. The EsxA-mediated intracellular survival is presumably achieved by its pH-dependent membrane-permeabilizing activity (MPA). However, the data from other studies have generated a discrepancy regarding the role of EsxA MPA in mycobacterial intracellular survival, which has raised a concern that genetic manipulations, such as deletion of esxB-esxA operon or RD-1 locus, may affect other codependently secreted factors that could be also directly involved cytosolic translocation, or stimulate extended disturbance on other genes’ expression. To avoid the drawbacks of gene knockout, we first engineered a Mycobacterium marinum (Mm) strain, in which a DAS4+ tag was fused to the C-terminus of EsxB to allow inducible knockdown of EsxB (also EsxA) at the post-translational level. We also engineered an Mm strain by fusing a SpyTag (ST) to the C-terminus of EsxA, which allowed inhibition of EsxA-ST MPA at the post-secretional level through a covalent linkage to SpyCatcher-GFP. Both post-translational knockdown and functional inhibition of EsxA resulted in attenuation of Mm intracellular survival in lung epithelial cells or macrophages, which unambiguously confirms the direct role of EsxA MPA in mycobacterial intracellular survival.
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影响因子：
3.6
作者：
Griffith KL;Grossman AD
通讯作者：
Grossman AD
DOI：
10.1074/mcp.m112.017533
发表时间：
2012-09
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Champion MM;Williams EA;Kennedy GM;Champion PA
通讯作者：
Champion PA
影响因子：
3.2
作者：
Chen, Jeffrey M.;Zhang, Ming;Cole, Stewart T.
通讯作者：
Cole, Stewart T.
影响因子：
5.8
作者：
Fine-Coulson, Kari;Giguere, Steeve;Reaves, Barbara J.
通讯作者：
Reaves, Barbara J.
影响因子：
30.8
作者：
通讯作者：
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