ABSTRACT

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.

摘要

目前的遗传研究（例如基因敲除）表明 EsxA 和 EsxB 作为分泌毒力因子发挥作用，这些毒力因子对结核分枝杆菌(Mtb) 细胞内存活至关重要，特别是在介导吞噬体破裂和 Mtb 易位至宿主细胞的细胞质中，这进一步促进Mtb 细胞内复制和细胞间传播。EsxA 介导的细胞内存活可能是通过其 pH 依赖性膜通透活性 (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，例如esxB-esxA 的缺失操纵子或 RD-1 基因座，可能会影响其他相互依赖的分泌因子，这些因子也可能直接涉及细胞质易位，或刺激其他基因表达的扩展干扰。为了避免基因敲除的弊端，我们首先设计了一种海洋分枝杆菌(Mm) 菌株，其中 DAS4+ 标签融合到 EsxB 的 C 端，以允许在翻译后水平诱导敲除 EsxB（也称为 EsxA）。我们还通过将 SpyTag (ST) 融合到 EsxA 的 C 端设计了 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 的翻译后敲低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。