State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Front Immunol. 2022 Feb 24;13:829410. doi: 10.3389/fimmu.2022.829410. eCollection 2022.
To reveal functions of novel () proteins responsible for modulating host innate immunity is essential to elucidation of mycobacterial pathogenesis. In this study, we aimed to identify the role of a putative protein Rv0309 encoded within RD8 of genome in inhibiting the host inflammatory response and the underlying mechanism, using and experiments. A recombinant strain Ms_rv0309 expressing Rv0309 and a mutant Bacillus Calmette-Guérin (BCG)ΔRS01790 strain with deletion of BCG_RS01790, 100% homologue of Rv0309 in BCG, were constructed. Rv0309 was found to localize in the cell wall and be able to decrease cell wall permeability. Purified recombinant rRv0309 protein inhibited lipopolysaccharide-induced IL-6 release in RAW264.7 cells. BCG_RS01790 in BCG or Rv0309 in Ms_rv0309 strain greatly inhibited production of IL-6, IL-1β, and TNF-α in RAW264.7 cells. Similarly, BCGΔRS01790 strongly induced expression of these cytokines compared with wild-type BCG and complement strain, cBCGΔRS01790::RS01790. Further BCG_RS01790 or Rv0309 suppressed cytokine production through NF-κB p65/IκBα and MAPK ERK/JNK signaling. Importantly, BCG_RS01790 in BCG and Rv0309 in Ms_rv0309 strain enhanced mycobacterial survival in macrophages. Mice infected with BCGΔRS01790 exhibited high levels of IFN-γ, TNF-α and IL-1β, and large numbers of neutrophils and lymphocytes in the early stage, and minimal lung bacterial load and inflammatory damage in late stage of the experiment. In conclusion, the cell wall protein Rv0309 or BCG_RS01790 enhanced mycobacterial intracellular survival after infection likely through inhibition of the pro-inflammatory response and decrease of bacterial cell wall permeability, thereby contributing to mycobacterial pathogenesis.
揭示新型（）蛋白在调节宿主固有免疫中的作用对于阐明分枝杆菌的发病机制至关重要。在这项研究中，我们旨在使用（）和（）实验来鉴定基因组 RD8 中编码的假定蛋白 Rv0309 在抑制宿主炎症反应及其潜在机制中的作用。构建了表达 Rv0309 的重组 菌株 Ms_rv0309 和缺失了 BCG_RS01790 的减毒卡介苗（BCG）ΔRS01790 菌株，BCG_RS01790 是 BCG 中 Rv0309 的 100%同源物。发现 Rv0309 定位于细胞壁中，能够降低细胞壁通透性。纯化的重组 rRv0309 蛋白抑制脂多糖诱导的 RAW264.7 细胞中 IL-6 的释放。BCG 中的 BCG_RS01790 或 Ms_rv0309 菌株中的 Rv0309 极大地抑制了 RAW264.7 细胞中 IL-6、IL-1β 和 TNF-α 的产生。类似地，与野生型 BCG 和互补菌株 cBCGΔRS01790::RS01790 相比，BCGΔRS01790 强烈诱导这些细胞因子的表达。进一步的研究表明，BCG_RS01790 或 Rv0309 通过 NF-κB p65/IκBα 和 MAPK ERK/JNK 信号通路抑制细胞因子的产生。重要的是，BCG 中的 BCG_RS01790 和 Ms_rv0309 菌株中的 Rv0309 增强了分枝杆菌在巨噬细胞中的存活。感染 BCGΔRS01790 的小鼠在实验的早期阶段表现出高水平的 IFN-γ、TNF-α 和 IL-1β，以及大量的中性粒细胞和淋巴细胞，而在后期阶段肺部细菌载量和炎症损伤最小。总之，细胞壁蛋白 Rv0309 或 BCG_RS01790 通过抑制促炎反应和降低细菌细胞壁通透性来增强感染后分枝杆菌的细胞内存活，从而有助于分枝杆菌的发病机制。
