[Effect of Mycobacterium tuberculosis protein Rv0309 on intracellular survival of Mycobacterium smegmatis by inhibiting macrophage autophagy via protein STUB1].

Objective: To investigate the molecular regulatory mechanism of Mycobacterium tuberculosis (MTB) protein Rv0309 to promote the survival of Mycobacterium smegmatis (Ms) in macrophages. Methods: Using Ms as a model to study Mycobacterium tuberculosis, recombinant Ms transfected with pMV261 and PMV261-RV0309 in the control group and RAW264.7 cells were constructed. The effect of Rv0309 protein on intracellular survival of Ms was investigated by counting colony forming units (CFUs). Mass spectrometry was used to screen proteins interacting with host protein Rv0309, and immunocoprecipitate (Co-IP) was used to verify that host protein STUB1 could interact with host protein Rv0309. STUB1 gene knock-out RAW264.7 cells were infected with Ms, and CFUs were counted to explore the effect of protein Rv0309 on intracellular survival of Ms after STUB1 gene knock-out. STUB1 gene knock-out RAW264.7 cells were infected with Ms, and after obtaining samples, Western blotting assay was performed to explore the effect of protein Rv0309 on autophagy function of macrophages after STUB1 gene knock-out. Statistical analysis was performed using GraphPad Prism 8 software. T-test was selected for analysis in this experiment, with P<0.05 was considered statistically significant. Results: Western blotting showed that Rv0309 was expressed in M. smegmatis and secreted extracellularly. The CFUs of the Ms-Rv0309 group was higher than that of Ms-pMV261 group at 24 h after THP-1 macrophage infection, and the difference was statistically significant (P<0.05). The trend of infected RAW264.7 macrophages was the same as that of infected THP-1 macrophages. The Co-IP results showed that the corresponding Flag and HA bands appeared in the results of immunoprecipitation (IP):Flag and IP: HA. The level of CFUs in the experimental group with STUB1 deletion was significantly higher than that in the control group without STUB1 deletion. Compared with Ms-pMV261, the CFUs in the Ms-Rv0309 group was significantly higher than that in the Ms-pMV261 group. The gray scale of LC3Ⅱ bands of Ms-Rv0309 in experimental group was lighter than that of Ms-pMV261 in the control group at the corresponding time point, and the result was most significant at 8 h (LC3Ⅱ/β-actin: 0.76±0.05 vs 0.47±0.07), the difference being statistically significant (P<0.05). After STUB1 genome knock-out, the gray level of LC3Ⅱ bands at the corresponding time was lighter than that without STUB1 genome knock-out. Comparison of the results of Ms-pMV261 and Ms-Rv0309 strains revealed that LC3Ⅱ band gray Rv0309 group was lighter at the corresponding time compared with pMV261 group. Conclusions: MTB protein Rv0309 can be successfully expressed in M. smegmatis and secreted extracellularly, which can inhibit the autophagy process of macrophages. Protein Rv0309 interacts with host protein STUB1 to inhibit macrophage autophagy and promote intracellular survival of Ms.目的： 探讨结核分枝杆菌（MTB）蛋白Rv0309促进耻垢分枝杆菌（Ms）在巨噬细胞胞内存活的分子调节机制。 方法： 以Ms为研究结核分枝杆菌的模型，构建带有对照组pMV261-3*flag空载质粒和实验组pMV261-Rv0309-3*flag质粒的重组Ms并感染RAW264.7细胞。通过计数菌落形成单位（CFU），探索蛋白Rv0309对Ms胞内存活的影响。通过质谱法筛选蛋白Rv0309与宿主相互作用的蛋白，采用免疫共沉淀法（Co-IP）验证宿主蛋白STUB1可与蛋白Rv0309相互作用。敲减RAW264.7细胞STUB1基因后感染Ms，计数CFU，探索STUB1基因敲减后，蛋白Rv0309对Ms胞内存活的影响。敲减RAW264.7细胞STUB1基因后感染Ms，收样后进行Western blot实验，探索STUB1基因敲减后，蛋白Rv0309对巨噬细胞自噬功能的影响。使用GraphPad Prism 8 软件进行统计分析，本实验选择t检验进行分析，以P<0.05为差异有统计学意义。 结果： Western blot显示蛋白Rv0309表达于耻垢分枝杆菌体内并分泌到胞外。感染THP-1巨噬细胞实验在24 h时实验组Ms-Rv0309的CFU高于对照组Ms-pMV261，差异有统计学意义（P<0.05）。感染RAW264.7巨噬细胞实验结果趋势与感染THP-1巨噬细胞相同。免疫共沉淀（Co-IP）结果显示免疫沉淀（IP）：Flag和IP：HA结果中出现对应的Flag和HA条带。敲减STUB1的实验组（siRNA-STUB1组）CFU水平显著高于未敲减STUB1对照组（NC组）CFU；与对照组Ms-pMV261相比，Ms-Rv0309组CFU均显著高于Ms-pMV261组。实验组Ms-Rv0309的LC3Ⅱ条带在对应时间灰度均浅于对照组Ms-pMV261，8 h时结果最显著（LC3Ⅱ/β-actin：0.76±0.05 vs 0.47±0.07），差异有统计学意义（P<0.05）。siRNA-STUB1组LC3Ⅱ条带在对应时间灰度均浅于NC组；对比Ms-pMV261和Ms-Rv0309菌株感染的CFU结果发现，与Ms-pMV261组相比，在对应时间LC3Ⅱ条带灰度Ms-Rv0309组更浅。 结论： MTB蛋白Rv0309能够成功表达于耻垢分枝杆菌并分泌到胞外，可抑制巨噬细胞的自噬过程。蛋白Rv0309与宿主蛋白STUB1相互作用，抑制巨噬细胞自噬促进Ms胞内存活。.