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

Abstract

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.