Abstract:

Objective: To explore the gene function of Mycobacterium tuberculosis (MTB) Rv2333c by overexpressing Rv2333c in Mycolicibacterium (M.) smegmatis in the process of infected macrophages. Methods: Overexpression of Rv2333c in M.smegmatis was achieved based on the construction of pSUM-MCS2-Rv2333c. Samples were collected at different time points after infecting macrophages with the empty strain (Ms_Vec) or the overexpression strain (Ms_Rv2333c). Then, the bacterial intracellular viability was assessed by counting colony-forming units (CFU) and the cytotoxicity of Rv2333c to the cell was detected by measuring the lactate dehydrogenase (LDH) activity in the cell media. Total RNA was extracted and the supernatant of culture media and cell lysates was collected to investigate Rv2333c effect on cytokines secretion and NF-κB/MAPK pathway in macrophages through reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. Results: Rv2333c of MTB was successfully expressed in M.smegmatis. The cytotoxicity significantly decreased at the early stage (6 h) of macrophages infected with Ms_Rv2333c (LDH: Ms_Vec: (52.55±0.90) %, Ms_Rv2333c: (27.87±1.77) %; t=17.560, P<0.001), but the intracellular proliferation of Ms_Rv2333c was not affected (log₁₀CFU: Ms_Vec: 7.45±0.05, Ms_Rv2333c: 7.35±0.16; t=1.069, P=0.345). RT-PCR revealed that IL-1β mRNA was downregulated at the early stage (6 h, Ms_Vec: 657.43±20.96, Ms_Rv2333c: 521.36±25.97; t=5.767, P=0.005), but upregulated at the late stage of macrophages infected with Ms_Rv2333c (48 h, Ms_Vec: 1548.53±125.17, Ms_Rv2333c: 2168.13±130.00; t=4.855, P=0.008). However, IL-6 mRNA was upregulated at the early (6 h) and late stage (48 h) of macrophages infected with Ms_Rv2333c (Ms_Vec: 2.46±0.49 & 776.41±15.38, Ms_Rv2333c: 6.02±0.37 & 1642.76±51.15; t=8.234 & 22.940, P=0.001 & <0.001, respectively). And the interferon-γ (IFN-γ) mRNA was also upregulated (Ms_Vec: 2.52±0.09 & 2.76±0.32, Ms_Rv2333c: 17.24±0.47 & 8.52±0.08; t=43.760 & 25.090, all P<0.001, respectively). The results of Western blotting showed that the expression of NF-κB and phosphorylation of ERK1/2 significantly increased in macrophages 3 hours after infecting with Ms_Rv2333c and maintained at high level even at 24 h (3 h, Ms_Vec: (1.00±0.14) % & (1.00±0.06) %, Ms_Rv2333c: (1.85±0.29) % & (1.74±0.03) %; t=3.765 & 15.040, P=0.020 & <0.001, respectively; 24 h, Ms_Vec: (1.00±0.14) % & (1.00±0.02) %, Ms_Rv2333c: (1.45±0.15) % & (1.17±0.04) %; t=3.041 & 5.538, P=0.038 & 0.005, respectively). Conclusion: Rv2333c plays an important role in the pathogenesis of MTB. Rv2333c inhibited apoptosis at the early stage of infection, which is helpful for the survival of bacteria in macrophages. In addition, Rv2333c can promote the expression of inflammatory factors at the middle and late stages of infection to induce inflammatory reaction in cells. It can also indirectly participate in transferring of NF-κB to the nucleus and regulating the activity of transcription factors by affecting the ERK1/2 phosphorylation capacity.

Key words: Macrophages, Mycobacterium tuberculosis, Gene expression regulation, Chemokines, Gene, Rv2333c