Mycobacterium Tuberculosis Rv3084 Encodes Functional Esterase and Suppresses the Pro-inflammatory Cytokines in vivo

ZHANG Chun-xi, LUO Tao, MA Peng-jiao, WANG Chu-han, SUO Jing, ZHAI Xiao-qian, PENG Xuan, BAO Lang

Abstract

To explore the biological characteristics of the esterase LipR encoded by Mycobacterium tuberculosis (MTB) Rv3084 and its immunomodulatory function in vivo.  Methods  The LipR gene was amplified from MTB H37Rv strain to construct recombinant expression plasmid. After sequencing, the recombinant plasmid was transformed into E. coli for expression and purification of LipR protein. The expressed protein was confirmed with Western blotting assay. The hydrolyzing activity of LipR was detected and the factors affecting LipR enzyme activity were analyzed. Mice were intramuscularly injected with 0.1 mL (containing plasmid DNA 100 μg) recombinant eukaryotic plasmid three times (day 1, 8, and 15); seven days after the last injection, the mice were executed, and the lung and spleen were taken for cytokine detection.  Results  The recombinant expression plasmid was successfully constructed and it was found that LipR protein was mainly expressed in the form of inclusion bodies in E. coli with the relative molecular mass of about 33×103. LipR was demonstrated as an alkaline eurythermic esterase, due to the preference of hydrolyzing short carbon chain esters with optimal hydrolyzing activity on pNP-acetate (pNPA, C2) and the capability in tolerance of high pH and temperature; in the presence of different detergents or metal ions, the activity of LipR hydrolyzing pNP-butyrate (pNPB, C4) was inhibited to some extent. In the mouse model, it was found that LipR could inhibit the secretion of interferon-γ (IFN- γ) and interleukin-2 (IL-2), but to stimulate the secretion of IL-10.  Conclusion  The esterase LipR may be one of the esterases help M. tuberculosis withstand harsh environment inside the host in collaboration, and simultaneously act as an immune modulator to inhibit the secretion of pro-inflammatory cytokines and consequently impact the killing effect of host immune system against M. tuberculosis.

 

Key words: Mycobacterium tuberculosis, Lip family, α/β hydrolase Esterase/lipase, Cytokine

 

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