Rv0774c, an iron stress inducible, extracellular esterase is involved in immune-suppression associated with altered cytokine and TLR2 expression

- Mycobacterium tuberculosis Rv0774c protein contains two domains with N-terminal PE like domain and C-terminal esterase domain.
- Both N and C-terminal domains work independently. N-terminal imparts thermostability to the full length protein, while C-terminal domain demonstrated esterase activity.
- Rv0774c was identified in culture filtrate in in vitro grown M. tuberculosis H37Ra culture. The protein was expressed in iron limited conditions.
- Recombinant Rv0774c down regulated the NO production through down regulating the iNOS expression.
- Rv0774c involved in immuno-suppression via modulation of TLR2 and cytokine expression and inhibited the LPS induced phosphorylation of p38.

Tuberculosis, one of the leading cause of death from infectious diseases, is caused by Mycobacterium tuberculosis. The genome of M. tuberculosis has been sequenced and nearly 40% of the whole genome sequence was categorized as hypothetical. Rv0774c was annotated as membrane exported hypothetical protein in TB database. In silico analysis revealed that Rv0774c is a paralog of PE-PGRS multi gene family with 100 aa N-terminal domain similar to PE domain of PE-PGRS proteins. Its C-terminal domain is quite different from PGRS domain, having characteristic lipase signature GXSXG & HG and catalytic residues predicted for lipolytic activity. Therefore, DNA coding for Rv0774c (303 aa), its N-terminal (1-100 aa) and C- terminal domain (100-303 aa) were separately cloned from M. tuberculosis and were over expressed in E. coli. Rv0774c gene and its C-terminal lipolytic domain preferably hydrolyzed short chain esters. Though no enzyme activity was observed in N-terminus PE like domain, it was demonstrated to enhance the thermostability of full length Rv0774c. Tetrahydrolipstatin inhibited the enzyme activity and predicted catalytic residues (Ser-185, Asp-255 and His-281) were confirmed by site directed mutagenesis. Rv0774c was secreted out in culture media by M. tuberculosis and was up-regulated in iron limiting conditions. Treatment of THP-1 cells with rRv0774c resulted in a decline in the LPS induced production of NO and expression of iNOS. rRv0774c treated THP-1 cells also showed an enhanced expression of IL-10 and TLR2. On contrary, it suppressed the LPS induced production of IL-12, chemokines MCP-1 and IL-8. Rv0774c inhibited the LPS induced phosphorylation of p38. These observations suggested that Rv0774c could modulate the pro-inflammatory immune response to support intracellular survival of the mycobacterium.