Kumar Arbind, Singh Sukh Mahendra, Singh Ranvir, Kaur Jagdeep
Department of Biotechnology, Panjab University, Chandigarh 160014, India.
School of Biotechnology, Banaras Hindu University, Varanasi 221005, India.
Int J Med Microbiol. 2017 Feb;307(2):126-138. doi: 10.1016/j.ijmm.2017.01.003. Epub 2017 Jan 22.
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.
结核病是传染病致死的主要原因之一，由结核分枝杆菌引起。结核分枝杆菌的基因组已被测序，近40%的全基因组序列被归类为假设性序列。在结核病数据库中，Rv0774c被注释为膜输出假设蛋白。电子分析表明，Rv0774c是PE-PGRS多基因家族的旁系同源物，其100个氨基酸的N端结构域类似于PE-PGRS蛋白的PE结构域。其C端结构域与PGRS结构域有很大不同，具有特征性的脂肪酶特征序列GXSXG和HG以及预测具有脂解活性的催化残基。因此，分别从结核分枝杆菌中克隆了编码Rv0774c（303个氨基酸）及其N端（1-100个氨基酸）和C端结构域（100-303个氨基酸）的DNA，并在大肠杆菌中进行了过量表达。Rv0774c基因及其C端脂解结构域优先水解短链酯。虽然在N端类似PE的结构域中未观察到酶活性，但已证明它能增强全长Rv0774c的热稳定性。四氢脂抑素抑制酶活性，通过定点诱变证实了预测的催化残基（Ser-185、Asp-255和His-281）。Rv0774c由结核分枝杆菌分泌到培养基中，并在铁限制条件下上调。用重组Rv0774c处理THP-1细胞导致脂多糖诱导的NO产生和诱导型一氧化氮合酶表达下降。经重组Rv0774c处理的THP-1细胞还显示白细胞介素-10和Toll样受体2表达增强。相反，它抑制了脂多糖诱导的白细胞介素-12、趋化因子单核细胞趋化蛋白-1和白细胞介素-8的产生。Rv0774c抑制脂多糖诱导的p38磷酸化。这些观察结果表明，Rv0774c可以调节促炎免疫反应，以支持分枝杆菌在细胞内的存活。
