National Centre for Cell Science, Ganeshkhind Road, Pune - 411007, Maharashtra, India.
BMC Biochem. 2010 Sep 20;11:35. doi: 10.1186/1471-2091-11-35.
Mycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein.
The recombinant purified Rv1694 protein was found to exhibit hemolytic activity in vitro. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers) of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like β-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s). The Rv1694 generated de novo by in vitro transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of E. coli as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of E. coli expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, E. coli expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth was significantly slower than mock vector transformed E. coli. The S30 extract of E. coli expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [3H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity.
The Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by Mycobacterium tuberculosis.
结核分枝杆菌是一种烈性杆菌，可引起结核病，这种疾病每年在全球导致数百万人死亡。为了了解其在人类中的发病机制，并帮助控制结核病，人们正在对结核分枝杆菌的许多基因的功能进行特征描述。在这项研究中，我们报告了结核分枝杆菌 tlyA 基因产物的双重功能，该基因产物被注释为 Rv1694，是一种 268 个氨基酸长的碱性蛋白。
发现重组纯化的 Rv1694 蛋白在体外具有溶血活性。它表现出对兔和人红细胞的浓度和时间依赖性溶血。在裂解的红细胞膜上可见这种蛋白的多种寡聚形式（二聚体到七聚体）。与传统的、众所周知的、形成孔的毒素的寡聚体一样，Rv1694 的寡聚体对热和 SDS 具有抗性，但对还原剂如β-巯基乙醇敏感，因为它消除了 Rv1694 的溶血活性，表明二硫键的作用。通过体外转录和翻译生成的 Rv1694 也表现出明确的溶血作用，证实了该蛋白的自我组装和寡聚化特性。有限的蛋白水解消化表明，这种蛋白的氨基末端在溶液中是易受攻击的，但在膜存在时受到保护。Rv1694 的一个显著特征是它在大肠杆菌的细胞壁上存在，这一点通过共聚焦显微镜观察到。表面表达与表达该蛋白的大肠杆菌的接触依赖性溶血能力一致。此外，针对该蛋白的免疫血清抑制了接触依赖性溶血。此外，Rv1694 蛋白与巨噬细胞吞噬体膜结合并形成稳定的寡聚体。除了所有这些特性之外，还发现表达 Rv1694 的大肠杆菌对抗生素卷曲霉素敏感，因为其生长速度明显慢于模拟载体转化的大肠杆菌。在卷曲霉素存在的情况下，表达 Rv1694 的 S30 提取物在翻译活性方面较差，进一步证实了其甲基化活性。最后，在分离的核糖体中掺入 [3H]-S-腺苷甲硫氨酸也证实了其甲基化活性。
Rv1694 具有一种不寻常的双重活性。它似乎包含两种不同的功能，如溶血活性和核糖体 RNA 甲基化活性。这种溶血活性可能与吞噬体等细胞内隔室有关，而不是红细胞的细胞溶解，其自我组装特性可能在结核分枝杆菌成功进入巨噬细胞后具有潜在作用。
