Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: a non-conventional hemolysin and a ribosomal RNA methyl transferase.
Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: a non-conventional hemolysin and a ribosomal RNA methyl transferase.
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DOI：
10.1186/1471-2091-11-35
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发表时间：
2010-09-20
期刊：
影响因子：
--
通讯作者：
Krishnasastry MV
中科院分区：
文献类型：
--
作者：
Rahman A;Srivastava SS;Sneh A;Ahmed N;Krishnasastry MV
结核分枝杆菌是一种引起结核病的致命杆菌，每年全球有数百万人死于结核病。为了了解结核分枝杆菌在人类中的发病机制，帮助控制结核病，人们正在研究结核分枝杆菌众多基因的功能。在这项研究中，我们报道了结核分枝杆菌tlyA基因产物Rv1694的双重功能，Rv1694是一个268个氨基酸长的碱性蛋白。重组纯化的Rv1694蛋白具有体外溶血活性。结果表明，兔红细胞和人红细胞的溶血具有浓度和时间依赖性。在裂解的红细胞膜上可以看到这种蛋白的多种寡聚体形式(二聚体到七聚体)。与传统的致孔毒素低聚体一样，Rv1694的寡聚体被发现耐热和耐十二烷基硫酸钠，但由于它取消了Rv1694的溶血活性，表明其具有二硫键的作用，因此对β-巯基乙醇等还原剂敏感(S)。通过体外转录和翻译产生的Rv1694也表现出明确的溶血作用，证实了该蛋白的自组装和寡聚特性。对该蛋白的有限蛋白降解表明，氨基末端在溶液中是敏感的，但在有膜的情况下是受保护的。Rv1694的显著特征是通过共聚焦显微镜观察到它存在于大肠杆菌的细胞壁上。其表面表达与表达该蛋白的大肠杆菌的接触性溶血能力相一致。此外，针对该蛋白的免疫血清可抑制接触性溶血。此外，Rv1694蛋白结合在巨噬细胞吞噬体膜上并在其上形成稳定的寡聚体。除了这些特性外，表达Rv1694的大肠杆菌对卷曲霉素敏感，因为它的生长速度明显慢于模拟载体转化的大肠杆菌。表达Rv1694的大肠杆菌S30提取物在卷曲霉素存在下的翻译活性较差，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-腺苷蛋氨酸的甲基化基团也证实了其甲基化活性。Rv1694具有不同寻常的双重活动。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小体等细胞内隔膜有关，而不是与红细胞的细胞裂解有关，这种自组装特性在结核分枝杆菌成功进入巨噬细胞后可能具有潜在的作用。
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.
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