Ahmad S, El-Shazly S, Mustafa A S, Al-Attiyah R
Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait.
Scand J Immunol. 2005 Jul;62(1):16-24. doi: 10.1111/j.1365-3083.2005.01639.x.
The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. The mammalian cell entry (mce)3 operon is one of four homologous mce operons that encodes six putative invasin-like exported proteins (Mce3A-F), possibly involved in entry and survival of M. tuberculosis inside macrophages. We have recently shown that Mce3A, Mce3D and Mce3E are expressed and elicit antibody responses in a majority of human subjects during natural infection with M. tuberculosis. In this study, we demonstrate the expression of Mce3A-F proteins and their mRNA during in vitro growth of M. tuberculosis. To demonstrate the expression of mce3A-F proteins, the antibodies were raised in rabbits against three pure proteins (Mce3A, Mce3D and Mce3E), and their specificity was checked by immunoblotting with recombinant Mce1A-F proteins encoded by mce1 operon. The antibodies were also generated against all the six Mce3 proteins, which were expressed and purified as fusion proteins with glutathione S-transferase (GST) as the fusion partner (GST-Mce3A-F). The antibodies reacted, in each case, with a protein of expected molecular mass (Mr) for the corresponding Mce3 protein in the cell wall fraction but not in the soluble fraction of in vitro-grown M. tuberculosis cells. The presence of mRNA for mce3A-F genes was also shown by using mce3A-F gene-specific primers, and total RNA isolated from in vitro-grown M. tuberculosis cells by reverse transcription-polymerase chain reaction (RT-PCR). Pretreatment of the RNA preparation with RNase A abolished amplification in RT-PCR confirming that mce3A-F mRNA rather than genomic DNA was being amplified. The data show that Mce3A-F encoded by the mce3 operon are expressed during in vitro growth of M. tuberculosis.
结核分枝杆菌的发病机制很大程度上归因于其进入人体巨噬细胞并在其中存活的能力。哺乳动物细胞进入（mce）3操纵子是四个同源mce操纵子之一，它编码六种假定的侵袭素样输出蛋白（Mce3A - F），可能参与结核分枝杆菌在巨噬细胞内的进入和存活。我们最近发现，在结核分枝杆菌自然感染期间，大多数人类受试者体内会表达Mce3A、Mce3D和Mce3E并引发抗体反应。在本研究中，我们证明了结核分枝杆菌体外生长期间Mce3A - F蛋白及其mRNA的表达。为了证明mce3A - F蛋白的表达，用针对三种纯蛋白（Mce3A、Mce3D和Mce3E）的兔抗体制备抗体，并通过用mce1操纵子编码的重组Mce1A - F蛋白进行免疫印迹来检查其特异性。还针对所有六种Mce3蛋白产生了抗体，这些蛋白作为与谷胱甘肽S - 转移酶（GST）作为融合伴侣的融合蛋白（GST - Mce3A - F）进行表达和纯化。在每种情况下，这些抗体都与体外培养的结核分枝杆菌细胞壁组分中相应Mce3蛋白预期分子量（Mr）的蛋白发生反应，但不与可溶性组分中的蛋白反应。通过使用mce3A - F基因特异性引物以及通过逆转录 - 聚合酶链反应（RT - PCR）从体外培养的结核分枝杆菌细胞中分离的总RNA，也证明了mce3A - F基因mRNA的存在。用核糖核酸酶A预处理RNA制剂可消除RT - PCR中的扩增，证实扩增的是mce3A - F mRNA而非基因组DNA。数据表明，mce3操纵子编码的Mce3A - F在结核分枝杆菌体外生长期间表达。
