Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter.
Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter.
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作者：
Binet R;Fernandez RE;Fisher DJ;Maurelli AT
甲基化对于所有细胞的生理学至关重要，包括专性细胞内细菌衣原体。然而，衣原体的甲基化循环面临着强大的还原进化压力。仅棘衣原体和 Waddlia chondrophila 基因组序列与 metK 和 sahH 具有同源性，metK 编码合成 SAM 所需的 S-腺苷甲硫氨酸 (SAM) 合成酶，而 sahH 编码 S-腺苷高半胱氨酸 (SAH) 水解酶，用于解毒从 SAM 转移甲基后形成的 SAH 到甲基化底物。用沙眼衣原体 L2 基因组文库转化大肠杆菌的条件致死 ΔmetK 突变体，将 CTL843 鉴定为假定的 SAM 转运蛋白，因为它能够让突变体仅在细胞外 SAM 存在的情况下存活于 metK 缺陷。 CTL843 属于药物/代谢物转运蛋白超家族，允许大肠杆菌转运 S-腺苷-1-[甲基-14C]甲硫氨酸，表观 Km 为 5.9 µM，Vmax 为 32 pmol min−1 mg−1。此外，CTL843 赋予了失去解毒 SAH 能力的 Δpfs 大肠杆菌突变体的生长优势，而竞争和反向运输实验进一步表明 SAH 是 CTL843 的额外底物。我们提出 CTL843 作为 SAM/SAH 转运蛋白 (SAMHT)，通过允许衣原体从宿主细胞获取 SAM 并排出有毒副产物 SAH，发挥双重功能。功能性 SAMHT 的演示进一步深入了解与衣原体专性细胞内生活方式相关的还原进化，并确定了一个极好的化疗靶点。像衣原体这样的专性细胞内寄生虫遵循的是还原进化路径，这使得它们几乎完全依赖宿主细胞获取营养。在这项工作中，我们确定了所有甲基化反应所必需的代谢物的独特转运蛋白，它可能绕过衣原体中两种酶促反应的需要。转运蛋白 CTL843 允许沙眼衣原体 L2 从真核宿主细胞质中窃取 S-腺苷甲硫氨酸 (SAM)，并可能去除 SAM 失去甲基时形成的有毒 S-腺苷高半胱氨酸 (SAH)，充当 SAM/SAH 转运蛋白 (SAMHT)。除了反映衣原体对专性细胞内生活方式的适应之外，SAMHT 在衣原体代谢中的特定和核心作用为开发用于治疗衣原体感染的治疗剂提供了目标。
Methylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-l-[methyl-14C]methionine with an apparent Km of 5.9 µM and a Vmax of 32 pmol min−1 mg−1. Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target. Obligate intracellular parasites like Chlamydia have followed a reductive evolutionary path that has made them almost totally dependent on their host cell for nutrients. In this work, we identify a unique transporter of a metabolite essential for all methylation reactions that potentially bypasses the need for two enzymatic reactions in Chlamydia. The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT). In addition to reflecting the adaptation of Chlamydia to an obligate intracellular lifestyle, the specific and central roles of SAMHT in Chlamydia metabolism provide a target for the development of therapeutic agents for the treatment of chlamydial infections.