Thiamin transport in Helicobacter pylori lacking the de novo synthesis of thiamin
Thiamin transport in Helicobacter pylori lacking the de novo synthesis of thiamin
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DOI：
10.1099/mic.0.000765
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发表时间：
2019-02-01
期刊：
影响因子：
2.8
通讯作者：
Mimuro, Hitomi
中科院分区：
文献类型：
--
作者：
Nosaka, Kazuto;Uchiyama, Ryosuke;Mimuro, Hitomi
幽门螺杆菌缺乏参与硫胺素从头合成的基因，因此是一种硫胺素缺乏症。PnuT转运蛋白是Pnu转运蛋白家族的一员，可介导硫胺跨膜的摄取。在幽门螺杆菌基因组中，pnuT基因与硫胺焦磷酸激酶基因thi80聚集在一起。在本研究中，我们发现[H-3]硫胺素通过促进扩散进入H. pylori SS1菌株，其K-m值为28 μ m。放射性硫胺素的掺入在一定程度上受到2-甲基-4-氨基-5-羟甲基嘧啶或硫胺素的抑制，但在很大程度上不受磷酸硫胺或焦磷酸硫胺的影响。RT-PCR分析表明，pnuT和thi80基因共转录为一个转录物。重组Thi80蛋白对硫胺素焦磷酸激酶活性的Km值估计为0.40 μ M，远低于H. pylori细胞中硫胺素运输的K-m值。这些发现表明，从环境中加入的硫胺素通过焦磷酸化有效地捕获，使运输定向。此外，当胞外硫胺素浓度为1 μ M时，缺乏pnut的幽门螺杆菌菌株的硫胺素运输活性不到野生型菌株的20%，而添加100 nM [H-3]硫胺素的pnut -缺陷菌株的掺入闪烁信号接近背景水平。我们还发现，缺乏pnut的菌株需要比野生型多100倍的硫胺素才能达到与野生型相同的生长。这些发现反映了硫胺素进入幽门螺杆菌的多种途径，PnuT可能负责高纯度的硫胺素运输，并作为抗幽门螺杆菌抗菌药物的靶点。
Helicobacter pylori lacks the genes involved in the de novo synthesis of thiamin, and is therefore a thiamin auxotroph. The PnuT transporter, a member of the Pnu transporter family, mediates the uptake of thiamin across the membrane. In the genome of H. pylori, the pnuT gene is clustered with the thiamin pyrophosphokinase gene thi80. In this study, we found that [H-3]thiamin is incorporated into the H. pylori SS1 strain via facilitated diffusion with a K-m value of 28 mu M. The incorporation of radioactive thiamin was inhibited to some extent by 2-methyl-4-amino-5-hydroxymethylpyrimidine or pyrithiamine, but was largely unaffected by thiamin phosphate or thiamin pyrophosphate. RT-PCR analysis demonstrated that the pnuT and thi80 genes are cotranscribed as a single transcript. The estimated Km value for thiamin in the thiamin pyrophosphokinase activity exerted by the recombinant Thi80 protein was 0.40 mu M, which is much lower than the K-m value of thiamin transport in H. pylori cells. These findings suggested that the incorporated thiamin from the environment is efficiently trapped by pyrophosphorylation to make the transport directional. In addition, the thiamin transport activity in the pnuT-deficient H. pylori strain was less than 20% of that in the wild-type strain at extracellular thiamin concentration of 1 mu M, but the incorporated scintillation signals of the pnuT-deficient strain with 100 nM [H-3] thiamin were nearly at the background level. We also found that the pnuT-deficient strain required 100-times more thiamin to achieve growth equal to that of the wild-type. These findings reflect the presence of multiple routes for entry of thiamin into H. pylori, and PnuT is likely responsible for the highaffinity thiamin transport and serves as a target for antimicrobial agents against H. pylori.