Genetic Evidence for SecY Translocon-Mediated Import of Two Contact-Dependent Growth Inhibition (CDI) Toxins.
Genetic Evidence for SecY Translocon-Mediated Import of Two Contact-Dependent Growth Inhibition (CDI) Toxins.
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Secy易位介导的两种接触依赖性生长抑制（CDI）毒素的遗传证据。
DOI：
10.1128/mbio.03367-20
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发表时间：
2021-02-02
期刊：
影响因子：
6.4
通讯作者：
Koskiniemi S
中科院分区：
文献类型：
--
作者：
Jones AM;Virtanen P;Hammarlöf D;Allen WJ;Collinson I;Hayes CS;Low DA;Koskiniemi S
许多细菌物种通过CDI系统通过细胞间的直接接触相互作用，这提供了一种将毒素注入抑制细菌生长的机制。在这里，我们发现两种CDI毒素，一种使膜去极化，另一种降解RNA，利用普遍保守的SecY易位机制，用于输出蛋白质进入靶细胞。接触依赖性生长抑制（CDI） CdiA蛋白的c端（CT）毒素结构域以革兰氏阴性菌为靶点，必须突破靶细胞的内外膜才能发挥生长抑制活性。在这里，我们研究了两种CdiA-CT毒素，它们在进入外周质后利用细菌的总蛋白分泌机制。SecY是Sec转位子中普遍保守的通道形成亚基，其跨膜段7的Ser281Phe氨基酸取代降低了肠出血性大肠杆菌EC869的膜去极化孤儿10毒素的细胞毒性。靶细胞表达secYS281F，缺乏PpiD或YfgM（两种SecY辅助因子），完全免受cdi介导的CdiA介导的抑制，无论是CdiA- cto10ec869还是CdiA- ctgn05224，后者是来自产气克雷伯菌GN05224的EndoU RNase CdiA毒素，具有相关的细胞质进入域。竞争共培养过程中，CdiA-CTGN05224在secYS281F靶细胞中RNase活性降低，在secYS281F ΔppiD或secYS281F ΔyfgM靶细胞中不存在RNase活性。重要的是，secY的等位基因特异性突变（secYG313W）使ΔppiD或ΔyfgM靶细胞对CdiA-CTGN05224具有特异性抗性，但对CdiA-CTo10EC869没有特异性抗性，进一步表明secY与CDI毒素之间存在直接相互作用。我们的研究结果为未折叠多肽的主要细胞输出途径和两种CDI毒素的输入途径之间的独特融合提供了遗传证据。
Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secYS281F and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CTo10EC869 or by CdiA-CTGN05224, the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CTGN05224 was reduced in secYS281F target cells and absent in secYS281F ΔppiD or secYS281F ΔyfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY (secYG313W) renders ΔppiD or ΔyfgM target cells specifically resistant to CdiA-CTGN05224 but not to CdiA-CTo10EC869, further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins.