Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.
Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.
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DOI：
10.1371/journal.ppat.1004295
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发表时间：
2014-07
期刊：
影响因子：
6.7
通讯作者：
Bradley KA
中科院分区：
文献类型：
--
作者：
Eshraghi A;Dixon SD;Tamilselvam B;Kim EJ;Gargi A;Kulik JC;Damoiseaux R;Blanke SR;Bradley KA
由细菌和植物产生的细胞内作用蛋白外毒素是驱动许多人类疾病的重要分子决定因素。这些毒素的一个子集，细胞致死性膨胀毒素（CDTs），由几种革兰氏阴性病原体编码，并已提出通过允许逃避免疫系统来增强毒力。CDT以逆行方式从细胞表面通过高尔基体并进入内质网（ER），然后最终到达宿主细胞核。然而，CDT退出ER的机制尚不清楚。在这里，我们表明，主机ER相关的降解（ERAD）机制，Derlin-2（Derl 2），E3泛素蛋白连接酶Hrd 1，和AAA ATP酶p97的三个核心组件，所需的一些CDTs中毒。Derl 2缺陷细胞与Derl 2：Derl 1嵌合体的互补鉴定了Derl 2中的两个先前未表征的功能结构域，N-末端88个氨基酸和第二个ER-管腔环，这是杜克雷嗜血杆菌编码的CDT（Hd-CDT）中毒所需的。与此相反，两个图案所需的ERAD底物，一个保守的WR基序和SHP盒介导的AAA ATP酶p97相互作用的Derlin依赖性的逆转录，被发现是Hd-CDT中毒。有趣的是，这种以前未描述的机制与植物毒素蓖麻毒素相同。这些数据揭示了CDT中毒需要ERAD途径的多个组分，并提供了对逆向运输毒素所利用的Der 12依赖性途径的深入了解。细胞致死性膨胀毒素（CDTs）是由几种细菌病原体产生的，并增加这些细菌引起疾病的能力。在被宿主细胞吸收后，CDTs被运输到内质网（ER），在那里它们必须易位穿过ER膜以接近其细胞内靶标;然而，对于CDTs的这种易位过程知之甚少。在这里，我们提供的证据表明，CDTs需要的ER相关降解（ERAD）途径的组件，一个正常的细胞过程，用于易位终端错误折叠的ER内腔和膜蛋白跨ER膜在胞质溶胶中的降解。该途径的关键成员Derl 2的缺失使细胞对多种CDT具有抗性。有趣的是，错误折叠蛋白质的ERAD所需的Der 12内的两个结构域被CDT毒害。此外，我们报告了两个先前未表征的域Derl 2内，每个都需要中毒。与Der 12的作用一致，ERAD途径的另外两个成员Hrd 1和p97的废除导致CDT在ER中的保留和对中毒的抵抗。总之，这些数据提供了新的见解CDTs如何退出ER，从而获得他们的细胞目标。
Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins. Cytolethal distending toxins (CDTs) are produced by several bacterial pathogens and increase the ability of these bacteria to cause disease. After being taken up by host cells, CDTs are trafficked to the endoplasmic reticulum (ER) where they must translocate across the ER membrane to gain access to their intracellular target; however, this translocation process is poorly understood for CDTs. Here we provide evidence that CDTs require components of the ER-associated degradation (ERAD) pathway, a normal cellular process utilized to translocate terminally misfolded ER lumenal and membrane proteins across the ER membrane for degradation in the cytosol. Deletion of a key member of this pathway, Derl2, makes cells resistant to multiple CDTs. Interestingly, two domains within Derl2 which are required for ERAD of misfolded proteins are dispensable for intoxication by CDT. Further, we report two previously uncharacterized domains within Derl2 that are each required for intoxication. Consistent with a role of Derl2, abrogation of two other members of the ERAD pathway, Hrd1 and p97, results in retention of CDT in the ER and resistance to intoxication. Taken together, these data provide novel insight into how CDTs exit the ER and therefore gain access to their cellular targets.
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