Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.
Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.
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DOI：
10.1371/journal.ppat.1006603
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发表时间：
2017-09
期刊：
影响因子：
6.7
通讯作者：
Bier E
中科院分区：
文献类型：
--
作者：
Guichard A;Jain P;Moayeri M;Schwartz R;Chin S;Zhu L;Cruz-Moreno B;Liu JZ;Aguilar B;Hollands A;Leppla SH;Nizet V;Bier E
各种细菌毒素通过过量生产cAMP来绕过宿主的防御。在之前的研究中，我们发现炭疽芽孢杆菌的腺苷环化酶--水肿因子(EF)可以破坏由小的GTP酶Rab11介导的内吞循环。因此，钙粘附素等货运蛋白无法到达细胞间连接。在本研究中，我们使用果蝇和哺乳动物系统对EF抑制Rab11的进一步机制进行了剖析。EF在GTP加载步骤后阻止Rab11的运输，阻止组成活性形式的Rab11将货物囊泡运送到质膜。两个主要的cAMP效应通路-PKA和EPAC/Rap1-都有助于抑制Rab11介导的贩运，但在传递过程的不同步骤发挥作用。PKA作用较早，阻止Rab11与其效应器Rip11和Sec15结合。相反，EPAC随后通过小GTPase Rap1发挥作用，阻止循环内小体与质膜的融合，并似乎是EF毒性的主要影响因素。同样，在哺乳动物系统中进行的实验表明，EPAC，而不是PKA，在细胞培养和体内都介导EF的活性。小的GTPase Arf6启动细胞黏附成分的内吞恢复，也通过抵消细胞-细胞接触部位Rab11依赖的货物蛋白的递送而促进连接稳态。这些研究具有潜在的重要实际意义，因为化学抑制Arf6或EPAC可以阻断EF在细胞培养和体内的作用，为治疗cAMP诱导毒素或相关屏障破坏病理引起的症状开辟了新的潜在治疗途径。最近在赞比亚和俄罗斯北部爆发的炭疽病疫情和生物防御准备突出表明，需要新的疗法来对抗感染炭疽杆菌的患者的致命晚期病理。事实上，这种病原体分泌的两种毒素--水肿毒素(ET)和致死毒素(LT)--在有效的抗生素治疗下可以导致死亡。ET是一种高效的腺苷酸环化酶，通过过量生产普遍存在的第二信使cAMP而严重影响宿主细胞和组织。此前，我们发现Rab11是一个被ET抑制的关键宿主因子。阻断依赖Rab11的内吞循环导致细胞间连接中断，可能是炭疽病人观察到威胁生命的血管渗出的原因之一。在这里，我们提出了一个多系统的分析机制，通过EF抑制Rab11和外囊依赖的运输。在果蝇的上位性实验表明，cAMP效应器PKA和EPAC/Rap1的过度激活在两个不同的步骤上干扰了Rab11介导的转运。我们进一步描述了EPAC和小的GTP酶Arf6在ET介导的囊泡运输中断中的保守作用，并说明了化学抑制这两条途径如何极大地减轻了ET诱导的水肿。因此，我们的研究将EPAC和Arf6定义为治疗感染性疾病和其他涉及cAMP超载或相关屏障破坏的病理的有希望的药物靶点。
Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies. Recent anthrax outbreaks in Zambia and northern Russia and biodefense preparedness highlight the need for new therapies to counteract fatal late-stage pathologies in patients infected with Bacillus anthracis. Indeed, two toxins secreted by this pathogen—edema toxin (ET) and lethal toxin (LT)—can cause death in face of effective antibiotic treatment. ET, a potent adenylate cyclase, severely impacts host cells and tissues through an overproduction of the ubiquitous second messenger cAMP. Previously, we identified Rab11 as a key host factor inhibited by ET. Blockade of Rab11-dependent endocytic recycling resulted in the disruption of intercellular junctions, likely contributing to life threatening vascular effusion observed in anthrax patients. Here we present a multi-system analysis of the mechanism by which EF inhibits Rab11 and exocyst-dependent trafficking. Epistasis experiments in Drosophila reveal that over-activation of the cAMP effectors PKA and Epac/Rap1 interferes with Rab11-mediated trafficking at two distinct steps. We further describe conserved roles of Epac and the small GTPase Arf6 in ET-mediated disruption of vesicular trafficking and show how chemical inhibition of either pathway greatly alleviates ET-induced edema. Thus, our study defines Epac and Arf6 as promising drug targets for the treatment of infectious diseases and other pathologies involving cAMP overload or related barrier disruption.
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影响因子：
21.3
作者：
Jones CA;Nishiya N;London NR;Zhu W;Sorensen LK;Chan AC;Lim CJ;Chen H;Zhang Q;Schultz PG;Hayallah AM;Thomas KR;Famulok M;Zhang K;Ginsberg MH;Li DY
通讯作者：
Li DY
影响因子：
11.4
作者：
Fukai, S;Matern, HT;Brunger, AT
通讯作者：
Brunger, AT
影响因子：
2.5
作者：
Farkas, Robert;Benova-Liszekova, Denisa;Mechler, Bernard M.
通讯作者：
Mechler, Bernard M.
影响因子：
5.8
作者：
Guichard A;Nizet V;Bier E
通讯作者：
Bier E
影响因子：
3.4
作者：
Costes, SV;Daelemans, D;Lockett, S
通讯作者：
Lockett, S