Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.
Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.
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DOI：
10.1371/journal.ppat.1007005
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发表时间：
2018-04
期刊：
影响因子：
6.7
通讯作者：
Heinzen RA
中科院分区：
文献类型：
--
作者：
Miller HE;Larson CL;Heinzen RA
伯内特柯克斯体是一种细胞内细菌，在广阔的吞噬溶酶体样液泡内复制。含柯克斯体的液泡 (CCV) 和晚期内涵体/多泡体之间的融合需要 Rab7、HOPS 束缚复合物和 SNARE 蛋白，据推测肌动蛋白也发挥了作用。在这里，我们研究了肌动蛋白在 CCV 融合中的重要性。 CCV 膜周围形成丝状肌动蛋白斑块，这是囊泡融合的首选位点。因此，内溶酶体融合的介质 Rab7、VAMP7 和突触蛋白 8 集中在 CCV 肌动蛋白斑块中。肌动蛋白斑块的生成需要伯内特梭菌 4B 型分泌和宿主逆转录酶功能。贴片装饰有 VPS29 和 VPS35（逆转录酶的成分）、FAM21 和 WASH（参与逆转录酶的 WASH 复合物的成员）以及 Arp3（Arp2/3 复合物的成分，可产生分支肌动蛋白丝）。通过 siRNA 去除 VPS35 或 VPS29 会减少 CCV 肌动蛋白斑块，并导致 Rab7 均匀分布在 CCV 膜中。 C.burnetii 在 VPS35 或 VPS29 耗尽的细胞以及 WASH 敲除的小鼠胚胎成纤维细胞中正常生长，其中 CCV 缺乏肌动蛋白斑块。在感染细胞中，葡萄糖转运蛋白 1 (GLUT1) 和阳离子独立的甘露糖 6-磷酸受体 (CI-M6PR) 分别循环到质膜和跨高尔基体的内体循环是正常的。然而，retromer 的 siRNA 敲低导致 GLUT1 的异常运输，但不会导致 CI-M6PR，这表明典型的逆行运输不受 retromer 破坏的影响。使用特异性 Arp2/3 抑制剂 CK-666 进行治疗可强烈抑制 CCV 形成，这种作用与转铁蛋白受体的内体运输改变有关。总的来说，我们的结果表明，retromer、WASH 和 Arp2/3 生成的 CCV 肌动蛋白斑块对于 CCV 生物发生和稳定性来说是可有可无的。然而，CCV 的产生需要 Arp2/3 介导的内体系统内货物运输所需的肌动蛋白丝的产生。这些发现描绘了塑造内体区室的许多肌动蛋白相关事件中的哪些对于 CCV 形成很重要。伯内特柯克斯体是人类 Q 热细菌，在一个严酷的溶酶体样区室（称为含柯克斯体液泡 (CCV)）中复制。在此，病原体通过分泌的 4B 型 (T4B) 效应蛋白的活性来指导 CCV 的形成。关于宿主细胞骨架和受体循环途径如何促进 CCV 的产生和功能的问题仍然存在。我们发现丝状肌动蛋白（F-肌动蛋白）斑块以 T4B 依赖性方式在 CCV 周围形成。斑块是内体-CCV 融合的首选位点，这种行为与斑块富集内体融合蛋白（例如 VAMP7 和 Rab7）相关。斑块的形成需要共定位的逆转录酶-WASH-Arp2/3 回收复合物。在耗尽逆转录体或 WASH 的细胞中，CCV 缺乏肌动蛋白斑块，并显示出 Rab7（也参与逆转录体募集），均匀地重新分布在 CCV 膜周围。 C. burnetii 在这些细胞中正常生长，表明逆转录酶介导的蛋白质回收和 CCV 肌动蛋白斑块对于生产性感染来说是可有可无的。相比之下，Arp2/3 生成的 F-肌动蛋白的整体破坏通过与缺陷内体运输相关的过程严重限制了 CCV 的形成。我们认为逆转录酶分选和 CCV 肌动蛋白斑块的形成对病原体生长无关紧要。这些研究加深了我们对 CCV 生物发生所需的囊泡运输途径的理解。
Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation. Coxiella burnetii, the human Q fever bacterium, replicates in a harsh, lysosome-like compartment termed the Coxiella-containing vacuole (CCV). Here, the pathogen directs formation of the CCV through the activities of secreted type 4B (T4B) effector proteins. Questions remain concerning how the host cytoskeleton and receptor recycling pathways contribute to creation and function of the CCV. We found that filamentous actin (F-actin) patches formed around the CCV in a T4B-dependent manner. Patches were preferred sites of endosome-CCV fusion, a behavior that correlated with patch enrichment of endosome fusion proteins, such as VAMP7 and Rab7. Patch formation required colocalized retromer-WASH-Arp2/3 recycling complexes. In cells depleted of retromer or WASH, CCV lacked actin patches and displayed Rab7, also involved in retromer recruitment, uniformly redistributed around the CCV membrane. C. burnetii grew normally in these cells, indicating retromer-mediated protein recycling and CCV actin patches are dispensable for productive infection. In contrast, global disruption of Arp2/3-generated F-actin severely restricted CCV formation through a process associated with defective endosome trafficking. We propose that retromer sorting and formation of CCV actin patches are inconsequential to pathogen growth. These studies refine our understanding of vesicular trafficking pathways required for CCV biogenesis.
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影响因子：
6.7
作者：
Carey KL;Newton HJ;Lührmann A;Roy CR
通讯作者：
Roy CR
影响因子：
6.4
作者：
Beare PA;Gilk SD;Larson CL;Hill J;Stead CM;Omsland A;Cockrell DC;Howe D;Voth DE;Heinzen RA
通讯作者：
Heinzen RA
影响因子：
6.7
作者：
Colonne PM;Winchell CG;Graham JG;Onyilagha FI;MacDonald LJ;Doeppler HR;Storz P;Kurten RC;Beare PA;Heinzen RA;Voth DE
通讯作者：
Voth DE
影响因子：
3.7
作者：
Derivery, Emmanuel;Helfer, Emmanuele;Gautreau, Alexis
通讯作者：
Gautreau, Alexis
DOI：
10.1073/pnas.1410552111
发表时间：
2014-09-02
影响因子：
11.1
作者：
Gallon, Matthew;Clairfeuille, Thomas;Cullen, Peter J.
通讯作者：
Cullen, Peter J.