Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis
Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis
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DOI：
10.1073/pnas.1309195110
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发表时间：
2013-12-03
影响因子：
11.1
通讯作者：
Heinzen, Robert A.
中科院分区：
文献类型：
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作者：
Larson, Charles L.;Beare, Paul A.;Heinzen, Robert A.
伯内特立克次体（人类 Q 热的病因）成功定植于巨噬细胞中，需要病原体定向生物发生，产生具有吞噬溶酶体特征的大型、允许生长的寄生液泡 (PV)。 C.burnetii 为 PV 发育而选择的囊泡运输途径尚不明确；然而，据预测，膜募集需要通过细胞器运输/细胞内增殖 (Dot/Icm) 型 4B 分泌系统缺陷将效应蛋白递送至胞质溶胶。在这里，我们描述了网格蛋白介导的囊泡运输在 PV 产生中的参与，以及伯内特衣原体 4B 型分泌系统底物柯克斯体液泡蛋白 A (CvpA) 对该途径的参与。 CvpA 包含多个基于二亮氨酸 [DERQ]XXXL[LI] 和酪氨酸 (YXX Phi) 的内吞分选基序，如网格蛋白接头蛋白 (AP) 复合物 AP1、AP2 和 AP3 识别的基序。 C.burnetii Delta cvpA 突变体在复制和 PV 发育中表现出显着缺陷，证实了 CvpA 在感染中的重要性。异位表达的 mCherry-CvpA 定位于 Rab11 和转铁蛋白受体阳性的中心体周围回收内体的管状和囊泡结构域，并且 CvpA 膜相互作用因内吞分选基序的突变而丢失。与内吞循环系统的 CvpA 参与一致，异位表达减少了转铁蛋白的摄取。在 Pull-down 测定中，含有 CvpA 分选基序和全长 CvpA 的肽与 AP2 亚基和网格蛋白重链相互作用。此外，通过 siRNA 处理去除 AP2 或网格蛋白可显着抑制伯氏梭菌的复制。因此，我们的结果揭示了网格蛋白包被的囊泡运输在伯内特衣原体感染中的重要性，并确定了 CvpA 在破坏这些运输机制中的作用。
Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXX Phi)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii Delta cvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms.