Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation.
Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation.
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DOI：
10.1073/pnas.1806904115
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发表时间：
2018-09-18
影响因子：
11.1
通讯作者：
Rikihisa Y
中科院分区：
文献类型：
--
作者：
Yan Q;Lin M;Huang W;Teymournejad O;Johnson JM;Hays FA;Liang Z;Li G;Rikihisa Y
当病原体所在的吞噬体与溶酶体融合时，吞噬作用和随后的病原体破坏是真核天然免疫防御的支柱。因此，逃避运输到吞噬溶酶体是大多数在真核宿主细胞内复制的细胞内病原体的基本生存策略。专性细胞内细菌查菲埃里希体也避免路由到宿主细胞吞噬溶酶体，但以一种独特的方式：埃里希体分泌一种蛋白质，埃里希体易位因子-2（Etf-2），具有缺乏RAB-GTf 3激活蛋白（GAP）活性的Tre 2-Bub 2-Cdc 16（TBC）样基序。Etf-2结合埃里希体包涵体上的RAB 5，并干扰RAB 5特异性GAP与RAB 5的接合，从而使RAB 5在细菌包涵体上保持GTP结合的活性形式。Etf-2是具有缺乏RABGAP活性的TBC样基序的RAB相关调节蛋白的独特实例。查菲埃里希体是一种专性细胞内细菌，通过在其膜结合的内含物上隔离内体运输的调节剂小GT3 RAB 5来感染单核细胞/巨噬细胞，以避免路由到宿主细胞吞噬溶酶体。然而，RAB 5如何被隔离在埃里希体包涵体上还知之甚少。我们发现，天然埃里希体易位因子-2（Etf-2），以前预测的效应埃里希体IV型分泌系统，和重组Etf-2（克隆到埃里希体基因组）分泌到宿主细胞的细胞质和本地化埃里希体包涵体。异位表达的Etf-2-GFP也定位于标记有RAB 5的早期内体的内含物和膜，并与GTP结合的RAB 5相互作用，但不与GDP结合的RAB 5相互作用。Etf-2虽然缺乏RAB GTP酶激活蛋白（GAP）Tre 2-Bub 2-Cdc 16（TBC）结构域，但含有两个保守的TBC结构域基序，即Arg指和Gln指，并且定点诱变揭示Arg 188和Gln 245都是Etf-2定位于早期内体所必需的。酵母双杂交试验和微量热电泳显示，Etf-2紧密结合GTP结合的RAB 5，但不GDP结合的RAB 5。然而，Etf-2缺乏RAB 5特异性GAP活性。Etf-2定位于含有珠粒的吞噬体以及含有包被有EtpE（埃里希体的进入触发蛋白）的C-末端片段的珠粒的内体，所述EtpE是埃里希体外膜侵袭素，并且显著延迟RAB 5从吞噬体/内体的解离和RAB 7定位于吞噬体/内体以及RABGAP 5定位于内体。因此，Etf-2与RAB 5-GTP的结合似乎通过阻碍RABGAP 5定位于内体来延迟RAB 5失活。这表明RAB 5被隔离在埃里希体包涵体上以有利于细菌存活和复制的独特机制。
Phagocytosis and subsequent destruction of pathogens when the phagosomes in which they reside are fused with lysosomes are pillars of the eukaryotic innate immune defense. Consequently, evading trafficking to phagolysosomes is a fundamental survival strategy of most intracellular pathogens that replicate inside eukaryotic host cells. The obligatory intracellular bacterium Ehrlichia chaffeensis also avoids routing to host-cell phagolysosomes, but in a unique way: Ehrlichia secretes a protein, Ehrlichia translocated factor-2 (Etf-2), that has a Tre2-Bub2-Cdc16 (TBC)-like motif lacking RAB-GTPase–activating protein (GAP) activity. Etf-2 binds RAB5 on Ehrlichia inclusions and interferes with the engagement of RAB5-specific GAP with RAB5, thereby maintaining RAB5 in a GTP-bound active form on bacterial inclusions. Etf-2 is a unique example of a RAB-associated regulatory protein with a TBC-like motif lacking RABGAP activity. Ehrlichia chaffeensis, an obligatory intracellular bacterium, infects monocytes/macrophages by sequestering a regulator of endosomal traffic, the small GTPase RAB5, on its membrane-bound inclusions to avoid routing to host-cell phagolysosomes. How RAB5 is sequestered on ehrlichial inclusions is poorly understood, however. We found that native Ehrlichia translocated factor-2 (Etf-2), a previously predicted effector of the Ehrlichia type IV secretion system, and recombinant Etf-2 (cloned into the Ehrlichia genome) are secreted into the host-cell cytoplasm and localize to ehrlichial inclusions. Ectopically expressed Etf-2–GFP also localized to inclusions and membranes of early endosomes marked with RAB5 and interacted with GTP-bound RAB5 but not with a GDP-bound RAB5. Etf-2, although lacking a RAB GTPase-activating protein (GAP) Tre2-Bub2-Cdc16 (TBC) domain, contains two conserved TBC domain motifs, namely an Arg finger and a Gln finger, and site-directed mutagenesis revealed that both Arg188 and Gln245 are required for Etf-2 localization to early endosomes. The yeast two-hybrid assay and microscale thermophoresis revealed that Etf-2 binds tightly to GTP-bound RAB5 but not to GDP-bound RAB5. However, Etf-2 lacks RAB5-specific GAP activity. Etf-2 localized to bead-containing phagosomes as well as endosomes containing beads coated with the C-terminal fragment of EtpE (entry-triggering protein of Ehrlichia), an Ehrlichia outer-membrane invasin, and significantly delayed RAB5 dissociation from and RAB7 localization to phagosomes/endosomes and RABGAP5 localization to endosomes. Thus, binding of Etf-2 to RAB5-GTP appears to delay RAB5 inactivation by impeding RABGAP5 localization to endosomes. This suggests a unique mechanism by which RAB5 is sequestered on ehrlichial inclusions to benefit bacterial survival and replication.
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