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.

中文翻译：

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埃里希体 IV 型分泌系统效应子 Etf-2 与活性 RAB5 结合并延迟内体成熟。


恰菲埃里希体是一种必需的细胞内细菌，它通过将内体运输调节剂（小 GTP 酶 RAB5）隔离在其膜结合内含物上来感染单核细胞/巨噬细胞，以避免路由至宿主细胞吞噬溶酶体。然而，人们对 RAB5 如何隔离在埃里希体夹杂物上知之甚少。我们发现天然埃利希体易位因子 2 (Etf-2)（之前预测的埃利希体 IV 型分泌系统的效应子）和重组 Etf-2（克隆到埃利希体基因组中）被分泌到宿主细胞的细胞质中并定位于埃里希体包裹体。异位表达的 Etf-2-GFP 也定位于 RAB5 标记的早期内体的内含物和膜，并与 GTP 结合的 RAB5 相互作用，但不与 GDP 结合的 RAB5 相互作用。 Etf-2虽然缺乏RAB GTP酶激活蛋白（GAP）Tre2-Bub2-Cdc16（TBC）结构域，但包含两个保守的TBC结构域基序，即Arg指和Gln指，并且定点诱变显示Arg188和 Gln245 是 Etf-2 定位到早期内体所必需的。酵母双杂交测定和微量热泳表明，Etf-2 与 GTP 结合的 RAB5 紧密结合，但不与 GDP 结合的 RAB5 紧密结合。然而，Etf-2 缺乏 RAB5 特异性 GAP 活性。 Etf-2 定位于含有珠子的吞噬体以及含有埃利希体外膜侵入素 EtpE（埃利希体进入触发蛋白）C 末端片段包被的珠子的内体，并显着延迟 RAB5 解离和 RAB7 定位吞噬体/核内体和 RABGAP5 定位于核内体。因此，Etf-2 与 RAB5-GTP 的结合似乎通过阻止 RABGAP5 定位到核内体来延迟 RAB5 失活。 这表明 RAB5 被隔离在埃利希体包涵体上以有利于细菌存活和复制的独特机制。