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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埃希氏IV型分泌系统效应子Etf-2与活性RAB5结合并延迟内体成熟。

必需的胞内细菌埃里希氏菌通过在其膜结合的内含物上隔离内体运输的调节剂小GTPase RAB5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，如何将RAB5螯合在埃希氏夹杂物上却知之甚少。我们发现天然埃希氏菌易位因子2（Etf-2），以前预测的埃希氏菌IV型分泌系统的效应子，以及重组Etf-2（克隆到埃希氏菌基因组中）被分泌到宿主细胞的细胞质中并定位于埃希氏夹杂物。异位表达的Etf-2-GFP也定位于标记有RAB5的早期内体的包裹体和膜，并与GTP结合的RAB5相互作用，但与GDP结合的RAB5不相互作用。Etf-2，尽管缺少RAB GTPase激活蛋白（GAP）Tre2-Bub2-Cdc16（TBC）域，但包含两个保守的TBC域基序，即Arg指和Gln指，定点诱变表明Arg188和Gln245都需要Etf-2定位到早期内体。酵母双杂交检测和微尺度热泳表明，Etf-2与GTP结合的RAB5紧密结合，而与GDP结合的RAB5却不紧密结合。但是，Etf-2缺乏RAB5特异性的GAP活性。Etf-2定位于含珠粒的吞噬体，以及内含体，其内含被EtpE（埃希氏菌的进入触发蛋白）的C端片段包被的珠子，埃希氏菌外膜浸润素，并显着延迟了RAB5的解离和RAB7的定位吞噬体/内体和RABGAP5定位于内体。因此，Etf-2与RAB5-GTP的结合似乎通过阻止RABGAP5定位于内体来延迟RAB5的失活。这表明RAB5被螯合在埃希氏菌包涵体上以有益于细菌存活和复制的独特机制。