Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation

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.