SignificancePhagocytosis 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.
Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3−induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia. Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.
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