Iron robbery by intracellular pathogen via bacterial effector–induced ferritinophagy

Abstract

Iron is essential for survival and proliferation of <i>Ehrlichia chaffeensis,</i> an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how <i>Ehrlichia</i> acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the <i>Ehrlichia</i> genome) <i>Ehrlichia</i> translocated factor-3 (Etf-3), a previously predicted effector of the <i>Ehrlichia</i> 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 <i>Ehrlichia</i> Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced <i>Ehrlichia</i> proliferation, whereas knockdown of Etf-3 in <i>Ehrlichia</i> via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited <i>Ehrlichia</i> proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both <i>Ehrlichia</i> and host cells. However, during <i>Ehrlichia</i> proliferation, we observed concomitant up-regulation of <i>Ehrlichia</i> Fe-superoxide dismutase, which is an integral component of <i>Ehrlichia</i> T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in <i>Ehrlichia-</i>infected cells compared with uninfected cells. Thus, <i>Ehrlichia</i> 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.