Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

A RAB5 effector, EEA1, localizes on E. chaffeensis inclusion membranes. 9 Because membrane localization of EEA1 is dependent on PtdIns3P (phosphatidylinositol 3-phosphate), we examined whether a 2£FYVE finger protein probe with unique specificity for PtdIns3P in the absence of RAB5 and an ERlocalization signal 44 is localized to E. chaffeensis replicative inclusions. We used monkey endothelial RF/6A cells, which can be readily infected with E. chaffeensis, are adherent cells with a flattened morphology for unambiguous localization, and can be more effectively transfected than the nonadherent human acute leukemia cell line THP-1. 43 Ectopically expressed 2£FYVE-GFP produced large puncta (vesicles) and was localized on »80% of E. chaffeensis replicative inclusions at 2 d post-infection (p.i.) ( Fig. 1A and C). To further verify the presence of PtdIns3P on E. chaffeensis inclusions, we examined another RAB5 effector, ANKFY1/Rabankyrin-5 (ankyrin repeat and FYVE domain containing 1), the membrane localization of which is also dependent on PtdIns3P. 45 GFP-ANKFY1 produced large puncta (vesicles) and accumulated on E. chaffeensis inclusions (Fig. S1 ). In uninfected cells, GFP-ANKFY1 showed smaller puncta than in infected cells (Fig. S1 ).