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

Uninfected or E. chaffeensis-infected THP-1 cells (»2 £ 10 6 cells) at 1 d p.i. were collected. Alternatively, RF/6A cells (1 £ 10 6 cells) were transfected with EGFP (1 mg) or Etf-1-EGFP plasmids (5 mg) by electroporation (100 V) in 0.2-cm cuvettes. Cells were cultured in a T75 flask for 1 d for the Etf-1-transfected groups, before being collected. PtdIns3P lipids were purified according to the manufacturer's instructions (PtdIns3P Mass ELISA kit; Echelon Biosciences, K-3300). Cells were precipitated with 0.5 M trichloroacetic acid (Sigma, T0699), lipids were extracted with MeOH:CHCl 3 (2:1), and acidic lipids were extracted with MeOH:CHCl 3 :12 M HCl (80:40:1). The supernatant (»2.25 mL) was mixed with 0.75 mL of CHCl 3 and 1.35 mL of 0.1 M HCl, and the organic (lower) phase was collected and vacuum dried. The PtdIns3P amount was measured with the competitive ELISA using a SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA).