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

THP-1 cells (2 £ 10 6 ) were grown for 24 h in RPMI medium (with 10% FBS and 2 mM glutamine) containing 10 mCi/ml of [ 3 H] L-glutamine (PerkinElmer, NET551250UC), washed, resuspended in fresh growth medium, and infected with E. chaffeensis for 2 d. 128 One group of infected cells was treated with 2 mM 3-MA for 6 h. E. chaffeensis was purified from infected cells by sonication and differential centrifugation as described, 128 and uninfected THP-1 cells were processed in parallel as control for potential host protein contamination. The protein amount of purified samples was determined, and the incorporation of [ 3 H]glutamine into E. chaffeensis proteins was determined with a liquid scintillation counter, with data normalized to the total protein amount.