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

To examine the colocalization of Etf-1-DsRed with GFP-ATG5 or GFP-LC3, RF/6A or HEK293 cells at »80% confluence were used for transfection. Cells were treated with or without 10 nM BAF (Enzo, BML-CM110-0100) for 16 h at 1 d p.t., prior to fixation in PFA. To examine the distribution of NPEPPS-GFP and Etf-1, sequential transfections were performed. Etf-1 was first transfected into DH82 cells, and NPEPPS-GFP was transfected 1 d later. At 1 d p.t. with NPEPPS-GFP, cells were fixed and incubated with rabbit anti-Etf-1 and AF555-conjugated goat anti-rabbit IgG. To examine the effect of Etf-1 on the degradation of Q103-GFP, Q103-GFP was first transfected into RF/6A cells and, at 16 h p.t., Etf-1-DsRed or DsRed was transfected into RF/6A cells. At 2 d p.t., cells were fixed and incubated with rabbit anti-Etf-1 and AF555-conjugated goat anti-rabbit IgG. Images were captured with a DeltaVision Deconvolution microscope system (GE Healthcare, Marlborough, MA). For western blot analysis, cells were transfected with Q103-GFP or cotransfected with Q103-GFP and Etf-1. At 16 h p.t., the cells were treated with or without 2 mM 3-MA (Sigma, M9281) for 2 d.