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

Localization of Etf-1 in the host cytoplasm of E. chaffeensisinfected THP-1 cells was determined using SLO (Sigma, S0149) to selectively permeabilize the plasma membrane as described. 37 After SLO treatment, cells were immediately fixed in 4% PFA and incubated with rabbit anti-Etf-1 (1:100) and mouse anti-VirB6-2 (1:50), followed by incubation with AF488-conjugated goat anti-rabbit IgG and AF555-conjugated goat anti-mouse IgG in PBS supplemented with 0.5% bovine serum albumin. After washing with PBS, cells were further labeled with mouse anti-VirB6-2 and AF350-conjugated goat anti-mouse IgG diluted in PGS to permeabilize all membranes. 43 To examine the localization of DsRed-GAPDH in E. chaffeensis-infected RF/6A cells, infected cells cultured on coverslips in a 12-well plate at 1 d p.i. were transfected with pDsRed-N1 control or pDsRed-GAPDH plasmids using Fugene HD. At 1 d p.t., cells were treated with 0.1 mM DFP (Sigma, D0879) for 2 h and then fixed in 4% PFA and incubated with DAPI to stain bacteria.