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 determine the effect of 3-MA on E. chaffeensis internalization into THP-1 cells, E. chaffeensis was incubated with 1.5 £ 10 5 THP-1 cells at an multiplicity of infection (MOI) of 1,500:1 for 2 h in the presence of 2.5 mM 3-MA. The cells were washed to remove bacteria that were unbound and then were fixed with 4% PFA. The extracellular and intracellular bacteria were distinguished by immunofluorescence assay using rabbit anti-P28 and AF555-conjugated goat anti-rabbit IgG before permeabilization and rabbit anti-P28 and AF488-conjugated goat antirabbit IgG after permeabilization as described. 54 THP-1 cells (1.5 £ 10 5 ) were incubated with E. chaffeensis at an MOI of 50:1. 3-MA at a final concentration of 2 mM was added to E. chaffeensis-infected THP-1 cells at 1 h and 1 d p.i., and E. chaffeensis infection was assessed at 3 d p.i. To determine the reversibility of 3-MA inhibition of E. chaffeensis replication, THP-1 cells were infected with E. chaffeensis for 23 h, and then a portion of cells was incubated with 10 mM 3-MA for 6 h or 58 h. The cells that were treated with 3-MA for 6 h were washed to remove 3-MA, and the incubation was continued for an additional 52 h. Bacterial infection was assessed by Diff-Quik staining (Thermo Scientific, 9990700) and immunofluorescence staining with anti-P28.