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

For amino acid supplementation, 3 £ 10 5 THP-1 cells were infected with E. chaffeensis in 1 ml complete RPMI 1640 medium. At 1 d p.i., cells were treated with complete RPMI 1640 medium with 2 mM 3-MA, or complete RPMI 1640 medium with 2 mM 3-MA plus 5£ MEM essential amino acids solution (Invitrogen, 11130-051), and E. chaffeensis infection was assessed at 3 d p.i. THP-1 cells at 1.5 £ 10 5 were preincubated with 0.1 mM rapamycin (Sigma, R0395) or 0.1% DMSO (control) for 2.5 h, and E. chaffeensis was added at an MOI of 50:1 in the continued presence of rapamycin or DMSO at 2 d p.i. Bacterial infection was assessed by western blotting using antibodies against P28 and ACT/actin and by qPCR using specific primers for E. chaffeensis 16S rDNA, with data normalized against the level of expression of human GAPDH. 127 To study the effect of PAQ-22 (Wako Pure Chemical Industries, 165-23581) on bacterial infection, 3 £ 10 5 E. chaffeensisinfected THP-1 cells were incubated with 10 or 100 mM of PAQ-22 or 0.1% DMSO (control) for 2 d.