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

E. chaffeensis infection was significantly increased in cells treated with rapamycin, the inhibitor of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), 59 based on western blot analysis of E. chaffeensis P28 (Fig. 3C) and qPCR of the E. chaffeensis 16S rRNA gene (rDNA, Fig. 3D ). Rapamycin treatment under these conditions had no effect on the viability of the infected host cells (Table S1 ). These results are similar to A. phagocytophilum, 35 except that 3-MA treatment does not induce enlarged vacuoles containing A. phagocytophilum, and opposite to intracellular bacteria such as Salmonella and Mycobacterium: 3-MA inhibits intracellular mycobacterium killing by macrophages via autophagy, 31 and induction of autophagy by rapamycin, suppresses intracellular bacteria such as Salmonella and Mycobacterium. 31, 60 ATG5 is required for infection and localizes to E. chaffeensis inclusions To further investigate the involvement of autophagy induction in E. chaffeensis infection of primary macrophages, the requirement of an autophagy double-membrane initiation protein, ATG5, 61 was examined. Homozygous deletion of Atg5 gene results in neonatal lethality in mice. 62 Therefore, tissue-specific knockout (TSKO) mice were created by a site-specific recombinase technology using the third exon of Atg5 flanked by LoxP sites in combination with the Cre recombinase driven by tissue-specific promoter. 63 Because siRNA transfection of monocytes-macrophages is not efficient, and WT (wild-type) mouse bone marrow-derived macrophages (BMDMs) are readily infected with E. chaffeensis, 54 we used BMDMs from atg5 flox/flox -Lyz2-Cre mice 64 in which Lyz2 promoter-driven Cre is used for myeloid cell-specific KO of Atg5. 65 Peritoneal macrophages and BMDMs from these mice lack ATG5. 66 E. chaffeensis infection in BMDMs from atg5 flox/flox -Lyz2-Cre (atg5 TSKO) mice was markedly reduced compared with WT mice (Fig. 4A ), demonstrating the critical role of ATG5 in E. chaffeensis infection of primary macrophages. The viability of BMDMs from atg5 TSKO and WT mice, either uninfected or infected with E. chaffeensis, was >90% at 4 d p.i. (Fig. S4) . Moreover, GFP-ATG5 transfected at 1 d p.i. localized to E. chaffeensis inclusions (Fig. 4B ). Similar to ectopically expressed GFP-ATG5, immunostaining of E. chaffeensis-infected cells with anti-ATG5 to detect the endogenous protein, showed the localization of endogenous ATG5 in 54 § 12% of inclusions (n D 75 cells; Fig. S5B ).