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

The small GTPase RAB5 regulates endosome maturation to late endosomes, thereby regulating fusion of LC3-decorated autophagosomes with late endosomes to form intermediary compartments prior to fusion with lysosomes. 21 RAB5 also regulates autophagy upstream of LC3 conjugation. [95] [96] [97] Given that E. chaffeensis replicates in inclusions that contain endogenous RAB5, 9 we first examined whether endogenous RAB5 is required for E. chaffeensis infection. There are 3 RAB5 isoforms expressed in human cells, namely RAB5A, 5B, and 5C, which share similar subcellular localization and regulatory functions in the early endocytic pathway. 98 RAB5 isoforms are at least partially redundant because downregulation of expression of any single RAB5 isoform using siRNA has no effect on EGF or transferrin internalization, but downregulation of all 3 isoforms has a significant effect. 99 Similarly, silencing of all 3 RAB5 isoforms is required to block AKT activation after insulin treatment. 100 With this in mind, we tested the role of RAB5 in E. chaffeensis infection by silencing expression of all 3 RAB5 isoforms using siRNA. Western blotting results showed that E. chaffeensis infection was significantly reduced by RAB5 knockdown compared with the control scrambled-siRNA transfection (Fig. 9A ). Next, we examined whether exogenous RAB5 traffics to already established E. chaffeensis inclusions by transfecting GFP-RAB5. All 3 GFP-RAB5 isoforms produced puncta (vesicles) and accumulated on E. chaffeensis inclusions when transfected at 15 h p.i., and quantitation from 10 to 20 cells each in 4 independent experiments showed RAB5A positive E. chaffeensis inclusions were 94.0 § 5.5%. RAB5 delivery appeared to occur largely via fusion of the E. chaffeensis