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 proliferation requires class III PtdIns3K activation and BECN1, and is enhanced by induction of autophagy with rapamycin Because PtdIns3P was elevated during E. chaffeensis infection, we next examined whether class III PtdIns3K activation is required for E. chaffeensis endocytosis or proliferation by using 3-methyladenine (3-MA), an inhibitor of class III PtdIns3K. [49] [50] [51] At a late stage of infection, we used qPCR (quantitative realtime PCR) to analyze the effect, as there were too many bacteria to be counted accurately. 43 At the early stage of infection or to analyze binding and entry, a direct counting method by immunofluorescence microscopy was used since qPCR cannot distinguish bound vs. internalized bacteria. Western blot analysis was used to compare knocked-down host protein levels and E. chaffeensis major outer membrane protein P28 52 levels relative to controls. All these methods are comparable in quantifying bacteria. 53,54 3-MA addition at 0 h did not inhibit E. chaffeensis entry as determined at 2 h p.i. (Fig. 2A) . However, when 3-MA was added at 1 h p.i. (immediately after ehrlichial entry) or at 1 d p.i. (at the beginning of exponential growth) and the infected THP-1 cells were continuously incubated in the presence of 3-MA, infection-based on both the percent of infected cells and bacterial numbers per host cell-was greatly inhibited compared with untreated infected cells ( Fig. 2B and C) . When 3-MA was added to infected cells at 23 h p.i. and incubated for 6 h or 58 h, E. chaffeensis became condensed and were marginalized in enlarged vacuoles (Fig. 2D , panels iv and vi), which was confirmed by staining with an antibody against E. chaffeensis P28 52 (Fig. 2E ). The vacuoles found in infected cells after 3-MA treatment were specific to E. chaffeensis infection, because similar vacuoles were not seen in uninfected THP-1 cells after 3-MA treatment for 2 d (Fig. S2A ). 3-MA did not have direct toxicity on E. chaffeensis: when E. chaffeensis was pretreated i.), cells were fixed and stained with DAPI to indicate E. chaffeensis (pseudocolored in red). PIK3C3 was labeled with mouse anti-FLAG. Merged/DIC, fluorescence image merged with differential interference contrast (DIC) image. Each boxed area is enlarged 4-fold on the right. N, nucleus; scale bars: 10 mm. (C) The percent colocalization of E. chaffeensis inclusions with PIK3C3 or 2£FYVE was determined by counting 10 to 20 inclusions per cell in 5 to 10 cells per experiment from 3 independent experiments. (D) PtdIns3P levels are increased in E. chaffeensis-infected THP-1 cells. Uninfected or E. chaffeensis-infected THP-1 cells (2 £ 10 6 cells) at 1 d p.i. were collected, and PtdIns3P lipids were purified and the amount determined by competitive ELISA. Assays were carried out in triplicate. Data are presented as the mean § standard deviation. Ã Significantly different by the Student t test (P < 0.05). with 3-MA and then incubated with THP-1 cells in the absence of 3-MA, there was no inhibitory effect on its ability to infect the cells (Fig. S2B ). 3-MA did not have direct toxicity on host mammalian cells, as treatment with 2 mM 3-MA for 1 d had no effect on THP-1 cell viability (Fig. S2A , Table S1 ). 3-MA does not seem to block intracellular bacterial infection nonspecifically by impairing host cell metabolism, because 3-MA treatment enhances intracellular Mycobacterium growth inside macrophages. 31 In addition, 3-MA treatment did not induce lysosomal