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

We have previously shown that the T4S effector Etf-1 is produced and secreted at the start of ehrlichial exponential growth, which is required for E. chaffeensis replication in THP-1 cells, since bacteria amount was reduced while affinity-purified anti-Etf-1 IgG was delivered into infected cells using the Chariot protein delivery system at 1 d p.i. 43 Here, we examined whether Etf-1 has additional effects on E. chaffeensis infection. E. chaffeensis infection was significantly enhanced in cells transfected with Etf-1-GFP compared with control GFP-transfected cells as determined by qPCR (Fig. 6A ). This growth-stimulating activity of Etf-1-GFP was specific to E. chaffeensis because Etf-1-GFP did not enhance infection by A. phagocytophilum (Fig. S8A ), which replicates in autophagosomes. 35 Although our previous study shows that Etf-1 targets mitochondria, it also localizes in part to E. chaffeensis inclusions. 43 To determine whether Etf-1 is present on cytoplasmic side of the inclusion membrane, we selectively permeabilized the plasma membrane of host cells using streptolysin O (SLO). 86 SLO is a thiol-activated protein toxin that binds cholesterol to form membrane-penetrating channels in a temperature-dependent manner. 87 Cytoplasmic Etf-1 was labeled with anti-Etf-1 after SLO treatment (Fig. 6B) . In contrast, the intrainclusion ehrlichial protein VirB6-2 40 was not labeled with anti-VirB6-2 in SLO-treated cells (Fig. 6B) , indicating the inability of SLO to permeabilize E. chaffeensis inclusions. Saponin treatment permeabilizes all cholesterol-containing membranes. Both Etf-1 and VirB6-2 could be labeled with their respective antibodies and colocalized in saponin-permeabilized cells (Fig. 6B ). This result indicates that native Etf-1 is present on the cytoplasmic face of inclusions. Furthermore, ectopically expressed Etf-1-GFP at 2 d p.i. was recruited to E. chaffeensis inclusions, and Etf-1-GFP could be observed inside bacteria-containing inclusions (56.8 § 4.8% of E. chaffeensis inclusions as quantitated from 10 to 20 cells each in 3 independent experiments, Fig. 6C) . Similarly, immunogold labeling showed Etf-1-GFP on the E. chaffeensis inclusion membranes or inside inclusions (Fig. 6D) . Etf-1 targeting to bacteria-containing inclusions was specific to E. chaffeensis, as Etf-1-GFP was not present on A. phagocytophilum inclusions (Fig. S8B) .