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

Full-length Etf-1 that had been codon-optimized for mammalian expression 43 was cloned into pEGFP-N1 (Clontech, 6085-1) and pDsRed-N1 (Clontech, 632412) to create plasmids encoding Etf-1-GFP and Etf-1-DsRed fusion proteins. pEtf-1-GFP was further modified to create pEtf-1-HA. Full-length RAB5A was cloned into pET-41a(C) (Novagen, 70556-3) for GST-RAB5A expression. A double-FYVE finger (2£FYVE) of HGS (HGF-regulated tyrosine kinase substrate) was cloned from mouse cDNA as described 44 into vector pEGFP-N1 to create plasmid 2£FYVE-GFP for mammalian expression. pEGFP-C1-ATG5 (pEGFP-C1-hApg5) was obtained from Addgene (plasmid 22952, deposited by Dr. Noboru Mizushima). 18 Dominant negative GFP-RAB5A S34N and constitutive active GFP-RAB5A Q79L mutants were constructed from GFP-RAB5A by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, 200519). RF/6A, HEK293, and DH82 cells were transfected using Fugene HD according to the manufacturer's instructions (Promega, E2311).