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

In yeast, Atg5 is involved in the sequestration of the cytosolic precursor of aminopeptidase I and aminopeptidase IV, and the Atg12-Atg5 conjugate localizes only to phagophores (precursors of autophagosomes) and dissociates just before or after completion of autophagic vacuole formation. 67 Therefore, ATG5 and ATG12 are not associated with mature autophagosomes. LC3, also called LC3-I (cytosolic form), becomes conjugated to phosphatidylethanolamine (termed LC3-II) and localizes on autophagosomal membranes (appearing as small puncta by fluorescence microscopy) when autophagy is induced. 19 Thus LC3-II serves as a marker for canonical autophagosomes. ATG12-ATG5 conjugation is required for the conversion of LC3-I to LC3-II, 61 and A. phagocytophilum inclusions are heavily encased by LC3-II. 35, 37 However, GFP-LC3, as well as endogenous LC3B, did not encase individual E. chaffeensis inclusions ( Fig. 4C and S5B ). In contrast, both GFP-LC3 and endogenous LC3B were observed as puncta in E. chaffeensisinfected RF/6A cells at 2 to 3 d p.i. (Fig. 4C and S5B ). The conversion from LC3B-I to LC3B-II did not increase during exponential E. chaffeensis proliferation but was evident at 3 d p.i. in human promyelocytic leukemia HL-60 cells (Fig. 4D ) when infected cells began to rupture because of the heavy bacterial burden, as shown by fluorescence microscopy (Fig. 4C ), suggesting that the late onset of LC3B-II conversion may be caused by canonical autophagy: nutritional and ATP depletion that is due to overwhelming ehrlichial replication. Because treatment of E. chaffeensis-infected cells at 2 d p.i. with 10 nM bafilomycin A 1 (BAF) for 6 h significantly increased the ratio of endogenous LC3B-II to LC3B-I (Fig. S6A ), the basal autophagy flux was not blocked during the exponential growth phase (2 d p.i.) of intracellular E. chaffeensis. In addition, LC3B knockdown with siRNA did not reduce E. chaffeensis infection at 2 d p.i. (Fig. 4E ). Thus ATG5 autophagosome localization to E. chaffeensis inclusions was not followed by the LC3B-II localization, a process that is distinct from the development of GFP-LC3-II-loaded autophagosomes routed to A. phagocytophilum inclusions. 35, 37 E. chaffeensis increases cellular glutamine and glutamate and takes up host-incorporated amino acids Mammalian cellular free amino acid concentrations (those not incorporated into proteins) are tightly regulated by membrane transporters and the autophagy pathway. 68 Given that E. chaffeensis replication requires host cell autophagy proteins (BECN1 and ATG5) and the class III PtdIns3K activation, we examined cellular free amino acid levels in E. chaffeensisinfected cells by targeted metabolomics analysis using LC-MS/ MS. 69 Compared with uninfected THP-1 cells, L-glutamine and L-glutamate in E. chaffeensis-infected THP-1 cells at 2 d p.i. were notably increased, and cellular glutamine, glutamate, aspartate, and proline were profoundly depleted at 3 d p.i., because of the overwhelming consumption of cellular free amino acids by E. chaffeensis (Tables 1 and S2) .