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

Our results suggest that inactivation of RAB5 GTPase on inclusions not only protects E. chaffeensis from a microbicidal mechanism and expands inclusions via homotypic fusion but also enhances "RAB5-regulated autophagy" as a result of the increase in GTP-bound RAB5. Indeed, several previous studies point to the critical role of RAB5-GTP in early autophagosome development/maturation. 1) Ectopic expression of dominantnegative RAB5 or 3-MA treatment blocks the progression of early ATG5-positive phagophores to form LC3-positive autophagosomes. 97 2) Upon growth factor restriction, the class 1A phosphoinositide-3-kinase catalytic subunit b (PIK3CB/p110b) dissociates from the growth factor receptor and binds RAB5. 95 Such interaction blocks the RAB5 GTPase-activating protein PIK3R1/p85a (class 1A phosphoinositide-3-kinase regulatory subunit 1) from binding to RAB5, which consequently increases the amount of GTP-bound RAB5 and enhances the RAB5-PIK3C3 interaction to promote autophagy. 95 3) Hepatitis C virus NS4B protein forms a complex with RAB5 and PIK3C3 and induces RAB5-GTP-promoted autophagy, which is required for virus replication. 96 4) HTT binds RAB5 via the RAB5 effector F8A1/HAP40 (coagulation factor VIII-associated 1), and degradated by autophagy. 112 Similar to E. chaffeensis infection, such RAB5-regulated autophagy is independent from MTOR signaling. 95, 97 The present study revealed host cytoplasmic molecules (amino acids, GAPDH, and Etf-1-GFP) are delivered into ehrlichial inclusions. It is still unclear how host cytoplasmic molecules enter into ehrlichial inclusions. Unlike most other intracellular bacteria that are killed by autophagy, E. chaffeensis inclusions lack ubiquitination, LC3, and late endosome or lysosomal markers, although they do contain ATG5, PIK3C3, PtdIns3P, and RAB5. Although the classical view has been that autophagic and endocytic pathways converge at the phagolysosomal and lysosomal levels, autophagosomes have also been found to undergo fusion with earlier components of the endocytic pathway. 21, 22, 24, 113 The presence of ATG5 suggests that E. chaffeensis inclusions are large amphisomes, and Etf-1-, RAB5-, and ATG5-containing small vesicles might also be considered amphisomes. Multivesicular bodies (MVBs), morphologically distinctive endosomes, internally accumulate small membrane vesicles (60 to 80 nm), which contain cytosolic cargo molecules. 21, 114 Morphological evidence suggests that MVBs are the main endocytic fusion partner of the autophagosome, forming the amphisome. 21, 115 It is tempting to speculate that fusion of these vesicles with ehrlichial inclusions or invagination of the ehrlichial inclusion membrane would deliver host cytosolic cargo into the inclusion lumen. PIK3C3 and PtdIns3P are essential for the generation of the internal vesicles of MVBs, and RAB5 promotes the formation of PtdIns3P on 116 and phagosomes. 117 Most 2£FYVE labeling is associated with the internal membranes of MVBs, which have low levels of the late endosome and lysosome markers like lysosomal membrane glycoproteins and lyso-bisphosphatidic acid. 44, 118 PtdIns3P accumulates within ECVs (endosomal carrier vesicles) and MVBs, and PtdIns3P signaling regulates receptor sorting into ECVs/MVBs. 44, 119 Indeed, our results with 2£FYVE-GFP-transfected cells also showed strong intralumenal PtdIns3P in large vesicles docked onto E. chaffeensis inclusions, suggesting that these vesicles are MVBs (Fig. 1A, arrow) . Inhibition of