Ehrlichial vacuoles did not colocalize with the lysosomal marker LAMP2, and lysosomes were redistributed and dramatically reduced in level in the infected cells

Ehrlichial vacuoles did not colocalize with the lysosomal marker LAMP2, and lysosomes were redistributed and dramatically reduced in level in the infected cells. and 3 postinfection, and increased LC3II levels were detected during infection, reaching a maximal level on day 3. Ehrlichial vacuoles did not colocalize with the lysosomal marker LAMP2, and lysosomes were redistributed and dramatically reduced in level in the infected cells. An inhibitor specific for the Wnt receptor signaling component Dishevelled induced lysosomal fusion with ehrlichial inclusions corresponding to p62 degradation and promoted transcription factor EB (TFEB) nuclear localization. infection activated the phosphatidylinositol 3-kinase (PI3K)CAktCmTOR (mechanistic target Tirasemtiv (CK-2017357) of rapamycin) pathway, and activation was induced by three ehrlichial tandem repeat protein (TRP) effectors, with TRP120 inducing the strongest activation. Moreover, induction of glycogen synthase kinase-3 (GSK3) performed using a Wnt inhibitor and small interfering RNA (siRNA) knockdown of critical components of PI3K-GSK3-mTOR signaling decreased ehrlichial survival. This report reveals exploitation of the evolutionarily conserved Wnt pathway to inhibit autolysosome generation, thereby leading to evasion of this important innate immune defense mechanism. is an obligately intracellular Gram-negative bacterium that preferentially infects mononuclear phagocytes (1, 2) and causes the emerging life-threatening tick-borne zoonosis human monocytotropic ehrlichiosis (HME). HME manifests as a disease with moderate to high severity, with meningitis, acute respiratory distress syndrome, and multisystem organ failure common in many fatal cases (3, 4). In order to survive intracellularly and evade innate defense mechanisms of the mononuclear phagocyte, spp. have evolved sophisticated molecular strategies to reprogram the host cell defenses. Mononuclear phagocytes are abundant in lysosomes, and a key survival strategy of involves inhibition of phagolysosomal fusion associated with the autophagic process. The mechanisms whereby inhibits autophagy are not well understood; however, ehrlichial type 1 secreted tandem repeat protein (TRP) effectors activate Wnt and Notch signaling pathways (5, 6) that are associated with autophagy regulation (7, 8). In eukaryotes, autophagy is a conserved, highly regulated cellular degradation pathway that sequesters and digests intracellular components, including invading pathogens (9, 10). Autophagy is a primary innate host defense mechanism against intracellular pathogens such as servovar Typhimurium, (11,C15). In contrast, induction of autophagy benefits some Tirasemtiv (CK-2017357) intracellular bacteria such as through nutrient acquisition or by allowing cell-to-cell spread (16,C18). The autophagy process involves membrane nucleation, elongation, and formation of a unique double-membrane TSPAN12 vacuole called the autophagosome. Autophagosomes can fuse directly with lysosomes to form an autolysosome or can fuse with endosomes, creating a single-membrane amphisome that eventually fuses with lysosomes to form an autolysosome (19). Autophagy is normally induced as an innate immune response to pathogens, but this process is inhibited during infection. A role for the functional two-component system in inhibition of autophagosome/lysosome fusion during ehrlichial infection has been reported (20), but the mechanistic details are unknown. Recently, type IV secretion effector protein Etf-1 has been shown to interact with Rab5, phosphatidylinositol 3-kinase C3 (PI3KC3), and Beclin-1 and to induce autophagosome Tirasemtiv (CK-2017357) formation proximally to ehrlichial inclusions for nutrient acquisition. However, LC3 was not detected on inclusions, Tirasemtiv (CK-2017357) suggesting that fusion with the autophagophore does not occur; and yet, ehrlichial vacuoles resemble amphisomes in other ways (21). Moreover, the related rickettsial pathogen also uses its type IV secretion effector, Ats-1, to initiate autophagy by binding Beclin-1 to activate class III PI3K, which, in contrast to the results seen with vacuole. The effector-driven autophagosome formation seen with both and appears to bypass normal starvation signal-induced mTOR (mechanistic target of rapamycin) inhibition (21,C23). Nevertheless, the mechanisms whereby lysosomal fusion is inhibited by are unknown. The central inhibitor of Tirasemtiv (CK-2017357) autophagy is the serine/threonine protein kinase mTOR. Under nutrient-deprived conditions, mTOR phosphorylates ULK1 (mammalian homolog of Atg1) and Atg13, inhibiting ULK1 complex coupling, which is the first step in autophagophore formation. Various host components such as LC3, Beclin-1, and p62 are involved in the autophagic pathway. Mammalian protein p62 (SQSTM1) directly binds to both ubiquitinated targets via its.