Mechanisms Of S. Typhimurium-induced Autophagy

Autophagy is a relatively conservative intracellular metabolic process.Damaged organelles,protein aggregates,and energy stress due to hypoxia and anticancer drug can all induce autophagy.In particular,autophagy functions to defend cells from invading pathogens.Double membrane autophagosomes mark the start of autophagy.The vesicles reach out and surround ambient cytoplasm and organelles to degrade them into small molecules for recycling through the effect of lysosome.Autophagy can work in both selective and nonselective ways.A selective degradation of foreign matters plays an indispensable role in maintaining normal metabolism of cells.By cooperating with innate imunity,autophagy helps to protect cells.AMP-activated protein kinase(AMPK)and mechanistic target of rapamycin(mTOR)sense energy stress and nutrient depletion,respectively,and play pivotal roles in regulating autophagy.AMPK directly phosphorylates ULK1 at S555 and activates it or indirectly activates ULK1 by inhibiting mTORC1 activity.mTOR,a serine/threonine kinasethat interacts with several adaptor proteins to form themTOR complex 1(mTORC1),phosphorylates ULK1/2 at S757,disrupts its interaction with AMPK and prevents it from activating the autophagy pathway.Inactivation of mTORC1 by nutrient insufficiency or by rapamycin,an inhibitor of mTOR,induces autophagy.S.Typhimurium is a facultative intracellular bacterium and causes many kinds of diseases to hosts.S.Typhimurium is responsible for the most cases of enteritis and shows an increasing trend of infection.Autophagy functions as a first line defense to fight against the invading parasitic bacteria.Autophagy cooperates with innate immunity to clear the intracellular bacteria.Better understanding of the mechanisms of S.Typhimurium-induced autophagy will help designing novel therapeutic drugs to limit or eliminate the deadly S.Typhimurium infection,in particular those multiple drug-resisntant "superbugs".Western blot was used to detect expression of LC3 and p62 in RAW264.7 and HeLa infected with various multiplicity of infection(MOI)of S.Typhimurium or 10 MOI of S.Typhimurium for various lengths of time.Co-localization of GFP-LC3-containing autophagsomes and RFP-labeled S.Typhimurium was examined under a confocal microscope.Chloroquine(CQ)and bafilomycin A1 were added into HeLa and RAW264.7 cells in the absence or presence of S.Typhimurium.We found that S.Typhimurim increased LC3-Ⅱ levels and the ratio of LC3-II/LC3-I but dereased p62 levels in a time-and dose-dependent manner in RAW264.7 and HeLa cells.Both bafilomacin A and CQ further increased the levels of LC3-II and the ratio of LC3-II/LC3-Ⅰ but restored p62 levels.Confocal microscopic analysis revealed that LC3-GFP and RFP-labelled S.Typhimurium were co-localized and formed a larger autophagosome puncta than that in rapamycin-treated cells.We next terested if S.Typhimurim-induced autopahgy was mediated by suppression of mTOR activity or by activation of AMPK.We conducted Western blot and analyzed the activation of mTOR and AMPK by analyzing the status of phosphorylation of several key molecules related to mTOR and AMPK.We found that S.Typhimurim infection did not suppress but rather activated the mTOR pathway,as revealed by increased the phosphorylation of mTOR,ULK1S757,S6K1,and pS6 of cells in both HeLa and RAW264.7 cells in a time and dose-dependent manner.In contrast,S.Typhimurim infection increased the phosphorylation of AMPK and ULK1S555 in HeLa and RAW264.7 cells in a time and dose-dependent manner.Further studies revealed that Compound C(AMPK inhibitor)and 5Z-7-oxozeanol(TAK1 inhibitor)blocked S.Typhimurim-increased LC3-Ⅱ levels and the ratio of LC3-II/LC3-I and normalized the p62 levels.Finally,we tested whether inhibition of autophagy by an AMPK inhibitor was able to increase the growth of S.Typhimurim in HeLa cells.HeLa cells were infected with salmonella for various lengths of time in the absence or presence of Compound C.The cell lysates were prepared and analyzed for S.Typhimurium growth by counting the colonies grown on agar plates.We found that Compound C significantly increased the colonies of S.Typhimurium inoculated from AMPK-treated HeLa cells,compared to untreated control.In summary,our results showed that S.Typhimurium infection of both RAW247.6 and HeLa cells leads to a complete and degradative autophagy.Autophagy is induced by AMPK activation in RAW264.7 and HeLa cells and plays a critical role in restricting the growth of intracellular S.Typhimurium.In addition,inhibiton of mTOR in HeLa cells also contributes to S.Typhimurium-induced autophagy.