Mechanism Of Leflunomide-meidated Restriction Of Intracellular Salmonella Typhimurium Growth

Autophagy is a concerved cellular process that involves the self-digestion of cytoplasmic materials including misfolded proteins,damaged organelles,and invading parasitic microbes.Autophagy begins by forming the cresenct-like double membrane that expands to enclose the unneeded matierals and forms autophagosomes.Autophagosomes fuse with lysosomes to form autophagosomes in which proteases and peroxidases in lysosomes degrade the enclosed contents.One of the hallmark changes in autophagy is that LC3-? binds to PE(phosphatidylethanolamine)in the cytosol to form LC3-?,and the level of LC3-? positively correlates with autophagy.mTOR and AMPK are two kinases that sense nutrients and energy deprivation,respectively.mTOR phosphorylates ULK1 at S757 and inhibits its activity and autophagy,whereas AMPK phosphorylates ULK1 at multiple sites,including S555,S317,and S777.Phosphorylation at these sites activates ULK1 and induces autophagy.Salmonella(S)Typhimurium is a Gram-negative facultative intracellular parasite.Once the bacteria enter the host cell,it resides in the Salmonella-containing vesicle(SCV).The type ?secretion system of the bacteria releases some effector proteins to damage the SCV.Salmonella breaks through the SCV and grows rapidly in the nutrient-rich cytosol.However,S.Typhimurium are encounted by rapid ubiquination and marked for selective autophagy.Intriguingly,Salmonella can escape autophagy by activating mTOR and degrading AMPK.Since most antibiotics cannot enter the cytoplasm of host cells to kill intracellular bacteria,there has been strong interest in using autophagy to induce autophagy and kill intracellular bacteria as a novel strategy.Leflunomide is an anti-inflammatory drug used for the treatment of rheumatoid arthritis.Its active metabolite A77 1726 inhibits pyrimidine nucleotide synthesis and inhibits protein tyrosine kinase and S6K1 kinase activity.A recent study in our laboratory found that A77 1726 activates AMPK by inhibiting S6K1,a kinase downstream of mTOR.AMPK phosphorylates ULK1 and induces autophagy.Our present study was designed to investigate whether A77 1726 inhibits intracellular Salmonella growth by activating autophagy.RAW264.7 and HeLa cells were treated with A77 1726,followed by Western blot analysis of autophagy-related proteins and protein phosphorylation levels in the autophagy pathway.The results showed that A77 1726 increased the expression of LC3-? in a time-and dose-dependent manner,and feedback activated the PI-3 kinase signaling pathway to up-regulate the phosphorylation levels of AMPKT172,ULK1S757 and ULK1S555.Confocal microscopy showed that A77 1726 significantly increased the number of autophagosomes and autophagosomes in RAW264.7 and HeLa cells.We further investigated whether A77 1726 can enhance autophagy in Salmonella Typhimurium-infected cells.The results showed that in A77 1726-treated Salmonella-infected RAW264.7 cells,the levels of LC3-II and AMPKT172 and ULK1S555 phosphorylation were significantly higher than those treated with Salmonella or A77 1726 alone.The number of autophagosomes and autophagosomes was also significantly higher in cells treated with Salmonella plus A77 1726 than those treated with Salmonella or A77 1726 alone.These results indicate that A77 1726 can promote Salmonella-induced autophagy.Finally,we investigated whether autophagy induced by A77 1726 affected intracellular gorwth of S.Typhimurium.A771726 significantly reduced the number of Salmonella in RAW264.7 cells.Confocal microscopy revealed that A77 1726 significantly reduced the number of red fluorescence protein-labeled Salmonella in RAW264.7 cells.However,A771726 did not affect Salmonella grown in LB medium.In conclusion,our study showed that A77 1726 activates AMPK and ULK1,and induces autophagy in RAW264.7 and HeLa cells by inhibiting S6K1 kinase activity.A77 1726 further enhances autophagy by promoting AMPK and ULK1 phosphorylation in Salmonella-infected RAW264.7 cells.A77 1726 can effectively inhibit the intracellular proliferation of Salmonella in macrophages.These results indicate that A77 1726,as an autophagy activator,can inhibit the proliferation of intracellular bacteria.Leflunomide has potential to be developed as a novel antibacterial drug.