Autophagy is a conserved cellular process that functions as a first-line defense to restrict the growth of invading parasitic bacteria. As an intracellular pathogen, Salmonella (S) Typhimurium invades host cells through two Type III secretion systems (T3SS) and resides in the Salmonella-containing vacuole (SCV). When the SCV membrane is perforated and ruptured by T3SS-1, a small portion of the Salmonella egresses from the SCV and replicates rapidly in the nutrient-rich cytosol. Cytosolic Salmonella and those residing in the membrane-damaged SCV are tagged by ubiquitination and marked for autophagy through the ubiquitin-binding adaptor proteins such as p62, NDP52, and optineurin. Prior studies suggest that transient intracellular amino-acid starvation and subsequent inactivation of the mechanistic target of rapamycin (mTOR), a key molecule that phosphorylates Unc-51 like autophagy activating kinase (ULK1) and inhibits its activity, can trigger autophagy in S. Typhimurium-infected cells. Other studies suggest that energy stress in S. Typhimurium-infected cells leads to AMP-activated protein kinase (AMPK) activation and autophagy. In the present study, we report that autophagy was rapidly induced in S. Typhimurium-infected cells, as evidenced by increased LC3 lipidation and decreased p62 levels. However, S. Typhimurium infection drastically increased AKT phosphorylation but decreased S6K1^T389, 4E-BP^T37/46, and ULK1^S757 phosphorylation, suggesting that mTOR activation by AKT is subverted. Further studies showed that AMPK was activated in S. Typhimurium-infected cells, as evidenced by increased ULK1^S317 and ACC^S79 phosphorylation. AMPK activation was mediated by Toll-like receptor-activated TAK1. Functional studies revealed that AMPK and TAK1 inhibitors accelerated S. Typhimurium growth in HeLa cells. Our results strongly suggest that TAK1 activation leads to AMPK activation, which activates ULK1 by phosphorylating ULK1^S317 and suppressing mTOR activity and ULK1^S757 phosphorylation. Our study has unveiled a previously unrecognized pathway for S. Typhimurium-induced autophagy.

Influenza A virus (IAV) primarily infects the airway and alveolar epithelial cells and disrupts the intercellular junctions, leading to increased paracellular permeability. Although this pathological change plays a critical role in lung tissue injury and secondary infection, the molecular mechanism of IAV-induced damage to the alveolar barrier remains obscure. Here, we report that Gli1, a transcription factor in the sonic hedgehog (Shh) signaling pathway, is cross-activated by the MAP and PI3 kinase pathways in H1N1 virus (PR8)-infected A549 cells and in the lungs of H1N1 virus-infected mice. Gli1 activation induces Snail expression, which downregulates the expression of intercellular junction proteins, including E-cadherin, ZO-1, and Occludin, and increases paracellular permeability. Inhibition of the Shh pathway restores the levels of Snail and intercellular junction proteins in H1N1-infected cells. Our study suggests that Gli1 activation plays an important role in disrupting the intercellular junctions and in promoting the pathogenesis of H1N1 virus infections.

Janus kinase (JAK) inhibitors have been developed as novel immunomodulatory drugs and primarily used for treating rheumatoid arthritis and other inflammatory diseases. Recent studies have suggested that this category of anti-inflammatory drugs could be potentially useful for the control of inflammation “storms” in respiratory virus infections. In addition to their role in regulating immune cell functions, JAK1 and JAK2 have been recently identified as crucial cellular factors involved in influenza A virus (IAV) replication and could be potentially targeted for antiviral therapy. Gingerenone A (Gin A) is a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase (S6K1). Our present study aimed to determine the antiviral activity of Gin A on influenza A virus (IAV) and to understand its mechanisms of action. Here, we reported that Gin A suppressed the replication of three IAV subtypes (H1N1, H5N1, H9N2) in four cell lines. IAV replication was also inhibited by Ruxolitinib (Rux), a JAK inhibitor, but not by PF-4708671, an S6K1 inhibitor. JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity. In vivo experiments revealed that Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice, alleviated their body weight loss, and prolonged their survival. Our study suggests that Gin A restricts IAV replication by inhibiting JAK2 activity; Gin A could be potentially useful for the control of influenza virus infections.

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1^S757phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPK^T172and ULK1^S555phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1^G93Aprotein aggregates with autophagosomes and accelerated SOD1^G93Aprotein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.

The H5N1 subtype of the avian influenza virus causes sporadic but fatal infections in humans. H5N1 virus infection leads to the disruption of the alveolar epithelial barrier, a pathologic change that often progresses into acute respiratory distress syndrome (ARDS) and pneumonia. The mechanisms underlying this remain poorly understood. Here we report that H5N1 viruses downregulate the expression of intercellular junction proteins (E-cadherin, occludin, claudin-1, and ZO-1) in several cell lines and the lungs of H5N1 virus-infected mice. H5N1 virus infection activates TGF-β-activated kinase 1 (TAK1), which then activates p38 and ERK to induce E3 ubiquitin ligase Itch expression and to promote occludin ubiquitination and degradation. Inhibition of the TAK1-Itch pathway restores the intercellular junction structure and function in vitro and in the lungs of H5N1 virus-infected mice. Our study suggests that H5N1 virus infection impairs the alveolar epithelial barrier by downregulating the expression of intercellular junction proteins at the posttranslational level.