Activation of TGF-β-activated kinase 1 (TAK1) restricts Salmonella Typhimurium growth by inducing AMPK activation and autophagy

Abstract

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 S6K1T389, 4E-BPT37/46, and ULK1S757 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 ULK1S317 and ACCS79 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 ULK1S317 and suppressing mTOR activity and ULK1S757 phosphorylation. Our study has unveiled a previously unrecognized pathway for S. Typhimurium-induced autophagy.

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrictSalmonellaTyphimurium growth in macrophages

“…It is highly likely that simultaneous phosphorylation at these two sites should lead to effective ULK1 activation. Of note, ULK1 is phosphorylated only at S317 by activated AMPK in S. Typhimurium-infected HeLa cells, whereas ULK1 phosphorylation at S555 is even decreased in this epithelial cell line (Liu et al, 2018).…”

“…Of note, ULK1 is phosphorylated only at S317 by activated AMPK in S . Typhimurium‐infected HeLa cells, whereas ULK1 phosphorylation at S555 is even decreased in this epithelial cell line (Liu et al, ).…”

“…In contrast, our recent study showed that indeed AMPK activation in S . Typhimurium‐infected HeLa cells is able to block the phosphorylation and activation of these kinases, including ULK1 S757 and S6K1 (Liu et al, ). It is not clear if this is due to the difference in cell types: RAW264.7 cells are macrophages, whereas HeLa cells are an epithelial tumour cell line.…”

“…Typhimurium differentially induces ULK1 phosphorylation in these two cell lines: AMPK activation led to increased ULK1 phosphorylation at S555 and S317 in RAW264.7 cells. In contrast, AMPK activation leads to increased ULK1 S317 phosphorylation but decreased ULK1 S555 in HeLa cells (Liu et al, ).…”

“…Analysis of autophagosome formation in RAW264.7 cells stably expressing GFP–RFP–LC3 was conducted as recently reported (Liu et al, ). To further analyse if LC3 was colocalized with S .…”