Restricting intracellular Salmonella proliferation by coordinating p-TBK1 mediated mitophagy and xenophagy.

Mitophagy is essential for eliminating dysfunctional mitochondria and is closely implicated in the immune evasion of several pathogens, including S. typhimurium. However, the specific mechanisms regarding the interaction between S. typhimurium and host cells in relation to mitophagy and xenophagy and their contribution to pathogen survival are unclear. Herein, using both in vitro and in vivo systems, we found that S. typhimurium escaped host innate immunity by repressing mitophagy and xenophagy to facilitate its intracellular replication. Moreover, we identified a novel xenophagy modulator, fisetin that could activate mitophagy to restrict intracellular S. typhimurium replication in RAW264.7 and bone marrow-derived macrophages, which was abolished by mitophagy inhibitor Mdivi-1. RNA-Seq transcriptome and metabolomics analysis demonstrated the effectiveness of fisetin in alleviating S. typhimurium infection. Confocal microscopy analysis revealed that fisetin-induced mitophagy promoted xenophagy, whereas inhibiting mitophagy repressed xenophagy and facilitated the survival of S. typhimurium. Our study further demonstrates that fisetin-induced mitophagy requires the recruitment of phosphorylation of TBK1 to mitochondria, which is a protein implicated in mitophagy and xenophagy. Additionally, fisetin improved the body weight loss, relative spleen, kidney, and liver weights, hepatic damage, and S. typhimurium load, all of which were abrogated by Mdivi-1 or Pink1 siRNA treatment in S. typhimurium-infected mice. Collectively, our results suggest that S. typhimurium induces mitochondrial damage whilst inhibiting mitophagy, while fisetin promotes xenophagy and restrains S. typhimurium survival by facilitating PINK1-PRKN-mediated mitophagy and p-TBK1 mitochondrial recruitment. Fisetin proves effective as a xenophagy enhancer in reducing intracellular Salmonella burden.Abbreviations: BafA1: bafilomycin A(1); BMDM: mouse bone marrow-derived macrophage; CFU: colony-forming units; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LDH: lactate dehydrogenase; Mdivi-1: mitochondrial division inhibitor 1; OPTN: optineurin; PBS, phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; siRNA: interfering RNA; SQSTM1/p62: sequestosome 1; S. typhimurium: Salmonella enterica serovar typhimurium; T3SS: type III secretion system 1; TBK1: TANK-binding kinase 1.