Intracellular lipopolysaccharide binds RETREG1/FAM134B to regulate ER remodeling upon bacterial infection

Selective autophagy of the endoplasmic reticulum (ER), termed ERphagy or reticulophagy, plays a key role in organelle remodeling and cellular homeostasis. However, whether and how ERphagy is regulated during Gram-negative bacteria infection to influence host responses remains unclear. Here, we show that Salmonella enterica serovar Typhimurium releases lipopolysaccharide (LPS) that colocalizes with RETREG1/FAM134B, a reticulon-like ER-resident receptor for ERphagy. Cytosolic delivery of LPS, either during infection or via transfection, markedly increases RETREG1- and LC3B-decorated ER fragments. Mechanistically, affinity-isolation assays demonstrate that LPS directly binds RETREG1 through interactions between lipid A and positively charged residues within its amphipathic helices and C-terminal region. This interaction promotes RETREG1 oligomerization and drives ER membrane fragmentation, a process further amplified by the O-antigen moiety of LPS. The resulting ER fragments accumulate around LC3-positive Salmonella-containing vacuoles, facilitating bacterial clearance. Importantly, both intracellular and extracellular Salmonella exploit outer membrane vesicles (OMVs) to deliver LPS into the host cytosol, triggering RETREG1 activation and ER remodeling. Collectively, our findings reveal a previously unrecognized host response by which LPS of Gram-negative bacteria are sensed by the host ERphagy machinery to promote xenophagy and enhance antibacterial defense.Abbreviations: AH: amphipathic helix; BMDMs: bone-marrow-derived macrophages; Co-IP: co-immunoprecipitation; BafA1: bafilomycin A1; Cterm: C-terminal region (Cterm); CFU: colony-forming units; DAPI: 4’,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; EPEC: enteropathogenic Escherichia coli; GBP: guanylate binding protein; Gm12250/IRGB10: predicted gene 12250; KDO: keto-3-deoxy-octonate; LPR: lipid-to-protein ratio; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; mtLIR: LC3B-interacting region mutant; MDP: muramyl dipeptide; OMVs: outer membrane vesicles; O-Ag: O-antigen; OmpA: outer membrane protein A; RHD: reticulum homology domain; R-LPS: rough-LPS; S-LPS: smooth-LPS; SCVs: Salmonella-containing vacuoles; SFB: S-protein-FLAG-streptavidin binding peptide; TM: transmembrane domain; TEM: transmission electron microscopy; WT: wild-type.