The F rancisella O ‐antigen mediates survival in the macrophage cytosol via autophagy avoidance

Summary Autophagy is a key innate immune response to intracellular parasites that promotes their delivery to degradative lysosomes following detection in the cytosol or within damaged vacuoles. Like L isteria and S higella , which use specific mechanisms to avoid autophagic detection and capture, the bacterial pathogen F rancisella tularensis proliferates within the cytosol of macrophages without demonstrable control by autophagy. To examine how F rancisella evades autophagy, we screened a library of F . tularensis subsp. tularensis   Schu S 4 HimarFT transposon mutants in GFP ‐ LC3 ‐expressing murine macrophages by microscopy for clones localized within autophagic vacuoles after phagosomal escape. Eleven clones showed autophagic capture at 6 h post‐infection, whose HimarFT insertions clustered to fourgenetic loci involved in lipopolysaccharidic and capsular O ‐antigen biosynthesis. Consistent with the HimarFT mutants, in‐frame deletion mutants of two representative loci, FTT 1236 and FTT 1448c ( manC ), lacking both LPS and capsular O ‐antigen, underwent phagosomal escape but were cleared from the host cytosol. Unlike wild‐type F rancisella , the O ‐antigen deletion mutants were ubiquitinated, and recruited the autophagy adaptor p62/ SQSTM1 and LC3 prior to cytosolic clearance. Autophagy‐deficient macrophages partially supported replication of both mutants, indicating that O ‐antigen‐lacking F rancisella are controlled by autophagy. These data demonstrate the intracellular protective role of this bacterial surface polysaccharide against autophagy.