TheFrancisellaO-antigen mediates survival in the macrophage cytosol via autophagy avoidance

Abstract

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 Listeria and Shigella, which use specific mechanisms to avoid autophagic detection and capture, the bacterial pathogen Francisella tularensis proliferates within the cytosol of macrophages without demonstrable control by autophagy. To examine how Francisella evades autophagy, we screened a library of F. tularensis subsp. tularensis Schu S4 HimarFT transposon mutants in GFP-LC3-expressing murine macrophages by microscopy for clones localised within autophagic vacuoles after phagosomal escape. Eleven clones showed autophagic capture at six hours post-infection, whose HimarFT insertions clustered to four genetic loci involved in lipopolysaccharidic and capsular O-antigen biosynthesis. Consistent with the HimarFT mutants, in-frame deletion mutants of two representative loci, FTT1236 and FTT1448c (manC), lacking both LPS and capsular O-antigen, underwent phagosomal escape but were cleared from the host cytosol. Unlike wild type Francisella, 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 Francisella are controlled by autophagy. These data demonstrate the intracellular protective role of this bacterial surface polysaccharide against autophagy.

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

“…In chemically fixed samples, bacteria, both phagosomal and cytosolic, were surrounded with electron-translucent areas, so-called halos (see Fig. S4A and B in the supplemental material), which were previously suggested to represent a bacterial capsule (17,22). However, in cryofixed samples, we could observe an electron-pale area only around cytosolic bacteria (Fig.…”

“…noatunensis confirm that Francisella bacteria are indeed extremely polymorphic and indicate that the electron-translucent area around bacteria in chemically fixed samples is a fixation artifact. For chemically fixed F. tularensis in macrophages, a similar zone has been suggested to represent a capsule made of the polysaccharide O antigen (22). Nevertheless, in HPF samples, cytosolic bacteria were surrounded with an electron-pale area, which appeared to contain weakly contrasted material.…”

“…Inside the macrophage cytosol, Francisella has been shown to be targeted for autophagic degradation by ubiquitination and recruitment of p62 (21); however, the role of autophagy in Francisella infections is still disputed and varies between host cell models. In human macrophages, F. tularensis avoids autophagic degradation (21)(22)(23) but is able to exploit the autophagic process by harvesting amino acids from degraded material (24). In murine macrophages, bacteria can reenter autophagic vacuoles after cytosolic replication, which has been suggested to help bacterial egress (25).…”

Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines

“…Avoiding autophagy is proposed as one of the immune evasion mechanisms adopted by F. tularensis (68). Studies conducted with M. tuberculosis have shown that ROS is required for autophagy-mediated elimination of intracellular BCG.…”

Bordetella parapertussis Survives inside Human Macrophages in Lipid Raft-Enriched Phagosomes

“…Since our results revealed an involvement of the O side chain in bacterial survival upon macrophage phagocytosis, we next investigated whether the O antigen affects intracellular trafficking to avoid bactericidal mechanisms, as found before for other bacterial pathogens (32)(33)(34). To this end, bacterial colocalization with the acidotropic dye LysoTracker was investigated at different time points postinfection.…”