Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis.

Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.

Inhibition of p-IκBα Ubiquitylation by Autophagy-Related Gene 7 to Regulate Inflammatory Responses to Bacterial Infection.

BACKGROUND: Klebsiella pneumoniae causes serious infections and healthcare burdens in humans. We have previously reported that the deficiency of autophagy-related gene (Atg) 7 in macrophages (murine alveolar macrophage cell line [MH-S]) induced irregular host immunity against K. pneumoniae and worsened pathologic effects in the lung. In the current study, we investigated the molecular mechanism by which Atg7 influenced K. pneumoniae-induced inflammatory responses. METHODS: Expression levels of Atg7, ubiquitin (Ub), and tumor necrosis factor (TNF) α and phosphorylation of IκBα (p-IκBα) were determined with immunoblotting. Ubiquitylation of p-IκBα was determined with immunoprecipitation. RESULTS: We noted an interaction between Atg7 and p-IκBα, which was decreased in MH-S after K. pneumoniae infection, whereas the interaction between Ub and p-IκBα was increased. Knock-down of Atg7 with small interfering RNA increased p-IκBα ubiquitylation, promoted nuclear factor κB translocation into the nucleus, and increased the production of TNF-α. Moreover, knock-down of Ub with lentivirus-short hairpin RNA Ub particles decreased binding of p-IκBα to Ub and inhibited TNF-α expression in the primary alveolar macrophages and lung tissue of atg7-knockout mice on K. pneumoniae infection. CONCLUSIONS: Loss of Atg7 switched binding of p-IκBα from Atg7 to Ub, resulting in increased ubiquitylation of p-IκBα and intensified inflammatory responses against K. pneumoniae. Our findings not only reveal a regulatory role of Atg7 in ubiquitylation of p-IκBα but also indicate potential therapeutic targets for K. pneumoniae control.