Background: Autophagy is essential for effective bacterial clearance, in which the E3 ubiquitin ligase beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) participates. Herein, investigation was performed regarding the role of BTRC in post-traumatic brain injury (TBI)-associated bacterial pneumonia, alongside elucidation of the downstream mechanisms involving Unc-51 like autophagy activating kinase 1 (ULK1).

Methods: Using post-TBI mouse models and primary alveolar macrophages (PAM), we examined the effects of Pseudomonas aeruginosa K-strain (PAK) infection and BTRC silencing on pulmonary inflammation and bacterial clearance. Inflammatory cytokines, lung pathology and PAM viability were analyzed. Bacterial clearance in lung tissues, as well as intracellular killing and phagocytosis in PAM, was evaluated by colony-forming assays. Expressions of autophagy-related markers were measured employing western blot and immunofluorescence. ULK1 ubiquitination was assessed via immunoprecipitation. To confirm the involvement of ULK1, experiments with both BTRC and ULK1 knockdown were performed.

Results: PAK infection impaired bacterial clearance, suppressed autophagy and increased inflammatory cytokine levels in both lung tissues and PAMs, while these pathological changes were significantly reversed by BTRC knockdown (p < 0.01). Mechanistically, PAK infection promoted ULK1 ubiquitination in PAMs, which was offset by BTRC silencing; however, ULK1 knockdown neutralized the effects of BTRC silencing, as indicated by repressed autophagy activity and bacterial clearance, and enhanced inflammatory responses (p < 0.05).

Conclusion: BTRC silencing alleviates PAK-induced bacterial pneumonia after TBI by enhancing macrophage-mediated bacterial clearance through inhibition of ULK1 ubiquitination and activation of autophagy, a process critically dependent on ULK1 stability.