Competitive Binding of Pseudomonas aeruginosa to Kelch-Like ECH-Associated Protein 1 Inhibits Nuclear Factor Erythroid 2-Related Factor 2 Ubiquitination and Suppresses Ferroptosis in Macrophages

Abstract

The survival of Pseudomonas aeruginosa (PA) is a major factor in causing chronic or acute lung infections in individuals with compromised immune systems. Being the initial line of defense against infections, macrophages use a variety of tactics to fight intracellular bacteria, which are intimately linked to ferroptosis. It is yet unknown, nevertheless, what function ferroptosis serves in PA-infected macrophages. Initially, we established a macrophage infection model with PA to investigate the infection levels and duration using Cell Counting Kit-8 (CCK-8) and fluorescence microscopy and assessed the intracellular quantity of PA by counting colony forming units (CFUs). Subsequently, changes in ferroptosis-related characteristics in macrophages infected with PA were detected through quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot analysis, and fluorescence probes. Furthermore, the relationship between PA infection and ferroptosis in macrophages, as well as the specific mechanism regulating nuclear factor erythroid 2-related factor 2 (NRF2) protein stability, was validated by constructing NRF2 knockdown cells. Finally, the binding of PA to Kelch-like ECH-associated protein 1 (KEAP1) in macrophages was detected using Co-Immunoprecipitation (Co-IP) and protein thermal stability analysis. Under optimal conditions (multiplicity of infection (MOI) = 15:1, t = 72 h), it was demonstrated that macrophages infected with PA resisted ferroptosis, as confirmed by ferroptosis-related assays. Subsequent construction of NRF2 knockdown cells showed that PA-mediated resistance of macrophage ferroptosis depended on NRF2. Mechanistically, it was proved that PA stabilized NRF2 protein expression by inhibiting ubiquitin-proteasome-mediated protein degradation and competitively binding to KEAP1. In conclusion, this study demonstrated that PA stabilized NRF2 protein expression in macrophages, inducing resistance to ferroptosis through the ubiquitin-proteasome pathway and competitive binding to KEAP1.

Keywords: KEAP1; NRF2; Pseudomonas aeruginosa; ferroptosis; macrophages.