Abstract Rationale Pseudomonas aeruginosa infection severely impairs ciliary defense in respiratory diseases by subverting mucociliary clearance, a primary host defense mechanism. Although inflammatory responses in airway epithelial cells (AECs) are well-studied, how microbial metabolic hijacking disrupts ciliary motility via epitranscriptomic reprogramming remains unknown. Methods We used integrated in vivo and in vitro models to define a lactylation-N6-methyladenosine (m6A) axis regulating ciliary function. Wild-type and conditional YTH domain family member 1 (YTHDF1) knockout (YTHDF1pc/pc shhcre+) C57BL/6 mice were intranasally challenged with P. aeruginosa PAO1 or lipopolysaccharide (LPS). Lung bacterial burden (colony-forming units, CFU), ciliary beat frequency (CBF) (high-speed video microscopy), mucociliary transport (fluorescent bead tracking), and mucus hypersecretion (periodic acid-Schiff, PAS) were quantified. In vitro, primary mouse tracheal epithelial cells (mTECs), MLE-12, and BEAS-2B cells were infected or treated with lactic acid, rotenone, or p300 inhibitor C646. Key assays included m6A-sequencing (m6A-seq), RNA immunoprecipitation-qPCR (RIP-qPCR), polysome profiling, histone H3K18 lactylation (H3K18la) immunoblot, and dynein axonemal heavy chain 5 (DNAH5) translation efficiency. Results P. aeruginosa depleted host lactic acid via lactate dehydrogenase-mediated consumption and virulence factor-induced glycolysis suppression, reducing H3K18la and downregulating YTHDF1. This triggered global m6A hypomethylation, with DNAH5 messenger RNA (mRNA) showing the most significant loss. YTHDF1 bound m6A-modified DNAH5 to enhance translation elongation and protein stability—critical for axonemal structure and ciliary motility. YTHDF1 deficiency abolished CBF, stalled mucociliary transport, and increased bacterial colonization and IL-1β-driven inflammation. A lactate dehydrogenase-deficient mutant (ΔlldDΔlldA) failed to suppress H3K18la/YTHDF1, preserving ciliary function. Exogenous lactic acid or mitochondrial complex I inhibition (rotenone) restored the axis, reversible by p300 blockade. Conclusions The lactylation-YTHDF1-DNAH5 axis is a central metabolic-epitranscriptomic checkpoint safeguarding ciliary beating and mucociliary clearance during P. aeruginosa infection. Microbial lactate scavenging reprogrammes RNA methylation to dismantle ciliary defense, enabling persistence. This pathway represents a novel therapeutic target to restore airway ciliary integrity in refractory bacterial lung infections. This abstract is funded by: NONE
Wang et al. (Fri,) studied this question.