Michetti, E.; Secli, V.; Pacello, F.; Iacovelli, F.; Mellini, M.; Scribani Rossi, C.; Rinaldo, S.; Cutruzzola, F.; Rampioni, G.; Ammendola, S.; Battistoni, A.

Host nutritional immunity restricts microbial growth by altering metal availability. In patients with cystic fibrosis (CF), microorganisms inhabiting the thick airway mucus experience severe nutrient limitation, particularly zinc (Zn) restriction, which plays a critical role in limiting lung colonization. However, Pseudomonas aeruginosa, a major contributor to morbidity and mortality in CF, employs multiple strategies to overcome Zn deficiency and persist within the airways. Understanding how P. aeruginosa adapts to Zn deprivation may facilitate the development of antimicrobial approaches targeting Zn homeostasis. In this study, we characterized the physiological and transcriptional adaptations that support P. aeruginosa survival under Zn-limited conditions. Transcriptomic analysis of a znuAzrmB double mutant unable to efficiently acquire Zn revealed widespread repression of pathways involved in central carbon metabolism, motility and virulence. Notably, Zn limitation promoted extensive respiratory remodeling, characterized by a shift toward anaerobic metabolism, induction of denitrification pathways, altered terminal oxidase expression, and reduced oxygen consumption. These metabolic changes correlated with decreased ATP production, altered membrane potential, and increased aminoglycoside tolerance. Furthermore, the Zn-starved mutant exhibited reduced production of quorum-sensing molecules, redox imbalance and altered oxidative stress responses. Many of these adaptations resemble those observed in P. aeruginosa isolated from CF sputum, suggesting convergence towards a common host-adapted physiological state. Collectively, these findings identify Zn starvation as a major driver of bacterial physiological remodeling in CF conditions and reveal a previously unrecognized link between Zn limitation, respiratory reprogramming, and the emergence of persistence-associated traits in P. aeruginosa.