Essential role of cytochrome bc1 in Pseudomonas aeruginosa cell physiology and virulence

Pseudomonas aeruginosa is a multidrug-resistant Gram-negative nosocomial pathogen posing a major healthcare burden due to its intrinsic antibiotic resistance and environmental adaptability, which continue to significantly constrain therapeutic options. This pathogen is a leading cause of hospital-acquired respiratory and urinary tract infections that are often persistent and difficult to treat. The elucidation of metabolic adaptations permitting P. aeruginosa survival and supporting virulence in diverse host environments is essential for informing novel treatments. This study investigates the role of cytochrome bc 1 in P. aeruginosa PA14 growth, energetics, and virulence in vitro , under physiologically relevant conditions, and in vivo . The bc 1 deletion mutant exhibits significant growth defects in urinary- and lung-like media, demonstrating its importance in PA14 physiology, and displays reduced virulence in bladder epithelial cell monolayers, mediated by decreased type III secretion system (T3SS) toxin expression. In vivo , the mutant was nearly avirulent in a murine systemic infection model. Bioenergetic analysis revealed severe decline in PA14 Δ bc 1 ATP production, aligning with the enzyme’s central role in respiration and providing a mechanistic explanation for the mutant’s reduced T3SS function, as toxin secretion requires energy. This work provides compelling evidence of direct T3SS regulation by cytochrome bc 1 , revealing a previously unrecognized link between respiratory metabolism and virulence. Metabolic profiling indicates that to compensate for cytochrome bc 1 loss, the mutant relies on NDH-2 and bd oxidase, permitting cellular survival but substantially reducing ATP yield. Collectively, these findings establish cytochrome bc 1 as a target for antibiotic development against P. aeruginosa , impacting bioenergetics, physiology, and virulence.