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

<i>Pseudomonas aeruginosa</i> 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 <i>P. aeruginosa</i> survival and supporting virulence in diverse host environments is essential for informing novel treatments. This study investigates the role of cytochrome <i>bc</i> ₁ in <i>P. aeruginosa</i> PA14 growth, energetics, and virulence <i>in vitro</i>, under physiologically relevant conditions, and <i>in vivo</i>. The <i>bc</i> 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. <i>In vivo</i>, the mutant was nearly avirulent in a murine systemic infection model. Bioenergetic analysis revealed severe decline in PA14 Δ<i>bc</i> 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 <i>bc</i> ₁, revealing a previously unrecognized link between respiratory metabolism and virulence. Metabolic profiling indicates that to compensate for cytochrome <i>bc</i> ₁ loss, the mutant relies on NDH-2 and <i>bd</i> oxidase, permitting cellular survival but substantially reducing ATP yield. Collectively, these findings establish cytochrome <i>bc</i> ₁ as a target for antibiotic development against <i>P. aeruginosa</i>, impacting bioenergetics, physiology, and virulence.