Uncovering the Multifaceted Role of PA2649 (nuoN) in Type III Secretion System and Other Virulence Production in Pseudomonas aeruginosa PAO1

<i>Pseudomonas aeruginosa</i> is a multi-drug-resistant opportunistic pathogen that adapts to challenging environments by deploying virulence factors, including the type III secretion system (T3SS). Emerging evidence points to a role for NADH dehydrogenase complexes in regulating virulence; however, their precise contributions remain unclear. Here, we identify <i>PA2649</i>, a component of the NADH dehydrogenase complex I (<i>nuo</i> operon), as a key regulator of T3SS-related activities. <i>PA2649</i> deletion resulted in a twofold increase in <i>exoS</i> expression and enhanced cytotoxicity in both A549 cell and Chinese cabbage models. Full revertant of the <i>nuo</i> operon was necessary to restore <i>exoS</i> expression to wild-type levels, suggesting a critical connection between NADH dehydrogenase activity and T3SS regulation. The <i>PA2649</i> mutation also disrupted the Rsm-Exs regulatory axis, downregulating <i>gacS</i>, <i>rsmY</i>, <i>rsmZ</i>, and <i>hfq</i> while upregulating <i>exsC</i>. Overexpression of <i>rsmY</i>, <i>rsmZ</i>, <i>gacA</i>, <i>hfq</i>, and <i>exsD</i> partially rescued T3SS function, confirming that <i>PA2649</i> influences T3SS via the Rsm-Exs pathway. Furthermore, <i>PA2649</i> deletion altered motility, biofilm formation, pyocyanin production, protease activity, and antibiotic susceptibility. These phenotypes could not be complemented with T3SS regulatory genes alone, indicating that <i>PA2649</i> modulates these traits through mechanisms independent of the Rsm-Exs axis, potentially involving NADH dehydrogenase-associated pathways. This study underscores the multifaceted role of <i>PA2649</i> in regulating <i>P. aeruginosa</i> pathogenicity and resistance, providing novel insights into its complex regulatory networks and highlighting new avenues for therapeutic targeting.