Uncovering the Multifaceted Role of () in Type III Secretion System and Other Virulence Production in PAO1.

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 , a component of the NADH dehydrogenase complex I ( operon), as a key regulator of T3SS-related activities. deletion resulted in a twofold increase in expression and enhanced cytotoxicity in both A549 cell and Chinese cabbage models. Full revertant of the operon was necessary to restore expression to wild-type levels, suggesting a critical connection between NADH dehydrogenase activity and T3SS regulation. The mutation also disrupted the Rsm-Exs regulatory axis, downregulating , , , and while upregulating . Overexpression of , , , , and partially rescued T3SS function, confirming that influences T3SS via the Rsm-Exs pathway. Furthermore, 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 modulates these traits through mechanisms independent of the Rsm-Exs axis, potentially involving NADH dehydrogenase-associated pathways. This study underscores the multifaceted role of in regulating pathogenicity and resistance, providing novel insights into its complex regulatory networks and highlighting new avenues for therapeutic targeting.