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

Pseudomonas aeruginosa 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 PA2649, a component of the NADH dehydrogenase complex I (nuo operon), as a key regulator of T3SS-related activities. PA2649 deletion resulted in a twofold increase in exoS expression and enhanced cytotoxicity in both A549 cell and Chinese cabbage models. Full revertant of the nuo operon was necessary to restore exoS expression to wild-type levels, suggesting a critical connection between NADH dehydrogenase activity and T3SS regulation. The PA2649 mutation also disrupted the Rsm-Exs regulatory axis, downregulating gacS, rsmY, rsmZ, and hfq while upregulating exsC. Overexpression of rsmY, rsmZ, gacA, hfq, and exsD partially rescued T3SS function, confirming that PA2649 influences T3SS via the Rsm-Exs pathway. Furthermore, PA2649 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 PA2649 modulates these traits through mechanisms independent of the Rsm-Exs axis, potentially involving NADH dehydrogenase-associated pathways. This study underscores the multifaceted role of PA2649 in regulating P. aeruginosa pathogenicity and resistance, providing novel insights into its complex regulatory networks and highlighting new avenues for therapeutic targeting.