The Putative RNA Methyltransferase Modulates T3SS Expression and Host NF-κB Activation via T6SS-Mediated Translocation in Pseudomonas aeruginosa

RNA methyltransferases are key regulators of bacterial physiology, yet their specific roles in virulence remain poorly defined. In this study, we characterize PA3840, a putative RNA methyltransferase in <i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>). Deletion of PA3840 specifically impaired twitching motility without affecting bacterial growth, swimming, or swarming. Notably, PA3840 was found to suppress the expression of Type III Secretion System (T3SS) genes, thereby reducing cytotoxicity and host cell rounding. Consistent with these observations, PA3840 expression attenuated pro-inflammatory cytokine production in epithelial cells by inhibiting NF-κB activation. Mechanistic analysis revealed that PA3840 is translocated into host cells in a Type VI Secretion System (T6SS)-dependent manner. This translocation was reduced by <i>hcp1</i> deletion and nearly abolished by a double deletion of <i>pscF</i> and <i>hcp3</i>, suggesting the involvement of multiple T6SS components and potential interplay with T3SS machinery. However, direct transfection of PA3840 into host cells failed to suppress cytokine expression, indicating that its immunomodulatory function is mediated by a bacterium-intrinsic mechanism rather than direct intracellular action. Collectively, these findings identify PA3840 as a translocated effector that modulates twitching motility and dampens host inflammation by repressing T3SS and NF-κB signaling, revealing a novel layer of post-transcriptional virulence regulation in <i>P. aeruginosa</i>.