The c-di-GMP-dependent transcriptional regulator Sfa4 controls H4-T6SS expression and virulence in Pseudomonas aeruginosa

The Type VI secretion system (T6SS) is a key nanoweapon in Gram-negative bacteria that mediates microbial competition and pathogenesis via toxic effector delivery. Three functionally distinct T6SS clusters (H1-H3) are known in Pseudomonas aeruginosa, yet the broader evolutionary diversity and regulatory networks of T6SS in this pathogen remain poorly defined. Here, we identify TsrF, a transcriptional regulator linked to a novel fourth T6SS (H4-T6SS) in clinical isolate LYSZa7. TsrF directly binds the amrZ and H4-T6SS cluster to activate their transcription. AmrZ, in turn, directly regulates all four T6SS clusters. TsrF also elevates intracellular c-di-GMP levels, which via FleQ coordinately enhance H4-T6SS activity, biofilm formation, and virulence in A549 alveolar epithelial cells and Galleria mellonella models. Phylogenetic analysis shows TsrF homologs are widespread in Gram-negative bacteria, implying conserved T6SS-regulatory functions. Collectively, our findings shed new light on T6SS diversity and regulatory cascades, and highlight the adaptive benefits of novel T6SS operons in bacterial pathogens.