The transcriptional regulator Sfa4 controls H4-T6SS expression and virulence in Pseudomonas aeruginosa through FleQ and c-di-GMP signaling.
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 Sfa4, a transcriptional regulator linked to a fourth T6SS (H4-T6SS) in clinical isolate LYSZa7. Sfa4 directly binds amrZ and H4-T6SS cluster to activate their transcription. AmrZ, in turn, directly regulates all four T6SS clusters. We further show that c-di-GMP receptor FleQ directly binds the promoters of all four T6SS clusters, revealing a direct regulatory link between c-di-GMP signaling and T6SS transcription. This regulation, together with Sfa4-mediated elevation of intracellular c-di-GMP levels, coordinately enhances H4-T6SS activity, biofilm formation, and virulence in A549 alveolar epithelial cells and Galleria mellonella models. Phylogenetic analysis shows Sfa4 homologs are present in Gram-negative bacteria, implying a potential T6SS-regulatory function. Collectively, our findings shed light on regulatory cascades and provide a mechanistic basis for understanding how clinically acquired T6SS clusters may be integrated into existing virulence networks.