Abstract Pseudomonas aeruginosa is a major nosocomial pathogen in which the type VI secretion system (T6SS) contributes to interbacterial competition and virulence. While most strains encode three T6SSs, additional T6SS clusters have been identified in clinical isolates through comparative genomics, but their functions and effector biology remain undefined. Here, we identify TseMt as a major antibacterial effector associated with an H4‐T6SS in a clinical P. aeruginosa isolate LYSZa7. TseMt is a periplasmically active toxin whose activity is neutralized by a cognate immunity protein, TsiMt. Biochemical assays show that TseMt binds membranes and forms ion‐conducting pores, establishing it as a pore‐forming effector. A 3.0 Å cryo‐electron microscopy structure reveals a distinct three‐domain architecture comprising an N‐terminal MIX‐like domain, a central α‐helical scaffold, and a C‐terminal toxin domain. Genetic analysis and structural modeling indicate that TseMt is delivered through a dedicated PAAR−VgrG−chaperone pathway. Together, these findings define the structural basis, functional mechanism, and delivery pathway of the H4‐T6SS effector TseMt from a clinical P. aeruginosa isolate and reveal its role in mediating bacterial competition.
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.
Yizhou Zhang, T. Ye, Jie Deng et al.· International Journal of Bio...· 0 citations
