Kingdom-wide Discovery of Type VI Secretion Effectors via AI Uncovers Three New Bacterial Toxins

The type VI secretion system (T6SS) is a molecular harpoon used by 19% of bacteria to inject toxic proteins into microbial or host cells. Despite its widespread distribution, many T6SS effector (T6Es) genes remain unidentified. Here, we developed an XGBoost classifier integrating 42 genetic and biochemical features to identify novel T6Es. Applying our model to 2,559 T6SS-encoding bacterial genomes revealed 19,958 T6E candidates. We validated our computational predictions and confirmed that three effector candidates, having new 3D structures, demonstrated cytotoxicity to bacteria and yeast, mostly in the bacterial periplasm. For two of these, cognate immunity proteins neutralizing toxicity have been validated. T6EC11, a new peptidoglycan degrading effector, quickly leads to spherical E. coli, with inner membrane separation, and gradual peptidoglycan degradation in a unipolar manner, demonstrating an intriguing moon-like structural phenotype. T6EC12, an effector toxic to bacteria and yeast, carries a conserved zinc-binding motif. Mutations the motif markedly reduced toxicity, suggesting that T6EC12 represents a novel metalloprotease. Our study expands the known T6E repertoire, and provides thousands of T6E predictions in all T6SS-encoding bacteria.