TseVF-TsiVF, a novel bacteriolytic effector-immunity pair of Vibrio fluvialis VflT6SS2, provides a fitness advantage in microbial competition

• T6SS effector TseVF demonstrates apparent bacterial killing activity in the periplasm. • TseVF interacts with phosphate lipid molecules and compromises the integrity of the cell membrane. • The C-terminal region of TseVF is responsible for its bactericidal activity. 4. TsiVF binds to and neutralizes TseVF toxicity. • TseVF secretion and bactericidality requires the C terminus of a cognate VgrG protein (TssI2_b). Vibrio fluvialis ( V. fluvialis ) is a halophilic Gram-negative bacterium regarded as an emerging pathogen that can cause severe cholera-like diarrhea and various extraintestinal infections. Type VI secretion systems (T6SSs) are nano-weapons used by bacteria to deliver toxic effectors into host cells or antagonizing competitors. VflT6SS2 of V. fluvialis is involved in bacterial pathogenicity and competitive environmental survival. Previously, we have identified some regulatory factors of VflT6SS2 and a pesticin domain-containing effector, TssI2. Here we reported a novel effector-immunity pair of VflT6SS2, namely Tse VF -Tsi VF , the homologs of which are widely distributed in Vibrio . Tse VF mediates killing effect toward co-existing bacteria while Tsi VF confers protection against Tse VF . Tse VF biologically functions in the periplasmic space and its C-terminal region (residues 965-1141), which adopts a helix-turn-helix fold composed of eight helices fully accounts for the bactericidal activity. Periplasmic expression of Tse VF965-1141 compromises the integrity of the cell membrane, leading to the leakage of cellular contents, and two highly conserved residues W1056 and W1091, are important for the bactericidality. The membrane-disrupting function of Tse VF was further supported by its interaction with phosphate lipid phosphatidylethanolamine and phosphocholine. The immunity Tsi VF directly binds to and neutralizes Tse VF toxicity through its TPR-like domain. The C terminus of TssI2_b specifically mediates the loading or secretion of Tse VF . In conclusion, our study identified a new class of T6SS pore-forming effector-immunity pair Tse VF -Tsi VF and revealed its molecular function and secretion mechanism in V. fluvialis , further enhancing our understanding of the diverse effectors of T6SS.