Tse(VF)-Tsi(VF), a novel bacteriolytic effector-immunity pair of Vibrio fluvialis VflT6SS2, provides a fitness advantage in microbial competition.

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 antagonize 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 and 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.