Paradoxical Activation of a Type VI Secretion System Phospholipase Effector by Its Cognate Immunity Protein

Type VI secretion systems (T6SSs) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce autopermeabilization through unopposed activity of the Tle phospholipase effector. This hyperpermeability phenotype is T6SS dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyperpermeability because Δtli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyperpermeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG β-spike protein. Together, these findings indicate that Tli has distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export. IMPORTANCE Gram-negative bacteria use type VI secretion systems deliver toxic effector proteins directly into neighboring competitors. Secreting cells also produce specific immunity proteins that neutralize effector activities to prevent autointoxication. Here, we show the Tli immunity protein of Enterobacter cloacae has two distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to block Tle lipase effector activity, while cytoplasmic Tli is required to activate the lipase prior to export. These results indicate Tle interacts transiently with its cognate immunity protein to promote effector protein folding and/or packaging into the secretion apparatus.