Nhan, D. Q. | Search bioRxiv

bioRxiv2026

Redox regulated auto-processing controls delivery of an antibacterial cysteine peptidase toxin

Contact-dependent growth inhibition (CDI) is a mechanism of inter-bacterial competition mediated by CdiA effectors, which deliver polymorphic C-terminal toxins (CT) into neighboring competitors. StbD from Citrobacter rodentium DBS100 is an unusual CdiA-like protein that carries a C-terminal cysteine peptidase toxin. Crystallography reveals that StbD-CT is composed of an N-terminal cytoplasm-entry domain connected to a C39 family peptidase by a flexible linker. The entry domain hijacks membrane-embedded YajC for translocation into the target-cell cytosol where the peptidase inactivates type II topoisomerases. Intoxication leads to a loss of DNA super-helicity, impaired chromosome segregation and cell filamentation. In addition to cleaving topoisomerases, StbD-CT exhibits auto-proteolytic processing under reducing conditions, and this activity is required for target cell intoxication. We propose that StbD-CT remains tethered to the cell periphery via interactions with YajC after delivery. Auto-processing releases the peptidase, enabling the domain to penetrate into the cell interior where it cleaves nucleoid-associated topoisomerases. Together, these findings identify a proteolytic effector that deactivates type II topoisomerases and reveal a redox regulatory strategy that coordinates toxin activation with intercellular delivery.

microbiology↗

bioRxiv2023

Paradoxical activation of a type VI secretion system (T6SS) phospholipase effector by its cognate immunity protein

Type VI secretion systems (T6SS) 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 auto-permeabilization through unopposed activity of the Tle phospholipase effector. This hyper-permeability 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 hyper-permeability because {Delta}tli 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 hyper-permeable 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 {beta}-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.

microbiology↗