Elongation Factor Tu Acts as a Chaperone to Activate an Antibacterial <scp>RNase</scp> Toxin

Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to deliver toxic proteins into neighboring competitors. CDI⁺ strains deploy CdiA effector proteins, which translocate their C-terminal toxin (CT) domains into target bacteria through a receptor-mediated delivery pathway. To protect against auto-intoxication, CDI⁺ bacteria also produce CdiI immunity proteins that neutralize CT toxin activity. Here, we present the crystal structure of the CT·CdiI^O32:H37 complex from Escherichia coli O32:H37. CT^O32:H37 adopts the same fold as the tRNase domain of colicin D, and the nucleases share similar catalytic centers. However, unlike colicin D, which cleaves the anticodon loops of tRNA^Arg isoacceptors, CT^O32:H37 exhibits nonspecific RNase activity. Notably, we find that endogenous elongation factor Tu (EF-Tu) co-purifies with the over-produced CT·CdiI^O32:H37 complex. Although EF-Tu does not bind stably to CT^O32:H37 in the absence of CdiI^O32:H37, the translation factor is required for toxic RNase activity in vitro. AlphaFold 3 modeling and site-directed mutagenesis indicate that CT^O32:H37 interacts with the N-terminal GTPase domain of EF-Tu. EF-Tu appears to stabilize residue Trp52 within the hydrophobic core of the toxin, which in turn supports the RNase active site through an unusual hydrogen-bonding interaction with the catalytic His67 residue. Thus, EF-Tu is hijacked as an essential co-factor to organize the toxin's catalytic center.