Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems

Abstract

Contact-dependent growth inhibition (CDI) systems encode polymorphic toxin/immunity proteins that mediate competition between neighboring bacterial cells. We present crystal structures of CDI toxin/ immunity complexes from Escherichia coli EC869 and Burkholderia pseudomallei 1026b. Despite sharing little sequence identity, the toxin domains are structurally similar and have homology to endonucleases. The EC869 toxin is a Zn 2+ -dependent DNase capable of completely degrading the genomes of target cells, whereas the Bp1026b toxin cleaves the aminoacyl acceptor stems of tRNA molecules. Each immunity protein binds and inactivates its cognate toxin in a unique manner. The EC869 toxin/immunity complex is stabilized through an unusual β-augmentation interaction. In contrast, the Bp1026b immunity protein exploits shape and charge complementarity to occlude the toxin active site. These structures represent the initial glimpse into the CDI toxin/immunity network, illustrating how sequence-diverse toxins adopt convergent folds yet retain distinct binding interactions with cognate immunity proteins. Moreover, we present visual demonstration of CDI toxin delivery into a target cell.structural biology | bacterial competition | β-complementation | tRNase activity

Unraveling the essential role of CysK in CDI toxin activation

“…Thus, the toxin does not induce the "closed" conformation observed when CysK contains substrate covalently bound to PLP in the active site (19). As we have found in other CdiA-CT toxin structures (5,6,20), only the C-terminal nuclease domain (residues Lys127-Ile227) is resolved in the final model. The CdiA-CT EC536 nuclease domain consists of four α-helices.…”

“…Protein crystallization was previously described (6). Briefly, crystals were grown by the hanging-drop, vapor-diffusion method at room temperature against a reservoir containing 0.2 M NaSO 4 , 0.1 M Bis-Tris propane (pH 7.9), and 20% (wt/vol) PEG 3350, for the binary complex, and 0.1 M sodium cacodylate (pH 7.1), 0.2 M ammonium sulfate, and 17% (wt/vol) PEG-8000, for the ternary complex.…”

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors

“…Because CdiA-CT EC869 cleaves near the 3′-end of tRNA CUG Gln , we hypothesize that EFTu positions the toxin active site near the scissile bond. However, EF-Tu is not necessary for such activity, because other CDI toxins cleave tRNA acceptor stems independently of the translation factor (5,15). One intriguing explanation is that toxin·EF-Tu interactions serve additional roles in intercellular signaling.…”

“…The duf-cdiA-CT/cdiI EC869 fragment was amplified from pCH10525 with CH2324/CH3834 and ligated to plasmid pCH6243 using SpeI/XhoI sites. The cdiA-CT/cdiI modules from E. coli 96.154 and O32:H37 were amplified with primers CH3170/CH3171 and CH3567/CH3568 (respectively) and combined with cdiA EC93 fragments amplified with 1527/2470 and 1663/2368 by overlapping-end PCR (15). The final products were electroporated together with plasmid pCH10163 into E. coli strain DY378 and recombinant plasmids selected as described (15).…”

“…CdiA-CT K96243 has anticodon nuclease activity on tRNA His , tRNA Asp , tRNA Asn , and tRNA Tyr isoacceptors, and CdiA-CT E479 cleaves the T-loop of tRNA molecules between conserved residues Ψ54 and T55 (13,14). CdiA-CT II Bp1026b preferentially cleaves within the aminoacyl acceptor stem of tRNA Ala to block translation (15). Other unrelated CdiA-CT toxins from E. coli isolates EC869 and 3006 also cleave tRNA acceptor stems but are specific for tRNA Gln and tRNA Ile , respectively (5,16).…”