Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.

Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to compete with neighboring cells. CDI⁺ strains express cell-surface CdiA effector proteins, which carry a toxic C-terminal region (CdiA-CT) that is cleaved from the effector upon transfer into the periplasm of target bacteria. The released CdiA-CT consists of two domains. The C-terminal domain is typically a nuclease that inhibits cell growth, and the N-terminal "cytoplasm-entry" domain mediates toxin translocation into the target-cell cytosol. Here, we use NMR and circular dichroism spectroscopic approaches to probe the structure, stability, and dynamics of the cytoplasm-entry domain from Escherichia coli STEC_MHI813. Chemical shift analysis reveals that the CdiA-CT^MHI813 entry domain is composed of a C-terminal helical bundle and a dynamic N-terminal region containing two disulfide linkages. Disruption of the disulfides by mutagenesis or chemical reduction destabilizes secondary structure over the N-terminus, but has no effect on the C-terminal helices. Although critical for N-terminal structure, the disulfides have only modest effects on global thermodynamic stability, and the entry domain exhibits characteristics of a molten globule. We find that the disulfides form in vivo as the entry domain dwells in the periplasm of inhibitor cells prior to target-cell recognition. CdiA-CT^MHI813 variants lacking either disulfide still kill target bacteria, but disruption of both bonds abrogates growth inhibition activity. We propose that the entry domain's dynamic structural features are critical for function. In its molten globule-like state, the domain resists degradation after delivery, yet remains pliable enough to unfold for membrane translocation.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition. CDI(+) bacteria deploy large CdiA effector proteins, which carry variable C-terminal toxin domains (CdiA-CT). CDI(+) cells also produce CdiI immunity proteins that specifically neutralize cognate CdiA-CT toxins to prevent auto-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(E479) toxin/immunity protein complex from Burkholderia pseudomallei isolate E479. The CdiA-CT(E479) tRNase domain contains a core α/β-fold that is characteristic of PD(D/E)XK superfamily nucleases. Unexpectedly, the closest structural homolog of CdiA-CT(E479) is another CDI toxin domain from B. pseudomallei 1026b. Although unrelated in sequence, the two B. pseudomallei nuclease domains share similar folds and active-site architectures. By contrast, the CdiI(E479) and CdiI(1026b) immunity proteins share no significant sequence or structural homology. CdiA-CT(E479) and CdiA-CT(1026b) are both tRNases; however, each nuclease cleaves tRNA at a distinct position. We used a molecular docking approach to model each toxin bound to tRNA substrate. The resulting models fit into electron density envelopes generated by small-angle x-ray scattering analysis of catalytically inactive toxin domains bound stably to tRNA. CdiA-CT(E479) is the third CDI toxin found to have structural homology to the PD(D/E)XK superfamily. We propose that CDI systems exploit the inherent sequence variability and active-site plasticity of PD(D/E)XK nucleases to generate toxin diversity. These findings raise the possibility that many other uncharacterized CDI toxins may belong to the PD(D/E)XK superfamily.

Contact-dependent growth inhibition (CDI) is a form of interbacterial competition mediated by CdiB-CdiA two-partner secretion systems. CdiA effector proteins carry polymorphic C-terminal toxin domains (CdiA-CT), which are neutralized by specific CdiI immunity proteins to prevent self-inhibition. Here, we present the crystal structures of CdiA-CT⋅CdiI complexes from Klebsiella pneumoniae 342 and Escherichia coli 3006. The toxins adopt related folds that resemble the ribonuclease domain of colicin D, and both are isoacceptor-specific tRNases that cleave the acceptor stem of deacylated tRNA_GAU^Ile. Although the toxins are similar in structure and substrate specificity, CdiA-CT^Kp342 activity requires translation factors EF-Tu and EF-Ts, whereas CdiA-CT^EC3006 is intrinsically active. Furthermore, the corresponding immunity proteins are unrelated in sequence and structure. CdiI^Kp342 forms a dimeric β sandwich, whereas CdiI^EC3006 is an α-solenoid monomer. Given that toxin-immunity genes co-evolve as linked pairs, these observations suggest that the similarities in toxin structure and activity reflect functional convergence.

Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Together, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.