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

Abstract

Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describe tsf mutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CT EC869 tRNase toxin from enterohemorrhagic Escherichia coli EC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CT EC869 binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CT EC869 only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.cell communication | protein synthesis | toxin-immunity proteins | ribonuclease | bacterial competition

Three Distinct Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition by the Cystic Fibrosis Pathogen Burkholderia dolosa

“…Conversely, E. coli producing BcpA-1-CT did not exhibit reduced viability, despite the likelihood that this polypeptide contains the toxic domain of BcpA-1. For some E. coli-type CDI systems, cytoplasmic "permissive factors" are required for activity of a delivered toxin within a target cell (32,36,37). A requirement for a toxicity-promoting factor specific to Burkholderia, or to BdAU0158, may explain the lack of toxicity of BdAU0158 BcpA-1-CT in E. coli.…”

A High-Throughput Interbacterial Competition Screen Identifies ClpAP in Enhancing Recipient Susceptibility to Type VI Secretion System-Mediated Attack by Agrobacterium tumefaciens

“…In the CDI system, non-immunity proteins in the recipient cell also participate in the bacterial competition outcome (Aoki et al, 2008(Aoki et al, , 2010Diner et al, 2012;Willett et al, 2015;Jones et al, 2017). For example, the CDI effector CdiA-CT EC93 utilizes recipient's outer membrane protein BamA and the inner membrane protein AcrB to enter the recipient cell (Aoki et al, 2008).…”

“…In the recipient cell, CdiA-CT EC536 binds to CysK to increase its thermostability and its tRNase activity (Johnson et al, 2016). Interestingly, this CysK.CdiA-CT EC536 complex mimics the CysK.CysE complex, which is typically formed during de novo cysteine biogenesis, with a higher binding affinity (Johnson et al, 2016;Jones et al, 2017). Other examples are the recipient elongation factor Tu (EF-Tu) in activating the toxicity of CdiA-CT EC869 and CdiA-CT NC101 (Jones et al, 2017;Michalska et al, 2017), and the involvement of recipient PtsG in CdiA-CT 3006 and CdiA-CT NC101 entry (Willett et al, 2015).…”

Can't you hear me knocking: contact-dependent competition and cooperation in bacteria

“…In this model, the binding partner ensures that the toxin refolds efficiently after delivery into the cytoplasm. More recently, we have discovered that diverse CdiA-CT toxins from E. coli strains EC869, NC101 and 96.154 interact functionally with translation factors EF-Tu and EF-Ts (46). CdiA-CT EC869 specifically cleaves within the aminoacyl acceptor stem of tRNA Gln and tRNA Asn molecules and binds to EF-Tu with high affinity.…”

“…CdiA-CT EC869 specifically cleaves within the aminoacyl acceptor stem of tRNA Gln and tRNA Asn molecules and binds to EF-Tu with high affinity. Remarkably, the toxin only cleaves substrate in the context of tRNA•EF-Tu•GTP ternary complexes (46), suggesting that the interaction with EF-Tu is required for nuclease activity. Moreover, EF-Ts is required for toxin activity in vivo , though guanine nucleotide exchange activity per se is not required for the nuclease reaction.…”