Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI)

Abstract

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI+ inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI+ cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK+ cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.

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

“…The critical role of EF-Ts in CdiA-CT EC869 toxicity appears to be similar to CdiA-CT EC536 , which only exhibits tRNA anticodon nuclease activity when bound to CysK (17,18). To determine whether EF-Ts activates CdiA-CT EC869 in the same manner, we tested CdiA-CT EC869 activity in defined in vitro reactions.…”

“…Most CDI nuclease domains efficiently cleave their substrates in vitro, but the CdiA-CT EC536 toxin deployed by uropathogenic E. coli 536 requires an additional factor to promote its tRNA anticodon nuclease activity (17). Using biochemical approaches, we discovered that the biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK) binds the toxin with high affinity and stimulates its nuclease activity.…”

“…Using biochemical approaches, we discovered that the biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK) binds the toxin with high affinity and stimulates its nuclease activity. This interaction is critical for toxin activity, and target bacteria deleted for cysK are fully resistant to CdiA-CT EC536 toxin (17). Because cysK mutations confer CDIresistance (CDI R ) to target bacteria, the advantage of an additional toxin-activation step is not clear.…”

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation ofO-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

“…Indeed, it has been demonstrated that deletion of genes belonging to the cysteine biosynthetic pathway leads to a bacterial phenotype with reduced virulence, compromised fitness and a susceptibility to antibiotics several fold higher than the wild strains 8,9 . The recent disclosure of a number of additional moonlighting activities of OASS, specifically for OASS-A isoform, that is, toxin activation in some strains of Escherichia coli (E. coli) 10 , gene expression in Bacillus subtilis (B. subtilis) 11 , and the involvement of the enzyme in some pathologically relevant processes as the formation of biofilm and swarming motility, has further focused the interest of researchers toward these proteins [11][12][13][14][15][16][17][18] . These activities span from toxin activation in contact-dependent growth inhibition of uropathogenic E. coli strains 10 , to gene expression in B. subtilis 11 and the involvement of the enzyme in some pathologically-relevant processes as the formation of biofilm and swarming motility 19 .…”

Unraveling the essential role of CysK in CDI toxin activation

“…The CdiA-CT EC536 region is composed of two domains that have distinct functions during CDI (9). The extreme C-terminal domain is an Ntox28 RNase family member (Pfam: PF15605) and is responsible for growth-inhibition activity (3,8). The N-terminal domain facilitates translocation of the tethered nuclease into the cytosol of target bacteria (9).…”