UC Irvine

Bacteria have developed complex mechanisms to survive and propagate within their environments. Contact-dependent growth inhibition (CDI) is a recently discovered mechanism of inter-bacterial competition and communication widespread amongst Gram-negative bacteria. CDI+ cells inhibit the growth of neighboring cells upon delivery of a toxic protein (CdiA-CT) into the target cell cytosol. To prevent auto-inhibition, CDI+ bacteria express an immunity protein (CdiI) to specifically bind and inactivate cognate toxin. Here we provide further structural characterization of CDI toxin/immunity proteins to elucidate the diversity of CdiA-CT toxin activity, their potential activation, and neutralization to repurpose CDI systems for novel antimicrobials.

Burkholderia pseudomallei isolates E479 and 1026b have been shown to utilize CDI to outcompete neighboring bacteria, signifying the pathway’s importance in growth and survival. The CdiA-CT toxins from these isolates are functional tRNases, yet have unique tRNA specificities and cleavage sites despite sharing no sequence similarity. We have solved the X-ray crystal structure of the B. pseudomallei E479 toxin/immunity protein complex and compared it to the previously solved 1026b complex. While the toxins are sequentially and functionally different, they share significant structural homology with nearly identical active sites, only differing in active site pocket size, correlating with the differing substrate cleavage sites of the two toxins. The toxin/tRNA interactions have been confirmed via molecular docking as well as small angle X-ray scattering.

The CDI system from uropathogenic E. coli 536 (UPEC536) is unusual, CdiA-CT exhibits toxic tRNase activity only when bound to target cell CysK, termed a “permissive factor”, involved in cysteine biosynthesis. We have solved the X-ray crystal structure of the CysK/toxin/immunity ternary complex. The UPEC536 toxin inserts its C-terminus into the active site cleft of CysK, mimicking its natural interaction with CysE. A homologous toxin from the Gram-positive Ruminococcus lactaris has been identified to perform the same enzymatic function in a CysK-independent manner. Characterization of this toxin has shown that it’s significantly more thermostable than the UPEC toxin, and is capable of binding tRNA substrate in the absence of CysK. This demonstrates a chaperone-like function for CysK, stabilizing the UPEC toxin fold and promoting its association with tRNA substrate.