Probing Structure-Function Relationships in the Yoeb Toxin From the Yefm-Yoeb Toxin-Antitoxin System in Escherichia Coli

Abstract

The emergence and dissemination of antibiotic-resistant bacteria are serious health threats. Certain pathogenic isolates of both Gram-negative and Gram-positive species are resistant to multiple antibiotics and cause infections that are increasingly difficult to treat. Investigation of alternative and innovative strategies is required urgently to develop novel therapies that combat infections of antibiotic-resistant species. Bacterial toxin-antitoxin (TA) systems are modules that typically consist of a pair of genes that encode for a stable toxin protein and an unstable cognate antitoxin molecule. TAs are abundant on plasmids and chromosomes, including multi-resistant pathogens. To date, six types of TAs are known in which the antitoxins use different mechanisms, typically by the formation of a non-toxic TA complexes, to neutralize the cognate toxins under normal growth conditions. However, the unstable antitoxins are rapidly degraded under stress conditions to liberate the toxins to exert intracellular damage. The toxins target a range of processes, including the translation machinery, DNA replication, cell division and membrane integrity, thereby causing reversible effects that dampen metabolism and cell growth. The YefM-YoeB complex is one of the most widespread and best-studied TA modules. Intriguingly, the complex is found in numerous species, including Escherichia coli as well as in multidrug resistant human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium tuberculosis. Artificial activation of the YoeB endoribonuclease toxin is a potential approach to promote bacterial suicide as a novel antibacterial strategy. This study explored structure-function relationships in the YoeB toxin of E. coli. First, the effects of random pentapeptide insertions on YoeB toxicity and the interaction with the YefM antitoxin were determined in vivo. Regions of the toxin that either were tolerant or intolerant of insertions were identified. Peptide insertions within loop regions of the protein generally were more innocuous for both toxicity and the YefM interaction than insertions in secondary structure elements. Insertions that specifically impaired the antitoxin interaction but not YoeB toxicity also were identified. Second, extended insertions were introduced within permissive loops in YoeB. Insertions at these positions were designed that increased the length of the protein as much as two-fold without impairing toxicity, but which inhibited the interaction with the antitoxin. These lengthy insertions are predicted to project away from the protein core and from residues that are key for endoribonuclease activity. Third, the identification of sites within YoeB that were highly tolerant of insertions, but which did not impact toxicity, permitted the construction of positive selection vectors. Here, the yoeB gene was placed under the control of a heterologous inducible promoter in a plasmid vector. Synthetic, in-frame multiple cloning sites were engineered into permissive sites in the gene without affecting the toxicity of the encoded protein. Insertion of cloned fragments in the multiple cloning sites was predicted to disrupt the yoeB reading frame and thereby specifically allow selection of recombinant plasmids in the presence of inducer, whereas non-recombinant plasmids that continue to express yoeB would be eliminated. Fragments from a variety of test sources were cloned which verified the utility of yoeB as a positive selection marker that can be manipulated further to construct a range of multifunctional cloning vectors. Fourth, spontaneous mutations within yoeB that allow cells to escape toxicity were discovered and characterized in a further strand of work. Analysis of the mutations indicated that, in addition to amino acids that previously were implicated in endoribonuclease activity, a quartet of previously uncharacterized residues are critical for YoeB function. The structure-function studies described here provide important insights into the regions of YoeB that are key for toxicity and for interaction with the YefM antitoxin. The data will be specially useful in developing strategies that promote artificial release of YoeB as a novel antibacterial approach in combatting multidrug resistant infections.