Antibiotics are no longer considered as miracle drugs because of therapeutic failure and the emergence of antibiotic-resistant bacteria which are difficult to treat. Therefore, there is an urgent need to develop new antimicrobial drugs to combat these resistant pathogenic bacteria. Toxin-antitoxin systems (TA) are found widely on plasmids and chromosomes of diverse bacteria, including pathogens, as genome maintenance and stress response modules, among other activities. TA modules typically consist of a pair of genes that encode a labile antitoxin and stable toxin. However, under stress conditions, the unstable antitoxin is degraded rapidly leading to the release of the toxin that interferes with crucial cellular processes and causes growth arrest or cell death. Thus, TA systems are potential targets for antimicrobial drugs by promoting toxin activation artificially. The aim of this work was to understand toxin activation mechanisms by studying the function and organization of the YefM-YoeB complex in Escherichia coli. YefM-YoeB is one of the most widespread TA systems which is found in a diverse range of bacterial species.The YoeB protein is a ribosome-dependent endonuclease toxin. The toxicity of YoeB is blocked by the dimeric YefM antitoxin under steady state conditions. However, YoeB is activated when stress-induced production of the Lon protease mediates degradation of YefM. Mutagenesis studies of YefM provide novel insights into antitoxin organization and function, and will signpost strategies for artificial release of the toxin. This study explored the function and organization of the YefM antitoxin in E. coli. Initially, the effects of pentapeptide insertions and alanine substitutions on self-association of the dimeric YefM antitoxin, on interaction with the YoeB toxin, and on transcriptional autoregulation of the yefMyoeB operon by YefM were assessed. Twenty insertions were constructed in YefM by pentapeptide scanning mutagenesis and ten by subsequent alanine scanning mutagenesis. The results defined mutations that selectively impacted dimerization of YefM, the interaction between YefM and YoeB, and transcriptional repression by the antitoxin. Mutations in the N-terminal domain affected YefM dimerization and transcriptional repression more strongly than C-terminal insertions. In contrast, the results emphasize that the C-terminal domain that interacts with YoeB toxin is not essential for self-association of YefM or transcriptional repression. Positions Ile-29 and Met-40 within the Î²-strands that form part of the core of the YefM dimer were identified as being particularly crucial for YefM function. Mutation of these residues disrupted dimerization and transcriptional autoregulation by YefM, as well as the interaction with YoeB toxin. Thus, these YefM core regions may be potential targets for novel antimicrobial agents that interrupt the YefM-YoeB complex thereby releasing YoeB toxin to promote bacterial suicide as a novel antibacterial strategy.
|Date of Award||1 Aug 2020|
- The University of Manchester
|Supervisor||Jeremy Derrick (Supervisor) & Finbarr Hayes (Supervisor)|