Allelic exchange technologies for Clostridioides difficile research and their application in toxin studies

The ability to precisely manipulate the genome of Clostridioides difficile allows for the generation of mutants that can be characterized to unveil critical aspects relating to pathogenesis and virulence. Such findings can ultimately direct the development of antivirulence agents to combat C. difficile infection, by small molecule inhibition. In this review, the mechanisms underpinning the most widely adopted allelic exchange technologies for generating precise genomic mutations in C. difficile, are discussed. The advantages and disadvantages of each system are considered. In addition, a comprehensive review of the application of these tools for the study of C. difficile toxins and genes residing within the toxin loci, is presented.