| Summary: | The emergence of vancomycin-resistant enterococci (VRE) since the 1980s has turned this Gram-positive bacterium into a serious and growing clinical challenge. Enterococci have acquired resistance to a number of different antibiotics and are intrinsically resistant to cephalosporin b-lactam drugs. As a result, they are now placed in the World Health Organisation list of priority pathogens for which new antibiotics are urgently needed. An important risk factor for the emergence of VRE is treatment with cephalosporins, suggesting a connection between the origins of resistance between these two different types of antibiotics, the mechanistic basis for which was unknown. In this study I demonstrate that cephalosporin resistance is significantly enhanced in the well characterized E. faecalis OG1RF strain containing the Tn1549 transposon conferring the VanB type vancomycin resistance. Cephalosporin resistance is shown to be enhanced to the presence of the Tn1549 transposon and a single, chromosomally encoded Serine/Threonine (ST) kinase gene called ireK. Complementation experiments and fluorescence microscopy with a range of biophysical techniques to characterise the protein. Deletion of the gene results in cephalosporin sensitivity. Moreover, this phenotype can be chemically simulated by treatment of E. faecalis with inhibitors of cytoplasmic ST kinases such as staurosporine, demonstrating a requirement for ST kinase activity for cephalosporin resistance. This also demonstrates a therapeutic potential for targeting ST kinases in Gram-positive pathogens with small molecule inhibitors to restore cephalosporin sensitivity which may have clear translational significance. IreK was identified as a key protein involved in enhanced cephalosporin resistance and led to examination of its extracellular and intracellular domains to understand its signal transduction mechanisms and its linkage to resistance. The extracellular domain of IreK was essential for cellular location. Further biophysical experiments identified ligands for the extracellular domains include sugar motifs such as the glycan backbone of peptidoglycans and sugar containing antibiotics. This has led to a new functional model for ST kinases in enterococci.
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