Multisite Phosphorylation Regulates GpsB Function in Cephalosporin Resistance of Enterococcus faecalis

Enterococci are normal human commensals and major causes of hospital-acquired infections. Enterococcal infections can be difficult to treat because enterococci harbor intrinsic and acquired antibiotic resistance, such as resistance to cephalosporins. In Enterococcus faecalis, the transmembrane kinase IreK, a member of the bacterial PASTA kinase family, is essential for cephalosporin resistance. The activity of IreK is boosted by the cytoplasmic protein GpsB, which promotes IreK autophosphorylation and signaling to drive cephalosporin resistance. A previous phosphoproteomics study identified eight putative IreK-dependent phosphorylation sites on GpsB, but the functional importance of GpsB phosphorylation was unknown. Here we used genetic and biochemical approaches to define three sites of phosphorylation on GpsB that functionally impact IreK activity and cephalosporin resistance. Phosphorylation at two sites (S80 and T84) serves to impair the ability of GpsB to activate IreK in vivo, suggesting phosphorylation of these sites acts as a means of negative feedback for IreK. The third site of phosphorylation (T133) occurs in a segment of GpsB termed the C-terminal extension that is unique to enterococcal GpsB homologs. The C-terminal extension is highly mobile in solution, suggesting it is largely unstructured, and phosphorylation of T133 appears to enable efficient phosphorylation at S80 / T84. Overall our results are consistent with a model in which multisite phosphorylation of GpsB impairs its ability to activate IreK, thereby diminishing signal transduction through the IreK-dependent pathway and modulating phenotypic cephalosporin resistance.& COPY; 2023 Elsevier Ltd. All rights reserved.

Automated summary

Enterococci are normal human commensals that often cause hospital-acquired infections. They exhibit antibiotic resistance, including resistance to cephalosporins. A study on Enterococcus faecalis revealed that the transmembrane kinase IreK plays a crucial role in cephalosporin resistance, with its activity being boosted by the cytoplasmic protein GpsB. Phosphorylation of specific sites on GpsB negatively regulates IreK activity, while phosphorylation at a unique site enables efficient phosphorylation at other sites and modulates cephalosporin resistance.