Faculty Collaboration Database

Multisite Phosphorylation Regulates GpsB Function in Cephalosporin Resistance of Enterococcus faecalis. J Mol Biol 2023 Sep 15;435(18):168216

Date

07/31/2023

Pubmed ID

37517789

Pubmed Central ID

PMC10528945

DOI

10.1016/j.jmb.2023.168216

Scopus ID

2-s2.0-85166951228 (requires institutional sign-in at Scopus site)   1 Citation

Abstract

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.

Author List

VanZeeland NE, Schultz KM, Klug CS, Kristich CJ

Authors

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin
Christopher J. Kristich PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin
Nicole E. VanZeeland Postdoctoral Fellow in the Pathology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Anti-Bacterial Agents
Bacterial Proteins
Cephalosporin Resistance
Cephalosporins
Enterococcus faecalis
Humans
Phosphorylation
Signal Transduction