IreK-Mediated, Cell Wall-Protective Phosphorylation in Enterococcus faecalis

Abstract

Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections due, in part, to its intrinsic resistance to cell wall-active antimicrobials. One critical determinant of this resistance is the transmembrane kinase IreK, which belongs to the penicillin-binding protein and serine/threonine kinase-associated kinase family of bacterial signaling proteins involved with the regulation of cell wall homeostasis. The activity of IreK is enhanced in response to cell wall stress, but direct substrates of IreK phosphorylation, leading to antimicrobial resistance, are largely unknown. To better understand stress-modulated phosphorylation events contributing to antimicrobial resistance, wild type E. faecalis cells treated with cell wall-active antimicrobials, chlorhexidine or ceftriaxone, were examined via phosphoproteomics. Among the most prominent changes was increased phosphorylation of divisome components after both treatments, suggesting that E. faecalis modulates cell division in response to cell wall stress. Phosphorylation mediated by IreK was then determined via a similar analysis with a E. faecalis ΔireK mutant strain, revealing potential IreK substrates involved with the regulation of peptidoglycan biosynthesis and within the E. faecalis CroS/R two-component system, another signal transduction pathway that promotes antimicrobial resistance. These results reveal critical insights into the biological functions of IreK.

PASTA‐kinase‐mediated signaling drives accumulation of the peptidoglycan synthesis protein MurAA to promote cephalosporin resistance in Enterococcus faecalis

“…Because of its design, the aforementioned selection had the potential to reveal other targets of IreK that were not necessarily downstream of IreB (given that IreK is known to have other substrates that play a role in cephalosporin resistance (Iannetta et al, 2021; Kellogg & Kristich, 2018; Minton et al, 2022)). As an alternative approach to more specifically identify factors downstream of IreB, in a second selection (selection 2, Figure S1b), we exploited a previous observation that an IreB expression vector could not be successfully introduced into an E. faecalis strain lacking IreK (Hall et al, 2013), suggesting that overexpression of unphosphorylated (i.e.…”

GpsB control of PASTA kinase activity inListeria monocytogenesinfluences peptidoglycan synthesis during cell wall stress and cytosolic survival

“…An important intrinsic resistance determinant conserved in Firmicutes and Actinobacteria is the penicillin-binding protein and serine/threonine-associated, or PASTA, kinase family. PASTA kinases have extracellular PASTA domains that sense perturbations in the cell envelope and mediate a global stress response via their cytoplasmic eukaryotic-like serine/threonine kinase domain, phosphorylating multiple downstream targets involved in cell wall homeostasis, central metabolism, and more (2)(3)(4)(5)(6). In a variety of bacteria, including important pathogens like Listeria monocytogenes, Staphylococcus aureus, Mycobacterium tuberculosis, Enterococcus faecalis, and Streptococcus pneumoniae, PASTA kinases are important for growth in the presence of cell wall-targeting agents (e.g., β-lactams and lysozyme) and also critically contribute to pathogenesis (7)(8)(9)(10)(11)(12)(13)(14).…”

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

“…The precise role of these phosphorylations in the control of the cell division process or its coupling with PG synthesis in C. difficile remains to be deciphered. We cannot exclude that a cell wall stress, which could be detected by the extracellular PASTA domains, could allow to identify additional PrkC targets in cell division and PG biosynthesis machinery as shown recently for E. faecalis and L. monocytogenes ( 94 , 95 ). It is also interesting to note that we do not identify two-component systems as targets of PrkC in C. difficile contrary to the data obtained in B. subtilis , E faecalis , and S. aureus ( 19 , 94 , 96 ).…”

“…We cannot exclude that a cell wall stress, which could be detected by the extracellular PASTA domains, could allow to identify additional PrkC targets in cell division and PG biosynthesis machinery as shown recently for E. faecalis and L. monocytogenes ( 94 , 95 ). It is also interesting to note that we do not identify two-component systems as targets of PrkC in C. difficile contrary to the data obtained in B. subtilis , E faecalis , and S. aureus ( 19 , 94 , 96 ). This is in agreement with the low impact of STK inactivation on global proteome even if one regulator of the Crp family and a diguanylate kinase are identified as candidate substrates of PrkC.…”