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

Abstract

The bacterial PASTA kinase, IreK, is required for intrinsic cephalosporin resistance in the Gram‐positive opportunistic pathogen, Enterococcus faecalis. IreK activity is enhanced in response to cell wall stress, such as cephalosporin exposure. The downstream consequences of IreK activation are not well understood in E. faecalis, but recent work in other low‐GC Gram‐positive bacteria demonstrated PASTA kinase‐dependent regulation of MurAA, an enzyme that performs the first committed step in the peptidoglycan synthesis pathway. Here, we used genetic suppressor selections to identify MurAA as a downstream target of IreK signaling in E. faecalis. Using complementary genetic and biochemical approaches, we demonstrated that MurAA abundance is regulated by IreK signaling in response to physiologically relevant cell wall stress to modulate substrate flux through the peptidoglycan synthesis pathway. Specifically, the IreK substrate, IreB, promotes proteolysis of MurAA through a direct physical interaction in a manner responsive to phosphorylation by IreK. MurAB, a homolog of MurAA, also promotes MurAA proteolysis and interacts directly with IreB. Our results therefore establish a connection between the cell wall stress sensor IreK and one critical physiological output to modulate peptidoglycan synthesis and drive cephalosporin resistance.

Secrets of getting started: Regulation of the first committed step of peptidoglycan synthesis by protein phosphorylation in Enterococcus and other Gram‐positive bacteria

“…The involvement of the IreK Ser/Thr-protein kinase, IreP Ser/ Thr-protein phosphatase, IreB regulatory protein, and ClpP protease from these new and past results (Kelliher et al, 2021;Sun et al, 2023;Wamp et al, 2020Wamp et al, , 2022 Notably, relative cellular MurAA amount also increased in cells treated with a sublethal concentration of ceftriaxone (Mascari et al, 2023), which activated protein phosphorylation by the IreK Ser/Thr-protein kinase (Labbe & Kristich, 2017;Minton et al, 2022).…”

“…Together, these results suggested that unphosphorylated IreB was a negative regulator of a downstream factor that contributed to cephalosporin resistance (Hall et al, 2013;Kristich et al, 2007). These findings also provided a framework for interpretation of IreB/ReoM function in the other F I G U R E 1 Regulatory pathway that leads to destabilization of MurAA by binding to unphosphorylated IreB in Enterococcus faecalis proposed in (Mascari et al, 2023). Cell wall stresses signal the IreK Ser/Thr-protein kinase to transfer a phosphoryl group from ATP to the IreB regulatory protein.…”

“…Furthermore, protein stability determinations after addition of the translation inhibitor chloramphenicol showed that ΔclpP or ΔireB stabilized MurAA. Last, absence of MurAB or the ReoY homolog (OG1RF_11272) increased the relative amount of MurAA in wild-type E. faecalis and increased cephalosporin resistance of a ΔireK mutant (Mascari et al, 2023). By contrast, the relative cellular amount of MurAB was not proteolytically regulated under any condition tested.…”

“…A new paper in this issue by Mascari, Little, and Kristich provides important new insights into this mechanism and its control of resistance to cephalosporin family β-lactam antibiotics in Enterococcus faecalis (Mascari et al, 2023). This paper builds on earlier work from the Kristich laboratory that linked absence of the E. faecalis IreK Ser/Thr-protein kinase or IreB regulatory protein to decreased or increased cephalosporin resistance, respectively (Hall et al, 2017;Kristich et al, 2007).…”

“…See text for additional details. (Mascari et al, 2023), which likely lacks PG turnover like S. pneumoniae (Boersma et al, 2015). This decrease in PG substrate flux was counteracted in ΔireK murAA(A115T) and ΔireK murAA(N188K)…”