Phosphorylation of the cell wall hydrolase MltG in response to cell wall stress modulates resistance toward cephalosporins in <i>Enterococcus faecalis</i>

<i>Enterococcus faecalis</i> is an opportunistic pathogen that colonizes the human gut microbiome. <i>E. faecalis</i> commonly establishes infection subsequent to antibiotic therapy in patients due to intrinsic resistance exhibited by <i>E. faecalis</i> toward cephalosporins and acquired resistance to many clinically used antibiotics. Intrinsic resistance toward cephalosporins in <i>E. faecalis</i> depends on the kinase activity of IreK, a transmembrane serine/threonine PASTA kinase that mediates responses to cell wall stress, including stress caused by cephalosporins, by phosphorylating downstream effector proteins. Our previous phosphoproteomics analysis identified MltG, a transmembrane protein with an extracellular catalytic domain that cleaves nascent peptidoglycan strands, as a putative substrate for phosphorylation by IreK in <i>E. faecalis</i>, suggesting the hypothesis that IreK-mediated phosphorylation of MltG might regulate cell wall homeostasis and possibly intrinsic cephalosporin resistance. Here we report that MltG is a bona fide direct substrate of IreK in <i>E. faecalis</i>. We found that MltG phosphorylation <i>in vivo</i> is enhanced in response to cell wall stress in an IreK-dependent manner, requiring a specific residue in the MltG cytoplasmic domain for phosphorylation by IreK both <i>in vivo</i> and <i>in vitro</i>. Finally, phosphoablative and phosphomimetic substitutions of MltG reciprocally influence resistance of <i>E. faecalis</i> to ceftriaxone, pointing to functional consequences of MltG phosphorylation. Collectively, our results reveal a novel pathway by which IreK senses antibiotic-mediated cell wall stress and responds by phosphorylating the cytoplasmic segment of MltG to enhance antibiotic resistance.IMPORTANCEInfections caused by <i>Enterococcus faecalis</i> are increasingly prevalent and difficult to treat due to the multi-drug resistance exhibited toward common antibiotics. A greater understanding of the mechanisms underlying antibiotic resistance can enable the development of new drugs or strategies to overcome antibiotic-resistant infections. <i>E. faecalis</i> exhibits intrinsic resistance toward cephalosporins. This intrinsic resistance requires activity of the PASTA kinase IreK; however, few substrates for phosphorylation by IreK have been rigorously identified. Here, we report that MltG is directly phosphorylated by IreK in response to cell wall stress. This phosphorylation event acts to promote cephalosporin resistance as part of the IreK signaling network. Our results thereby validate a new substrate and expand knowledge of the IreK signaling pathway contributing to cephalosporin resistance.