Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

Abstract

Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis. Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis. Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium. Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci.KEYWORDS enterococcus, antibiotic resistance, two-component regulatory systems E nterococcus faecalis and Enterococcus faecium represent serious opportunistic pathogens that are responsible for many nosocomial infections. Treatment of enterococcal infections is particularly challenging due to intrinsic and acquired resistance toward many clinically relevant antibiotics, including beta-lactams, aminoglycosides, glycopeptides, and trimethoprim (1). Because all clinical isolates of E. faecalis and E. faecium are intrinsically resistant to cephalosporins (a subset of beta-lactam antibiotics), disabling cephalosporin resistance with small molecule therapeutics may be a viable strategy to overcome antibiotic-resistant enterococcal infections. Both species use transpeptidase activity of a low-affinity penicillin-binding protein (Pbp5) in cooperation with the glycosyltransferase activity of the penicillin-binding proteins (PBPs) PonA or PbpF to continue transpeptidation and transglycosylation reactions required for cell wall assembly during cephalosporin exposure (2-5). However, additional determinants contributing to cephalosporin resistance have also been explored in E. faecalis and E. faecium.In E. faecalis, two enzymes involved in cell wall synthesis (the UDP-N-acetylglucosamine 1-carboxyvinyl transferase MurAA [6] and the alanine transferase BppA2 [7]) are known to be required for normal cephalosporin resistance. In addition, two signal transduction pathways mediate intrinsic resistance to cephalosporins and other cell wall-targeting antibiotics. One pathway includes a eukaryotic-like Ser/Thr kinase, IreK, and its cognate phosphatase, IreP, which act antagonistically to regulate a pathway leading to cephalosporin resistance (8, 9). An ortholog of IreK in E. faecium has

Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification

“…Mueller-Hinton broth (MHB) was used for growth of E. faecalis in all experiments. Markerless allelic exchange was used to construct chromosomal mutations in the genome of E. faecalis OG1, as described previously (42,43). Antibiotic resistance was determined by the broth microdilution method after 24 h at 37°C in 96-well plates.…”

Modulators of Enterococcus faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract

“…The method described by Varadarajan and colleagues (50,51) was used to identify V71G as a candidate substitution in RepA to provide temperature sensitivity. We previously showed that the V71G substitution provides temperature-sensitive replication of a closely related vector (48). Inverse PCR on pCJK245 was used to introduce the V71G substitution into RepA.…”

The Enterococcus: a Model of Adaptability to Its Environment

“…They are not, however, sufficient for resistance, since other proteins that are required for resistance expression have been found. In E. faecalis, the CroRS regulatory locus is required for cephalosporin resistance, as is a serine-threonine eukaryote-like kinase, IreK (also known as Stk) (160,161). The presence of genes for two of the three E. faecalis class A PBPs (ponA and pbpZ) is also required for resistance to cephalosporins in E. faecalis (141).…”