Oxidative Stress Enhances Cephalosporin Resistance of Enterococcus faecalis through Activation of a Two-Component Signaling System

Abstract

Enterococcus faecalis is a low-GC Gram-positive bacterium, a normal resident of the gastrointestinal (GI) tract, and an important hospital-acquired pathogen. An important risk factor for hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporins, antibiotics that impair cell wall biosynthesis by inhibiting peptidoglycan cross-linking. Enterococci are intrinsically resistant to cephalosporins; however, environmental factors that modulate cephalosporin resistance have not been described. While searching for the genetic determinants of cephalosporin resistance in E. faecalis, we unexpectedly discovered that oxidative stress, whether from external sources or derived from endogenous metabolism, drives enhanced intrinsic resistance to cephalosporins. A particular source of oxidative stress, H 2 O 2 , activates signaling through the CroR-CroS two-component signaling system, a known determinant of cephalosporin resistance in E. faecalis. We find that CroR-CroS is required for adaptation to H 2 O 2 stress and that H 2 O 2 potentiates the activities of cephalosporins against E. faecalis when the CroR-CroS signaling system is nonfunctional. Rather than directly detecting H 2 O 2 , our data suggest that the CroR-CroS system responds to cell envelope damage caused by H 2 O 2 exposure in order to promote cell envelope repair and enhanced cephalosporin resistance. Enterococci are ubiquitous inhabitants of the gastrointestinal tract in healthy animals, including humans. However, antibiotic-resistant enterococci are also major causes of hospital-acquired infections (1, 2) and therefore represent a serious public health problem. One well-known risk factor for the acquisition of enterococcal hospital-acquired infections is prior therapy with broad-spectrum cephalosporins (3), antibiotics that belong to the ␤-lactam family and interfere with cell wall biosynthesis by inhibiting the penicillin-binding proteins (PBPs) that cross-link peptidoglycan. Enterococci exhibit intrinsic resistance to cephalosporins, enabling them to proliferate and achieve abnormally high densities in the gastrointestinal (GI) tract of patients during cephalosporin therapy (4), thereby promoting dissemination to other sites, where they cause infection (5). This intrinsic cephalosporin resistance is a trait shared by essentially all isolates of Enterococcus faecalis; however, our understanding of the genetic and biochemical basis underlying cephalosporin resistance in enterococci remains incomplete. One important well-characterized factor that is required for cephalosporin resistance is a specialized low-affinity PBP (Pbp5) that does not get inactivated by cephalosporins and can therefore perform cross-linking of peptidoglycan to permit growth in the presence of the antibiotic (6, 7). Although required, Pbp5 is not sufficient for resistance, as mutations in other loci render E. faecalis susceptible to cephalosporin by mechanisms that have not been fully worked out (8-12).Among the additional determinants required for intrinsi...

Multiple Roles for Enterococcus faecalis Glycosyltransferases in Biofilm-Associated Antibiotic Resistance, Cell Envelope Integrity, and Conjugative Transfer

“…To quantify differences in cell envelope integrity, we performed ␤-galactosidase assays utilizing the nonpermeable reagent chlorophenyl red-␤-D-galactopyranoside (CPRG) (47,48). A constitutive lacZ reporter construct (47) was introduced into the E. faecalis isogenic mutants, and the strains were cultured in biofilm-conducive medium supplemented with CPRG.…”

Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

“…In fact, we were unable to find conditions under which ceftriaxone exposure led to the robust production of CroR-P in E. faecalis OG1; thus, we used vancomycin to stimulate signal transduction. Because a variety of cell wall-targeting agents with different modes of action can stimulate CroRS signaling, the input for CroS and output of CroR are thought to be involved in cell wall homeostasis (3,14). Specifically, the output of CroR has been proposed to be related to the function of penicillin binding proteins, which mediate resistance to ␤-lactam antibiotics (3).…”

“…However, changes in the morphology of the V583 croR mutant were not described. Although CroRS signaling has been proposed to respond to and elicit the repair of cell wall damage (3,14), molecular details of the output of CroRS signaling remain a mystery. CroR is capable of gene regulation, but thus far only three CroR-dependent genes have been described.…”

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis

“…Hydrolysis of CPRG was monitored to assess cell envelope integrity, similar to previous reports (49,50). Bacterial cultures were grown to stationary phase at 37°C in MHB supplemented with 40 g/ml CPRG and erythromycin (for the maintenance of pCJK205, which encodes ␤-galactosidase).…”

“…To test this, we analyzed cell envelope integrity using chlorophenol red ␤-D-galactoside (CPRG). CPRG is normally excluded from wild-type E. faecalis, but it enters cells with defects in cell envelope integrity and is subsequently hydrolyzed by ␤-galactosidase, releasing the chromophore chlorophenol red (49,50). Mutants lacking IreK entirely or expressing a catalytically impaired IreK exhibited ϳ10-fold increases in CPRG hydrolysis, compared with wild-type levels, when grown in the absence of stress (Fig.…”