Nutritional control of antibiotic resistance via an interface between the phosphotransferase system and a two-component signaling system

Abstract

Enterococci are ubiquitous inhabitants of the gastrointestinal (GI) tract. However, antibiotic-resistant enterococci are also major causes of hospital-acquired infections. Enterococci are intrinsically resistant to cephalosporins, enabling growth to abnormally high densities in the GI tract in patients during cephalosporin therapy, thereby promoting dissemination to other sites where they cause infection. Despite its importance, many questions about the underlying basis for cephalosporin resistance remain. A specific two-component signaling system, composed of the CroS sensor kinase and its cognate response regulator (CroR), is required for cephalosporin resistance in Enterococcus faecalis, but little is known about the factors that control this signaling system to modulate resistance. To explore the signaling network in which CroR participates to influence cephalosporin resistance, we employed a protein fragment complementation assay to detect protein-protein interactions in E. faecalis cells, revealing a previously unknown association of CroR with the HPr protein of the phosphotransferase system (PTS) responsible for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses indicate that association with HPr restricts the ability of CroR to promote cephalosporin resistance and gene expression in a nutrient-dependent manner. Mutational analysis suggests that the interface used by HPr to associate with CroR is distinct from the interface used to associate with other cellular partners. Our results define a physical and functional connection between a critical nutrient-responsive signaling system (the PTS) and a two-component signaling system that drives antibiotic resistance in E. faecalis, and they suggest a general strategy by which bacteria can integrate their nutritional status with diverse environmental stimuli.

FIG 1

mDHFR-based PCA specifically detects protein-protein interactions in E. faecalis cells. Coexpression of fusions between interacting E. faecalis proteins and the F_[1,2] and F_[3] fragments of mDHFR confers resistance to trimethoprim. E. faecalis strains coexpressing the indicated fusions were subjected to 10-fold serial dilutions and inoculated on MH agar supplemented with Cm and Em (for plasmid selection) in the presence or absence of trimethoprim. Coexpression of pairs of fusion proteins that are known or predicted to interact (HprK-HPr and CroR-CroR) led to enhanced growth in the presence of trimethoprim. The association between CroR and HPr was previously unknown. EF1714 and EF1039 represent randomly chosen E. faecalis control proteins that demonstrate the specificity of the CroR-HPr in vivo association. The host strain was E. faecalis OG1RF.

FIG 2

HPr and CroR coimmunoprecipitate from E. faecalis lysates. Association of CroR and HPr in whole-cell lysates of E. faecalis was assessed by coimmunoprecipitation with anti-F_[3] antiserum. CroR or a randomly chosen E. faecalis control protein (EF1714) fused to a Strep tag was coexpressed with wild-type HPr, an HPr point mutant, or a randomly chosen E. faecalis control protein (EF1039) fused to mDHFR F_[3], as indicated. Lysates and immunoprecipitates were subjected to immunoblot analysis with antiserum specific for the Strep or F_[3] fusions, revealing that CroR specifically coprecipitates with wild-type HPr. The host strain was E. faecalis OG1RF. “Lysates” corresponds to the whole-cell lysates used as input for coimmunoprecipitation; “IP” corresponds to the immune complexes recovered after coimmunoprecipitation.

FIG 3

Proximity of mutations in HPr that impair association with CroR. Positions of mutations in HPr that impair association with CroR, but not HPr, were mapped onto the nuclear magnetic resonance (NMR) structure of E. faecalis HPr (29) (side chains are depicted as sticks), revealing that most of the mutations are juxtaposed in adjacent structural elements. Several relevant secondary structural elements in the structure are indicated. Loops are abbreviated as L1, L2, etc. Note that the known sites of phosphorylation in HPr (His15 and Ser46) are positioned on the opposite (obscured) side of the structure.

FIG 4

HPr mutants impaired in association with CroR support normal growth on PTS substrates. E. faecalis strains were precultured in MHB until exponential phase, washed, and diluted 100-fold in chemically defined medium supplemented with the PTS carbohydrate glucose (open triangles) or N-acetylglucosamine (filled squares) at 0.2%. The defined medium was based on that described by Murray et al. (34), supplemented with sodium acetate and manganese chloride and containing 10 μg/ml inosine as the only nucleobase precursor. The kinetics of growth on both carbohydrates for mutants expressing noninteracting HPr mutants were nearly identical to that of the wild type. Strains: wild-type, OG1; ΔptsH2, JL395; ptsH E2G, JL422; ptsH E70G, JL425. Results are representative of 2 independent experiments.

FIG 5

Association with HPr influences the ability of CroR to promote gene expression. E. faecalis strains carrying a croR′-lacZ reporter were cultured in MM9YE supplemented with either 0.1% glucose, 0.1% GlcNAc, 0.2% pyruvate, or 0.1% citrate. Exponentially growing cells were treated with ceftriaxone at 4× MIC for 2 h, and β-galactosidase activity was determined, revealing that association with HPr inhibits the ability of CroR to promote its own gene expression in glucose-supplemented medium. Strains: WT, wild-type OG1 (open bars); E2G, JL422 (filled bars). Error bars represent standard deviations for 2 independent biological replicates.