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Differential Effects of Penicillin Binding Protein Deletion on the Susceptibility of Enterococcus faecium to Cationic Peptide Antibiotics.

ABSTRACT: Beta-lactam antibiotics sensitize Enterococcus faecium to killing by endogenous antimicrobial peptides (AMPs) of the innate immune system and daptomycin through mechanisms yet to be elucidated. It has been speculated that beta-lactam inactivation of select E. faecium penicillin binding proteins (PBPs) may play a pivotal role in this sensitization process. To characterize the specific PBP inactivation that may be responsible for these phenotypes, we utilized a previously characterized set of E. faecium PBP knockout mutants to determine the effects of such mutations on the activity of daptomycin and the AMP human cathelicidin (LL-37). Enhanced susceptibility to daptomycin was dependent more on a cumulative effect of multiple PBP deletions than on inactivation of any single specific PBP. Selective knockout of PBPZ rendered E. faecium more vulnerable to killing by both recombinant LL-37 and human neutrophils, which produce the antimicrobial peptide in high quantities. Pharmacotherapy targeting multiple PBPs may be used as adjunctive therapy with daptomycin to treat difficult E. faecium infections.

SUBMITTER: Sakoulas G

PROVIDER: S-EPMC4576044 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Differential Effects of Penicillin Binding Protein Deletion on the Susceptibility of Enterococcus faecium to Cationic Peptide Antibiotics.

Sakoulas George G Kumaraswamy Monika M Nonejuie Poochit P Werth Brian J BJ Rybak Micahel J MJ Pogliano Joseph J Rice Louis B LB Nizet Victor V

Antimicrobial agents and chemotherapy 20150720 10

Beta-lactam antibiotics sensitize Enterococcus faecium to killing by endogenous antimicrobial peptides (AMPs) of the innate immune system and daptomycin through mechanisms yet to be elucidated. It has been speculated that beta-lactam inactivation of select E. faecium penicillin binding proteins (PBPs) may play a pivotal role in this sensitization process. To characterize the specific PBP inactivation that may be responsible for these phenotypes, we utilized a previously characterized set of E. f ...[more]

PMID: 26195528

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Project description:Multidrug-resistant Enterococcus faecium strains are currently a leading cause of difficult-to-treat nosocomial infections. The emerging resistance of enterococci to last-resort antibiotics, such as daptomycin, prompts a search for alternative antimicrobials. Aureocin A53- and enterocin L50-like bacteriocins are potent antimicrobial agents that form daptomycin-like cationic complexes and have a similar cell envelope-targeting mechanism of action, suggesting their potential as next-generation antibiotics. However, to ensure their safe use, the mechanisms of resistance to these bacteriocins and cross-resistance to antibiotics need to be well understood. Here, we investigated the genetic basis of E. faecium's resistance to aureocin A53- and enterocin L50-like bacteriocins and compared it with that to antibiotics. First, we selected spontaneous mutants resistant to the bacteriocin BHT-B and identified adaptive mutations in the liaFSR-liaX genes encoding the LiaFSR stress response regulatory system and the daptomycin-sensing protein LiaX, respectively. We then demonstrated that a gain-of-function mutation in liaR increases the expression of liaFSR, liaXYZ, cell wall remodeling-associated genes, and hypothetical genes involved in protection against various antimicrobials. Finally, we showed that adaptive mutations or overexpression of liaSR or liaR alone results in cross-resistance to other aureocin A53- and enterocin L50-like bacteriocins, as well as antibiotics targeting specific components of the cell envelope (daptomycin, ramoplanin, gramicidin) or ribosomes (kanamycin and gentamicin). Based on the obtained results, we concluded that activation of the LiaFSR-mediated stress response confers resistance to peptide antibiotics and bacteriocins via a cascade of reactions, eventually leading to cell envelope remodeling. IMPORTANCE Pathogenic enterococci carry virulence factors and a considerable resistome, which makes them one of the most serious and steadily increasing causes of hospital epidemiological risks. Accordingly, Enterococcus faecium is classified into a top-priority ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of six highly virulent and multidrug-resistant (MDR) bacterial pathogens for which novel antimicrobial agents need to be developed urgently. Alternative measures, such as the use of bacteriocins, separately or in combination with other antimicrobial agents (e.g., antibiotics), could be a potential solution, especially since several international health agencies recommend and support the development of such interventions. Nevertheless, in order to exploit their efficacy, more basic research on the mechanisms of cell killing and the development of resistance to bacteriocins is needed. The present study fills some of the knowledge gaps regarding the genetic basis of the development of resistance to potent antienterococcal bacteriocins, pointing out the common and divergent features regarding the cross-resistance to antibiotics.

Project description:UnlabelledThe intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as "cephalosporins") is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.Importanceβ-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a resistant phenotype. The present work identifies a novel, previously uncharacterized, protein that interacts with Pbp5, whose expression increases in conjunction with stimuli that increase resistance to cephalosporins, and that confers increased resistance to cephalosporins when overexpressed. P5AP may represent a promising new target, inhibition of which could restore cephalosporin susceptibility to E. faecium.

Dataset Information

Differential Effects of Penicillin Binding Protein Deletion on the Susceptibility of Enterococcus faecium to Cationic Peptide Antibiotics.

Publications

Differential Effects of Penicillin Binding Protein Deletion on the Susceptibility of Enterococcus faecium to Cationic Peptide Antibiotics.

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