More than any other human pathogen, Enterococcus. faecium has grown in importance as a result of its resistance to commonly used antimicrobial agents, in particular to the ?-lactams. High-level ? lactam resistance expressed by E. faecium not only compromises therapy of bacterial infections, it promotes gastrointestinal colonization and further dissemination of resistant strains. The hallmark of high-level ?-lactam resistance in E. faecium is the expression of low affinity class B penicillin-binding protein Pbp5. As a class B Pbp with only transpeptidase activity, Pbp5 must coordinate its activities with that of glycosyltransferases to synthesize mature peptidoglycan. Class B Pbps most commonly coordinate with bifunctional (possessing both glycosyltransferase and transpeptidase activities) class A Pbps, which in E. faecium are PbpF, PbpZ and PonA. In work performed during the previous period of this grant, we described auxiliary and parallel mechanisms by which E. faecium expresses resistance to ?-lactam antibiotics. We have deleted the E. faecium class A pbps in every combination and have discovered that deletion of PbpF and PonA results in a heterogeneous susceptibility to ceftriaxone (suggesting that Pbp5 coordinated with either of these class A Pbps to confer resistance to ceftriaxone), from which homogeneous resistance can be selected by growth on ceftriaxone (selection of spontaneous mutants) or induced by exposure to penicillin. Loss of PbpF is also associated with the autolytic phenotype of E. faecium. We have also identified and characterized an L,D-transpeptidase (Ldtfm) that is able to confer ?-lactam and vancomycin resistance in the absence of Pbp5, in concert with the activity of a D,D-carboxypeptidase. The present proposal will continue with this important work in the following manner: 1) We will investigate the mechanisms underlying the class A Pbp deletion phenotypes by characterizing the E. faecium peptidoglycan synthesis complex, targeting a likely alternative glycosyltransferases identified through a genome search and using microarray analysis to analyze the regulatory framework of penicillin-inducible ceftriaxone resistance in the ponApbpF double mutant 2) We will investigate the mechanisms underlying PbpF ceftriaxone-moenomycin synergism vs. E. faecium D344R and the impact of PbpF on the autolytic phenotype through site-directed mutagenesis of PbpF and functional studies of E. faecium autolysins 3) We will explore the molecular basis for the substrate specificity of Ldtfm to understand the surprising pattern of inhibition of the enzyme by ?-lactams 4) We will characterize the physiological aspects of the L,D-transpeptidation pathway using a proteomic approach to identifying partners of Ldtfm in the peptidoglycan polymerization complexes. We will also use transcriptome analysis to identify differentially expressed genes and perform whole genome sequencing to identify all of the mutations leading to expression of high levels of ampicillin and vancomycin resistance through the Ldtfm pathway. These investigations will identify and characterize critical and redundant ?-lactam resistance mechanisms in a bacterial species in which ?-lactam resistance is absolutely essential to its propagation in the hospital setting. They will also enhance our understanding of cell wall synthesis mechanisms in Gram-positive cocci and reveal promising new targets for antibacterial therapy.

Public Health Relevance

Enterococcus faecium is a common and increasingly important cause of nosocomial infections. Its characteristic high level of resistance to ?-lactam antibiotics limits treatment and promotes dissemination. This project aims to identify and characterize the complex and redundant mechanisms of ?-lactam resistance in an effort to better understand enterococcal physiology and promote identification of novel therapeutic targets.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Huntley, Clayton C
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Rhode Island Hospital
United States
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García-Solache, Mónica; Rice, Louis B (2016) Draft Genome Sequence of Vancomycin-Susceptible, Ampicillin-Intermediate Enterococcus faecium Strain D344RRF. Genome Announc 4:
Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica et al. (2016) Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faeci MBio 7:e02188-15
García-Solache, Mónica; Rice, Louis B (2016) Genome Sequence of the Multiantibiotic-Resistant Enterococcus faecium Strain C68 and Insights on the pLRM23 Colonization Plasmid. Genome Announc 4:
Triboulet, Sébastien; Bougault, Catherine M; Laguri, Cédric et al. (2015) Acyl acceptor recognition by Enterococcus faecium L,D-transpeptidase Ldtfm. Mol Microbiol 98:90-100
Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole et al. (2014) Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales. Antimicrob Agents Chemother 58:1749-56
Triboulet, Sébastien; Dubée, Vincent; Lecoq, Lauriane et al. (2013) Kinetic features of L,D-transpeptidase inactivation critical for ?-lactam antibacterial activity. PLoS One 8:e67831
Lecoq, Lauriane; Dubée, Vincent; Triboulet, Sébastien et al. (2013) Structure of Enterococcus faeciuml,d-transpeptidase acylated by ertapenem provides insight into the inactivation mechanism. ACS Chem Biol 8:1140-6
Papp-Wallace, Krisztina M; Senkfor, Baui; Gatta, Julian et al. (2012) Early insights into the interactions of different ?-lactam antibiotics and ?-lactamase inhibitors against soluble forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3. Antimicrob Agents Chemother 56:5687-92
Galloway-Pena, Jessica R; Rice, Louis B; Murray, Barbara E (2011) Analysis of PBP5 of early U.S. isolates of Enterococcus faecium: sequence variation alone does not explain increasing ampicillin resistance over time. Antimicrob Agents Chemother 55:3272-7
Triboulet, Sébastien; Arthur, Michel; Mainardi, Jean-Luc et al. (2011) Inactivation kinetics of a new target of beta-lactam antibiotics. J Biol Chem 286:22777-84

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