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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI045626-12
Application #
8287158
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Huntley, Clayton C
Project Start
2001-03-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
12
Fiscal Year
2012
Total Cost
$373,233
Indirect Cost
$94,182
Name
Rhode Island Hospital
Department
Type
DUNS #
075710996
City
Providence
State
RI
Country
United States
Zip Code
02903
Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole et al. (2014) Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales. Antimicrob Agents Chemother 58:1749-56
Triboulet, Sebastien; Arthur, Michel; Mainardi, Jean-Luc et al. (2011) Inactivation kinetics of a new target of beta-lactam antibiotics. J Biol Chem 286:22777-84
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
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Sacco, Emmanuelle; Hugonnet, Jean-Emmanuel; Josseaume, Nathalie et al. (2010) Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faecium. Mol Microbiol 75:874-85
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Endimiani, Andrea; Hujer, Andrea M; Perez, Federico et al. (2009) Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA. J Antimicrob Chemother 63:427-37
Rice, Louis B; Carias, Lenore L; Rudin, Susan et al. (2009) Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faecium. J Bacteriol 191:3649-56
Rice, Louis B; Lakticova, Viera; Carias, Lenore L et al. (2009) Transferable capacity for gastrointestinal colonization in Enterococcus faecium in a mouse model. J Infect Dis 199:342-9

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