Enterococci are among the most common bacteria isolated in nosocomial infections in the United States. Optimal antimicrobial therapy for serious enterococcal infections requires the use of synergistic combinations of a cell wall-active agent, such as ampicillin or vancomycin,with an aminoglycoside, which results in bactericidal activity against enterococci. However, many enterococcal strains have acquired aminoglycoside resistance genes that encode aminoglycoside-modifying enzymes, which eliminate this synergistic bactericidal effect. Thus, in many medical centers resistance to aminoglycosides has emerged as a major therapeutic challenge, precluding optimal therapy for the majority of enterococcal infections. High-level resistance to the clinically important aminoglycoside gentamicin in enterococci is mediated by APH(2"""""""") enzymes. We propose to study these aminoglycoside phosphotransferases in detail by (1) performing kinetics studies and investigating chemical mechanisms of these aminoglycoside-modifying enzymes; (2) investigating the roles that conserved amino acid residues play in the catalytic mechanism; and (3) studying evolution of one of these enterococcal enzymes toward higher levels and broader spectrums of aminoglycoside resistance. A clearer understanding of the interaction between these enzymes and their substrates (both aminoglycosides and the ATP cofactor), and of the evolution of the enzymes to produce a more diverse array would facilitate the development of novel aminoglycosides, either as poor substrates of the enzymes or as enzyme inhibitors, to overcome resistance conferred by these enzymes. The evolution of one of these enterococcal aminoglycoside phosphotransferases toward higher levels and broader spectrums of aminoglycoside resistance will enable predictions of which aminoglycoside antibiotics are more likely to retain their activity in the future as resistance evolves toward other aminoglycosides. Progress in either of these directions would be an important advance in the treatment of serious enterococcal infections. Effective treatment of serious infections caused by antibiotic-resistant enterococci has become a major clinical challenge. We propose to study mechanisms of resistance in enterococci to a class of antibiotics called the aminoglycosides. Results from our study will help in the development of new antibiotics and approaches to treat enterococcal infections, which are emerging rapidly as a serious threat to public health.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI057393-03
Application #
7350197
Study Section
Special Emphasis Panel (ZRG1-DDR (01))
Program Officer
Perdue, Samuel S
Project Start
2006-02-15
Project End
2011-01-31
Budget Start
2008-02-01
Budget End
2009-01-31
Support Year
3
Fiscal Year
2008
Total Cost
$321,486
Indirect Cost
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha et al. (2014) Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia. Acta Crystallogr D Biol Crystallogr 70:1561-71
Smith, Clyde A; Toth, Marta; Weiss, Thomas M et al. (2014) Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia revealed by crystallographic and small-angle X-ray scattering analysis. Acta Crystallogr D Biol Crystallogr 70:2754-64
Bhattacharya, Monolekha; Toth, Marta; Smith, Clyde A et al. (2013) Bulky ""gatekeeper"" residue changes the cosubstrate specificity of aminoglycoside 2''-phosphotransferase IIa. Antimicrob Agents Chemother 57:3763-6
Smith, Clyde A; Frase, Hilary; Toth, Marta et al. (2012) Structural basis for progression toward the carbapenemase activity in the GES family of ?-lactamases. J Am Chem Soc 134:19512-5
Smith, Clyde A; Toth, Marta; Frase, Hilary et al. (2012) Aminoglycoside 2''-phosphotransferase IIIa (APH(2'')-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2'' kinases. J Biol Chem 287:12893-903
Toth, Marta; Vakulenko, Sergei B; Smith, Clyde A (2012) Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6'-acetyltransferase AAC(6')-Im. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:472-5
Frase, Hilary; Toth, Marta; Vakulenko, Sergei B (2012) Revisiting the nucleotide and aminoglycoside substrate specificity of the bifunctional aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2'')-Ia enzyme. J Biol Chem 287:43262-9
Toth, Marta; Frase, Hilary; Antunes, Nuno Tiago et al. (2010) Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2''-IVa. Protein Sci 19:1565-76
Toth, Marta; Frase, Hilary; Chow, Joseph W et al. (2010) Mutant APH(2'')-IIa enzymes with increased activity against amikacin and isepamicin. Antimicrob Agents Chemother 54:1590-5
Young, Paul G; Walanj, Rupa; Lakshmi, Vendula et al. (2009) The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] provide insights into substrate selectivity in the APH(2'') subfamily. J Bacteriol 191:4133-43

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