In the last two decades it has become increasingly clear that the efficacy of antibiotics for the treatment of infectious diseases is in jeopardy due to the common appearance of drug resistant strains of microorganisms. Understanding the mechanisms of antimicrobial resistance is crucial for effective patient care in the clinic and essential for developing strategies to enhance biodefense against intentionally disseminated of pathogens. Fosfomycin is a potent, broad-spectrum antibiotic effective against both Gram-positive and Gram-negative microorganisms. A decade after its introduction plasmid-mediated resistance to fosfomycin was observed in the clinic. Investigations supported by this project have established that the resistance is due to a metalloenzyme (FosA) that catalyzes the addition of glutathione to the antibiotic, rendering it inactive. Similar resistance elements have now been shown to exist in the genomes of several pathogenic microorganisms including, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus anthrasis, Brucella melitensis, Listeria monocytogenes and Clostridium botulinum. Genomic and biochemical analysis from this project suggest that there are three distinct subgroups of metalloenzymes, termed FosA, FosB and FosX, that confer resistance through somewhat different chemical mechanisms. The objectives of this research project are to identify plasmid and genomically encoded proteins involved in microbial resistance to fosfomycin and to elucidate the underlying structural and mechanistic enzymology of resistance. These objectives will be accomplished by integrating enzymological, biophysical and genomic analyses of the resistance problem. The three-dimensional structures of the FosA from Pseudomonas aeruginosa and its relatives FosB and FosX will be determined by X-ray crystallography. The chemical mechanisms of catalysis will be elucidated by: (i) examination of the inner coordination sphere of Mn 2+ in FosA and FosX by EPR and ENDOR spectroscopy; (ii) a steady state kinetic analysis of the thiol selectivity of FosA and FosB, and (iii) a mechanistic study of the unique hydration reaction catalyzed by FosX. Potential transition state inhibitors will investigated by structural, spectroscopic and kinetic techniques. The thermodynamics of the interaction of substrates and inhibitors with the enzymes will be examined by isothermal titration calorimetry Particular emphasis will be placed on the enzymes from the pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum. The intent of this investigation is to establish the mechanistic and structural bases for the design of drugs to counter both plasmid borne and genomically encoded resistance to fosfomycin.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI042756-05A2
Application #
6678647
Study Section
Biochemistry Study Section (BIO)
Program Officer
Perdue, Samuel S
Project Start
1998-02-01
Project End
2007-12-31
Budget Start
2003-07-01
Budget End
2003-12-31
Support Year
5
Fiscal Year
2003
Total Cost
$138,250
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Brown, Daniel W; Schaab, Matthew R; Birmingham, William R et al. (2009) Evolution of the antibiotic resistance protein, FosA, is linked to a catalytically promiscuous progenitor. Biochemistry 48:1847-9
Rigsby, Rachel E; Brown, Daniel W; Dawson, Eric et al. (2007) A model for glutathione binding and activation in the fosfomycin resistance protein, FosA. Arch Biochem Biophys 464:277-83
Fillgrove, Kerry L; Pakhomova, Svetlana; Schaab, Matthew R et al. (2007) Structure and mechanism of the genomically encoded fosfomycin resistance protein, FosX, from Listeria monocytogenes. Biochemistry 46:8110-20
Walsby, Charles J; Telser, Joshua; Rigsby, Rachel E et al. (2005) Enzyme control of small-molecule coordination in FosA as revealed by 31P pulsed ENDOR and ESE-EPR. J Am Chem Soc 127:8310-9
Rigsby, Rachel E; Rife, Chris L; Fillgrove, Kerry L et al. (2004) Phosphonoformate: a minimal transition state analogue inhibitor of the fosfomycin resistance protein, FosA. Biochemistry 43:13666-73
Pakhomova, Svetlana; Rife, Chris L; Armstrong, Richard N et al. (2004) Structure of fosfomycin resistance protein FosA from transposon Tn2921. Protein Sci 13:1260-5
Fillgrove, Kerry L; Pakhomova, Svetlana; Newcomer, Marcia E et al. (2003) Mechanistic diversity of fosfomycin resistance in pathogenic microorganisms. J Am Chem Soc 125:15730-1
Smoukov, Stoyan K; Telser, Joshua; Bernat, Bryan A et al. (2002) EPR study of substrate binding to the Mn(II) active site of the bacterial antibiotic resistance enzyme FosA: a better way to examine Mn(II). J Am Chem Soc 124:2318-26
Rife, Chris L; Pharris, Rachel E; Newcomer, Marcia E et al. (2002) Crystal structure of a genomically encoded fosfomycin resistance protein (FosA) at 1.19 A resolution by MAD phasing off the L-III edge of Tl(+). J Am Chem Soc 124:11001-3
Cao, M; Bernat, B A; Wang, Z et al. (2001) FosB, a cysteine-dependent fosfomycin resistance protein under the control of sigma(W), an extracytoplasmic-function sigma factor in Bacillus subtilis. J Bacteriol 183:2380-3

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