TY - JOUR
T1 - Exposing a β-lactamase "twist"
T2 - The mechanistic basis for the high level of ceftazidime resistance in the C69F variant of the Burkholderia pseudomallei PenI β-lactamase
AU - Papp-Wallace, Krisztina M.
AU - Becka, Scott A.
AU - Taracila, Magdalena A.
AU - Winkler, Marisa L.
AU - Gatta, Julian A.
AU - Rholl, Drew A.
AU - Schweizer, Herbert P.
AU - Bonomo, Robert A.
N1 - Funding Information:
We thank Michael E. Harris for helpful discussions of kinetics and the reviewers of the manuscript for their helpful suggestions. Research reported in this publication was supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs to K.M.P.-W. and R.A.B., the Veterans Affairs Career Development Program to K.M.P.-W., Veterans Affairs Merit Review Program Award 1I01BX002872 to K.M.P.-W., Veterans Affairs Merit Review Program Award 1I01BX001974 to R.A.B., Geriatric Research Education and Clinical Center grant VISN 10 to R.A.B., and National Institute of Allergy and Infectious Diseases of the National Institutes of Health award numbers R01 AI100560 and R01 AI063517 to R.A.B. and U54 AI03657 to H.P.S. M.L.W. was supported by Medical Scientist Training Program Training Grant Case Western Reserve University-T32 GM07250. HHS | National Institutes of Health (NIH) provided funding to Marisa L. Winkler, Herbert P. Schweizer, and Robert A. Bonomo under grant numbers AI100560, AI063517, AI03657, and GM07250. U.S. Department of Veterans Affairs (VA) provided funding to Krisztina M. Papp-Wallace and Robert A. Bonomo. The content is solely our responsibility and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PY - 2016/2
Y1 - 2016/2
N2 - Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a β-lactamase profoundly affects β-lactam resistance.
AB - Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a β-lactamase profoundly affects β-lactam resistance.
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U2 - 10.1128/AAC.02073-15
DO - 10.1128/AAC.02073-15
M3 - Article
C2 - 26596949
AN - SCOPUS:84957900117
SN - 0066-4804
VL - 60
SP - 777
EP - 788
JO - Antimicrobial Agents and Chemotherapy
JF - Antimicrobial Agents and Chemotherapy
IS - 2
ER -