The goal of the proposed research is to characterize at the molecular level how mutations can alter the substrate profile of the clinically important TEM-1 beta-lactamase. These specific questions will be addressed: 1. What beta-lactamase mutations cause an increase in resistance to beta-lactam antibiotics? Every amino acid position in TEM-1 beta-lactamase will be randomized to sample all possible amino acid substitutions using a novel mutagenesis technique. All of the random substitutions will then be screened to identify those amino acid substitutions that result in increased beta-lactamase activity towards a set of clinically relevant beta-lactam antibiotics. 2. How do mutations change the substrate spectrum of beta-lactamase? Biochemical methods will be used to further characterize the new hydrolytic capacities of the specificity mutants. These studies will include purification of the mutant proteins and determination of kinetic parameters. The conformational stability of the mutant proteins also will be determined by a variety of methods. 3. What beta-lactamase mutations result in increase antibiotic resistance in clinical isolates? The information obtained from the above studies will be used to design oligonucleotide probes that will specifically detect genes encoding specificity mutants among TEM beta-lactamase-containing clinical isolates. The studies will test the predictive capacity of the random substitution experiments and provide specific information about the mechanisms of beta-lactam antibiotic resistance. In total, these studies will assess systematically the ability of TEM-1 beta-lactamase to expand its substrate spectrum by mutation. The information gained in these studies will be of immediate empirical value in predicting the susceptibility of a given antibiotic to mutational changes in the beta-lactamase. In the long term we would like to use the data to predict how beta-lactamase will respond to the selective pressure of antibiotic therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032956-03
Application #
2067899
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1992-07-01
Project End
1996-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Sun, Zhizeng; Hu, Liya; Sankaran, Banumathi et al. (2018) Differential active site requirements for NDM-1 ?-lactamase hydrolysis of carbapenem versus penicillin and cephalosporin antibiotics. Nat Commun 9:4524
Palzkill, Timothy (2018) Structural and Mechanistic Basis for Extended-Spectrum Drug-Resistance Mutations in Altering the Specificity of TEM, CTX-M, and KPC ?-lactamases. Front Mol Biosci 5:16
Patel, Meha P; Hu, Liya; Brown, Cameron A et al. (2018) Synergistic effects of functionally distinct substitutions in ?-lactamase variants shed light on the evolution of bacterial drug resistance. J Biol Chem 293:17971-17984
Patel, Meha P; Hu, Liya; Stojanoski, Vlatko et al. (2017) The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M ?-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation. Biochemistry 56:3443-3453
Adamski, Carolyn J; Palzkill, Timothy (2017) BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A ?-Lactamases. Biochemistry 56:1075-1084
Adamski, Carolyn J; Palzkill, Timothy (2017) Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A ?-lactamases. BMC Biochem 18:2
Stojanoski, Vlatko; Adamski, Carolyn J; Hu, Liya et al. (2016) Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine ?-Lactamases by Relieving Steric Strain. Biochemistry 55:2479-90
Chow, Dar-Chone; Rice, Kacie; Huang, Wanzhi et al. (2016) Engineering Specificity from Broad to Narrow: Design of a ?-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC ?-Lactamase. ACS Infect Dis 2:969-979
Adamski, Carolyn J; Cardenas, Ana Maria; Brown, Nicholas G et al. (2015) Molecular basis for the catalytic specificity of the CTX-M extended-spectrum ?-lactamases. Biochemistry 54:447-57
Patel, Meha P; Fryszczyn, Bartlomiej G; Palzkill, Timothy (2015) Characterization of the global stabilizing substitution A77V and its role in the evolution of CTX-M ?-lactamases. Antimicrob Agents Chemother 59:6741-8

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