Bacterial resistance to antimicrobial agents is a significant threat to the efficacy of antibiotic therapy. The ?-lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobials. The most common source of resistance to ?-lactam antibiotics is the production of ?-lactamases, which cleave the drugs and render them ineffective. ?-lactamases have been grouped into four classes based on primary sequence homology. Classes A, C and D are active-site serine enzymes that catalyze the hydrolysis of ?-lactam antibiotics via a serine-bound acyl-enzyme intermediate. Class B enzymes require zinc for activity and catalysis does not proceed via a covalent intermediate. Because of the wide range of substrate specificities of these enzymes, virtually all ?-lactam antibiotics are susceptible to hydrolysis. Clearly, the design of new antibiotics that escape hydrolysis by the growing collection of ?-lactamase activities will be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of each class of ?-lactamase. The goal of this work is to understand how the amino acid sequence determines the structure, activity and evolution of class A ?-lactamases. The TEM-1 and CTX-M family of class A enzymes are an important cause of resistance to ?-lactam antibiotics worldwide. In a previous funding period in vitro mutagenesis was used to randomize the entire coding sequence of the class A TEM-1 ?-lactamase. The sets of random mutants were used to identify residues that are critical for the folding, stability and substrate specificity of the enzyme. The recent advent and application of ultra-high throughput sequencing technology has allowed the scope of this approach to be vastly extended to accurately determine the sequence requirements for hydrolysis of multiple classes of ?-lactam antibiotics for every residue position in TEM-1 ?-lactamase. In addition, preliminary studies suggest that amino acid substitutions which increase the stability of TEM-1 ?-lactamase play an important role in the evolution of TEM-1-mediated resistance to extended-spectrum ?-lactam antibiotics and ?-lactamase inhibitors. These studies will be continued to understand how these changes influence the stability and activity of evolved enzyme variants. Finally, the CTX-M family of ?-lactamases exhibits a high degree of sequence variation and it is hypothesized that many of the amino acid substitutions observed alter the stability or substrate specificity of the enzymes. Experiments are proposed to test this hypothesis by examining the role of a selected set of amino acid substitutions on the structure, activity and evolution of CTX-M enzymes.

Public Health Relevance

This project addresses how bacterial resistance to ?-lactam antibiotics such as the penicillins and cephalosporins evolves. ?-lactamases catalyze the destruction of ?-lactam antibiotics and are the most common mechanism of resistance to these drugs. The proposed experiments will determine how amino acid substitutions in the sequence of ?-lactamase enzymes alter their structure, function and evolution, which will facilitate the design of new antimicrobial therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032956-22
Application #
8464622
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Huntley, Clayton C
Project Start
1992-07-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
22
Fiscal Year
2013
Total Cost
$367,775
Indirect Cost
$132,775
Name
Baylor College of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
051113330
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
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
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
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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