Mutation S115T in IMP-Type Metallo-β-Lactamases Compensates for Decreased Expression Levels Caused by Mutation S119G

Area of Science:

• Microbiology
• Enzymology
• Drug Resistance

Background:

• Metallo-β-lactamases (MBLs) confer resistance to critical antibiotics like carbapenems.
• The rapid emergence of MBL variants poses a significant clinical challenge due to a lack of inhibitors.
• Horizontal gene transfer fuels the diversity and spread of MBL resistance genes.

Purpose of the Study:

• To investigate the functional impact of specific mutations (S115T and S119G) in IMP-family metallo-β-lactamases.
• To compare the resistance profiles and biochemical properties of single and double mutants relative to IMP-1.
• To understand the evolutionary mechanisms driving the development of enhanced MBL variants.

Main Methods:

• Site-directed mutagenesis was used to generate IMP-1 variants with S115T and/or S119G mutations.
• Antibiotic resistance levels, in-cell expression, and enzyme kinetics were determined for each variant.
• Circular dichroism and Zn(II) content analysis assessed enzyme structure and stability.

Main Results:

• All four MBL variants conferred resistance to most tested β-lactam antibiotics, except aztreonam.
• Single mutants exhibited deficiencies: IMP-1S115T showed reduced activity, while IMP-1-S119G had decreased stability and expression.
• The double mutant (S115T/S119G) lacked the defects observed in single mutants, indicating a compensatory role.

Conclusions:

• The S119G mutation likely enhances MBL enzyme activity.
• The S115T mutation appears to compensate for stability and expression defects caused by the S119G mutation.
• These findings provide insights into the adaptive evolution of drug-resistant metallo-β-lactamases.