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Asp50Glu mutation in MurA results in fosfomycin resistance in Enterococcus faecium
Ling Xin1, Zetao Hu2, Renru Han1
1Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China; Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, China.
Objectives:
Enterococcus faecium is one of the important pathogens causing nosocomial infection, which can be resistant to fosfomycin by obtaining the plasmid-encoded fosfomycin resistance genes, and the mutation of MurA protein encoded by chromosome is a newly discovered fosfomycin resistance mechanism in recent years.
Methods:
In this study, we found a fosfomycin-resistant clinical isolate of E. faecium Efm_1415 with fosfomycin MIC of 512 mg/L, carrying Asp50Glu mutant of MurA protein, which was never reported before. To study the role and mechanism of this mutant protein in fosfomycin resistance, we used gene cloning, protein expression, and purification, steady-state kinetic, fosfomycin inhibition assay, and next-generation sequencing (NGS) to investigate the functions, characters, and enzymatic kinetic properties of MurA protein.
Results:
The results revealed that the Asp50Glu MurA can mediate a 4-fold increase in the fosfomycin MIC of the host bacteria. Compared with the wild-type MurA, the affinity of the Asp50Glu MurA to the substrates was increased, and the enzyme activity cannot be inhibited by the concentration of fosfomycin less than 100 mg/L.
Conclusions:
The research on the mutant MurA had gained a new understanding of the fosfomycin resistance mechanisms and helped to find new antibiotics with MurA enzyme as the target of action.
Insights
A novel Asp50Glu mutation in the MurA protein of Enterococcus faecium confers significant fosfomycin resistance. This discovery deepens our understanding of antibiotic resistance mechanisms and aids in developing new MurA-targeted antibiotics.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Enterococcus faecium is a significant cause of hospital-acquired infections.
- Fosfomycin resistance in E. faecium can arise from plasmid-encoded genes or chromosomal mutations.
- Mutations in the MurA protein represent a recently identified mechanism of fosfomycin resistance.
Purpose of the Study:
- To investigate a novel Asp50Glu mutation in the MurA protein of a fosfomycin-resistant Enterococcus faecium clinical isolate.
- To elucidate the role and mechanism of the Asp50Glu MurA mutant in conferring fosfomycin resistance.
- To characterize the enzymatic properties of the mutant MurA protein.
Main Methods:
- Gene cloning, protein expression, and purification of wild-type and mutant MurA.
- Steady-state kinetic assays and fosfomycin inhibition studies.
- Next-generation sequencing (NGS) for comprehensive analysis.
Main Results:
- The Asp50Glu MurA mutant was identified in a fosfomycin-resistant E. faecium isolate (MIC = 512 mg/L).
- This mutation conferred a 4-fold increase in fosfomycin minimum inhibitory concentration (MIC).
- Asp50Glu MurA exhibited increased substrate affinity and resistance to fosfomycin inhibition (up to 100 mg/L).
Conclusions:
- The Asp50Glu MurA mutation is a novel mechanism contributing to fosfomycin resistance in E. faecium.
- Understanding this mechanism provides new insights into antibiotic resistance.
- This research facilitates the development of new antibiotics targeting the MurA enzyme.
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