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Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus
Abstract:
The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.
Insights
Inactivating the FemB enzyme in Staphylococcus aureus increases susceptibility to beta-lactam antibiotics. This is linked to reduced glycine in the cell wall, impacting its structure and function.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant public health threat.
- Beta-lactam antibiotics are crucial for treating S. aureus infections.
- The mechanisms underlying MRSA resistance and cell wall synthesis are complex and incompletely understood.
Purpose of the Study:
- To investigate the role of the FemB protein in Staphylococcus aureus cell wall synthesis.
- To determine the effect of FemB inactivation on susceptibility to beta-lactam antibiotics.
- To elucidate the contribution of FemB to peptidoglycan structure and function.
Main Methods:
- Genetic inactivation of the femB gene using Tn551 insertion.
- Assessment of beta-lactam antibiotic susceptibility in femB mutant strains.
- Quantification of glycine content in peptidoglycan.
- Analysis of peptidoglycan cross-linking using high-pressure liquid chromatography.
- Electron microscopy to examine cell wall morphology and cell division.
Main Results:
- FemB inactivation increased susceptibility to beta-lactam antibiotics, similar to femA inactivation.
- FemB inactivation reduced peptidoglycan glycine content by approximately 40%.
- Reduced glycine led to decreased peptidoglycan cross-linking and shorter peptide chains.
- Electron microscopy revealed aberrant cell wall formation and impaired cell separation in femB mutants.
Conclusions:
- The complete pentaglycine interpeptide bridge, involving FemB, is essential for S. aureus sensitivity to beta-lactam antibiotics.
- FemB plays a critical role in maintaining normal cell wall synthesis and integrity.
- FemB inactivation results in significant alterations in peptidoglycan structure, leading to phenotypic changes and increased antibiotic susceptibility.