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Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated
A M di Guilmi1, N Mouz, L Martin
1Institut de Biologie Structurale Jean-Pierre Ebel (CEA/CNRS), 38027 Grenoble Cedex 1, France.
Abstract:
Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.
Insights
Penicillin-binding proteins (PBPs) mediate bacterial cell wall synthesis. Studies reveal Streptococcus pneumoniae PBP 2a exhibits differential beta-lactam resistance, crucial for developing new antibiotics.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Penicillin-binding proteins (PBPs) are essential bacterial enzymes involved in peptidoglycan synthesis.
- Antibiotic resistance in Streptococcus pneumoniae is often linked to altered PBP structures with reduced affinity for beta-lactams.
- Streptococcus pneumoniae PBP 2a possesses both glycosyltransferase (GT) and transpeptidase (TP) activities, making it a key target for resistance studies.
Purpose of the Study:
- To investigate the structural and functional characteristics of Streptococcus pneumoniae PBP 2a, focusing on its domains involved in beta-lactam resistance.
- To determine the kinetic parameters of PBP 2a for various beta-lactams to understand resistance mechanisms.
- To elucidate the membrane interaction and solubility properties of PBP 2a and its domains.
Main Methods:
- Expression of the extracellular region of S. pneumoniae PBP 2a (PBP 2a*) as a glutathione S-transferase fusion protein in E. coli.
- Stopped-flow fluorometry to measure acylation kinetic parameters of PBP 2a* with beta-lactams.
- Limited proteolysis to purify the transpeptidase (TP) domain of PBP 2a.
- Interaction studies with lipid vesicles to assess membrane association.
Main Results:
- PBP 2a* showed significantly higher acylation efficiency with cefotaxime compared to benzylpenicillin.
- These findings suggest PBP 2a contributes to resistance against cefotaxime and related beta-lactams, but not benzylpenicillin.
- The PBP 2a* protein required detergents for solubility and interacted with lipid vesicles, while its TP domain was water-soluble.
Conclusions:
- PBP 2a plays a role in Streptococcus pneumoniae's resistance to specific beta-lactams like cefotaxime.
- The distinct solubility and membrane interaction properties of PBP 2a and its TP domain provide insights into its function.
- Understanding PBP 2a's interaction with the cytoplasmic membrane is vital for designing novel antibiotics targeting resistant bacterial strains.