Related Experiment Videos
Inhibition of peptidoglycan biosynthesis by ramoplanin
1Department of Biochemistry, University of Cambridge, United Kingdom.
Abstract:
Ramoplanin, a new lipoglycopeptide antibiotic, inhibits cell wall peptidoglycan biosynthesis in gram-positive bacteria. In both Staphylococcus aureus and Bacillus megaterium, UDP-N-acetylmuramyl-pentapeptides (UDP-MurNAc-pentapeptides) accumulated at concentrations of ramoplanin close to the MIC, indicating that inhibition of peptidoglycan biosynthesis occurred after formation of cytoplasmic precursors. Susceptible bacteria bound or accumulated approximately 5 x 10(4) molecules of ramoplanin per cell, only 1/100th of the amount of vancomycin which binds to groups within peptidoglycan conforming to the pattern L-alpha alpha (amino acid)-D-alpha alpha-D-alpha alpha, suggesting that ramoplanin has a different target site. This was confirmed by in vitro studies involving a wall-membrane particulate fraction from Gaffkya homari in which peptidoglycan synthesis from UDP-MurNAc-tetrapeptide was inhibited by ramoplanin but not by vancomycin. The incorporation of peptidoglycan precursors into nascent peptidoglycan of a toluenized cell preparation of B. megaterium was inhibited by ramoplanin, indicating that the antibiotic acts at a step before transpeptidation. In vitro studies of a wall-membrane particulate fraction of B. megaterium indicated that ramoplanin did not prevent the formation of lipid intermediate I (undecaprenyl-P-P-MurNAc-pentapeptide) but inhibited the next reaction in which N-acetylglucosamine is transferred to that lipid intermediate. The high concentrations required to inhibit in vitro peptidoglycan-synthesizing systems probably reflect the high concentrations of target sites present. High concentrations of ramoplanin also damage certain properties of the cell membrane, but low concentrations only affected wall synthesis in intact bacteria without perturbing membrane function. These studies indicate that the primary target of ramoplanin is peptidoglycan biosynthesis and that the probable reaction inhibited is the N-acetylglucosaminyltransferase-catalyzed conversion of lipid intermediate I to lipid intermediate II.
Insights
Ramoplanin, a lipoglycopeptide antibiotic, stops bacterial cell wall peptidoglycan biosynthesis. It targets the transfer of N-acetylglucosamine, crucial for forming lipid intermediate II in gram-positive bacteria.
Area of Science:
- Microbiology
- Antibiotic Resistance
- Biochemistry
Background:
- Ramoplanin is a novel lipoglycopeptide antibiotic with activity against gram-positive bacteria.
- Understanding the precise mechanism of action for new antibiotics is crucial for combating bacterial infections.
- Peptidoglycan biosynthesis is a validated target for antibacterial agents.
Purpose of the Study:
- To elucidate the specific molecular target and mechanism of action of ramoplanin.
- To compare ramoplanin's inhibitory site with that of vancomycin.
- To determine if ramoplanin affects cytoplasmic precursor synthesis or cell wall assembly.
Main Methods:
- Assessed the accumulation of UDP-N-acetylmuramyl-pentapeptides (UDP-MurNAc-pentapeptides) in Staphylococcus aureus and Bacillus megaterium.
- Quantified ramoplanin binding to susceptible bacterial cells.
- Performed in vitro studies using cell wall-membrane particulate fractions from Gaffkya homari and B. megaterium to assess inhibition of peptidoglycan synthesis.
- Investigated the effect of ramoplanin on lipid intermediate formation and precursor incorporation in toluenized B. megaterium cells.
Main Results:
- Ramoplanin inhibited peptidoglycan biosynthesis after the formation of cytoplasmic precursors, evidenced by UDP-MurNAc-pentapeptide accumulation.
- Ramoplanin bound to cells at significantly lower concentrations than vancomycin, suggesting a different target.
- In vitro studies confirmed ramoplanin inhibits peptidoglycan synthesis at a step before transpeptidation, specifically the N-acetylglucosaminyltransferase step.
- Ramoplanin inhibited the conversion of lipid intermediate I to lipid intermediate II, without affecting lipid intermediate I formation.
Conclusions:
- Ramoplanin's primary target is peptidoglycan biosynthesis in gram-positive bacteria.
- The antibiotic specifically inhibits the N-acetylglucosaminyltransferase-catalyzed transfer of N-acetylglucosamine to lipid intermediate I.
- Ramoplanin acts at a distinct site compared to vancomycin, offering a potential alternative in combating resistant strains.