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Reactions of the three AmpD enzymes of Pseudomonas aeruginosa
Weilie Zhang1, Mijoon Lee, Dusan Hesek
1Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Abstract:
A group of Gram-negative bacteria, including the problematic pathogen Pseudomonas aeruginosa, has linked the steps in cell-wall recycling with the ability to manifest resistance to β-lactam antibiotics. A key step at the crossroads of the two events is performed by the protease AmpD, which hydrolyzes the peptide in the metabolite that influences these events. In contrast to other organisms that harbor this elaborate system, the genomic sequences of P. aeruginosa reveal it to have three paralogous genes for this protease, designated as ampD, ampDh2, and ampDh3. The recombinant gene products were purified to homogeneity, and their functions were assessed by the use of synthetic samples of three bacterial metabolites in cell-wall recycling and of three surrogates of cell-wall peptidoglycan. The results unequivocally identify AmpD as the bona fide recycling enzyme and AmpDh2 and AmpDh3 as enzymes involved in turnover of the bacterial cell wall itself. These findings define for the first time the events mediated by these three enzymes that lead to turnover of a key cell-wall recycling metabolite as well as the cell wall itself in its maturation.
Insights
Pseudomonas aeruginosa possesses three AmpD proteases. AmpD recycles cell-wall components, while AmpDh2 and AmpDh3 degrade the cell wall itself, impacting antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas aeruginosa antibiotic resistance is linked to cell-wall recycling.
- The protease AmpD is crucial for cell-wall recycling and influences beta-lactam antibiotic resistance.
- P. aeruginosa has three AmpD paralogs: ampD, ampDh2, and ampDh3.
Purpose of the Study:
- To characterize the functions of the three AmpD paralogs in P. aeruginosa.
- To elucidate the roles of AmpD, AmpDh2, and AmpDh3 in cell-wall metabolism and antibiotic resistance.
Main Methods:
- Purification of recombinant AmpD, AmpDh2, and AmpDh3.
- Enzymatic assays using synthetic bacterial metabolites and peptidoglycan surrogates.
- Assessment of enzyme function in cell-wall recycling and turnover.
Main Results:
- AmpD was confirmed as the primary enzyme for cell-wall recycling.
- AmpDh2 and AmpDh3 were identified as enzymes involved in bacterial cell-wall turnover.
- The distinct roles of these three proteases in cell-wall metabolism were defined.
Conclusions:
- P. aeruginosa utilizes three distinct AmpD proteases with specialized functions.
- These findings clarify the mechanisms of cell-wall recycling and turnover in P. aeruginosa.
- Understanding these pathways may offer new strategies to combat antibiotic resistance.
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