Identifying peptidoglycan-related enzymes
The incorporation of D-amino acid (DAA)-based probes, such as FDAAs, into peptidoglycan (the main content of bacterial cell wall) is conducted by the peptidoglycan synthesis enzymes. There are different classes of enzymes involved in peptidoglycan synthesis and their roles can be reflected by the result of FDAA labeling. For enzymes that construct new peptidoglycan structures such as transpeptidases, up-regulation of their expression results in increased FDAA labeling intensity; while down-regulation (or inhibition using antibiotics) caused reduced labeling. Typical examples include the knocking out of penicillin-binding proteins or inhibition peptidoglycan precursor synthesis using antibiotics.
In contrast, for enzymes involved in peptidoglycan remodeling and degradation, such as autolysins, up-regulation of their expression gives lower FDAA labeling intensity; while down-regulation results in increased FDAA intensity. For instance, carboxypeptidases are small molecular weight penicillin-binding proteins that serve to trim the terminal amino acid residue from the stem peptide of peptidoglycan. Knocking out carboxypeptidases increases the intensity of FDAA intensity in both Gram-positive (B. subtilis) and Gram-negative species (E. coli). [1]
Based on these properties, DAA-based probes can potentially be employed to identify unknown enzymes involved in peptidoglycan synthesis pathways and their roles. One can manipulate the expression of the enzyme, such as overexpression or knocking out, and then perform FDAA labeling to compare the labeling intensity with the control group. However, a possible pitfall of this application is that enzymes that are not directly involved in the cell wall synthesis pathways could also have effects on FDAA labeling intensity (false-positive). This is because the cell wall synthesis is interrelated with many other cell growth mechanisms. For example, decreasing the rate of DNA duplication slows down cell growth processes, which also reduces the rate of new peptidoglycan formation. As a result, depleting DNA duplication enzymes very likely causes decreased FDAA labeling intensity. To address this issue, carefully designed control experiments are required.
References
[1] Kuru et al. In Situ Probing of Newly Synthesized Peptidoglycan in Live Bacteria with Fluorescent D-Amino Acids. Angew. Chem. 2012, 124, 12687-12691.
[Cover image] Radkov et al. Imaging Bacterial Cell Wall Biosynthesis. Annu. Rev. Biochem., 2018, 87:22.1-22.4.