Clickable D-Amino Acids
Fluorescent D-amino acids (FDAAs) are outstanding tools for bacterial peptidoglycan labeling. However, coupling a fluorophore to an amino acid significantly increases the size of the molecule, which leads to low membrane permeability and reduced peptidoglycan labeling efficiency in Gram-negative species. To address this issue, clickable D-amino acids have been designed and synthesized. Instead of fluorophores, clickable D-amino acids contain an alkyne tag that is small enough to circumvent the membrane barrier and also inert enough to prevent non-specific interactions to bio-molecules. Once clickable D-amino acids incorporate into peptidoglycan, one can perform a "click reaction" that conjugates the alkyne tag to an azide-containing fluorophore. As a result, these probes can be visualized using microscopy. (Fixation and permeabilizing the cells might be required if the fluorophore is too big to penetrate the cell membranes.)
There are two classes of clickable D-amino acids. The clickable monopeptide, ethynyl D-Alanine (EDA), incorporates into peptidoglycan through transpeptidase activity, the incorporation pathway of FDAAs. On the other hand, clickable dipeptide, ethynyl D-Alanine-D-Alanine (EDA-DA), incorporates into peptidoglycan through a very different mechanism as explained in the following sections. [1] [2]
Mechanism of EDA-DA incorporation
The synthesis of peptidoglycan precursor (lipid II) involves a ligation reaction where a D-Alanine-D-Alanine dipeptide is coupled to a saccharide backbone. [3] Inspired by the enzyme promiscuity of peptidoglycan synthases, we prepared a clickable dipeptide, EDA-DA, and hypothesized that it can incorporate into lipid II through the ligases' activity. This hypothesis was confirmed by labeling experiments where EDA-DA was found in the peptidoglycan polypeptide with the ethynyl group located at the 4th position. This result was also supported by the fact that EDA-DA treatment resued the E. coli cells whose D-Alanine-D-Alanine synthases were inhibited by antibiotics. [1] Based on these data, we concluded that EDA-DA incorporates into lipid II in the cytoplasm. Since its incorporation can only be detected (by coupling to fluorophores through click chemistry) after the modified lipid II is flipped across the inner membrane and polymerized into the existing peptidoglycan layers, EDA-DA becomes a powerful tool to detect the transglycosylation reaction that is essential and responsible for peptidoglycan polymerization.
Proposed mechanism of EDA-DA incorporation. [4]
Preventing CPase hydrolase activity
As mentioned above, EDA-DA incorporates into peptidoglycan with the ethynyl group located at the 4th position of the polypeptide. On the other hand, FDAA and EDA incorporate into the the 5th position of peptidoglycan polypeptide. In some bacterial species, such as Chlamydia trachomatis, carboxypeptidases trim off the 5th-position residue from the polypeptide for peptidoglycan remodeling. This could result in false-negative FDAA labeling signal in the experiments. However, having the fluorescent and/or clickable tag located at the 4th position of the polypeptide circumvents carboxypeptidases' activities, giving a stable, reliable labeling signal for studying peptidoglycan synthesis. [1]
References
[1] Liechti et al. A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis. Nature. 2013, 506, 507-510.
[2] Liechti et al. Pathogenic Chlamydia lack a ‘classical’ sacculus but synthesize a narrow, midcell peptidoglycan ring, regulated by MreB, for cell division. PLoS Pathog. 2016, 12(5):e1005590.
[3] Typas et al. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol. 2011. 10(2):123-36
[4] Hsu et al. D-Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis. Acc Chem Res. 2019. doi: 10.1021/acs.accounts.9b00311.