Assaying the anti-cell wall ability of antibiotics
D-amino acid-based probes, such as FDAAs, incorporate into peptidoglycan (the main content of bacterial cell wall) through its synthesis enzymes. When peptidoglycan synthesis is inhibited, FDAA incorporation does not occur and no fluorescence signal is detected. Since their incorporation is highly specific to the peptidoglycan structures, D-amino acid-based probes have become powerful tools to detect the activity of peptidoglycan synthesis activity. This, from another perspective, also makes them great tools to assay the peptidoglycan inhibition ability of antibiotics. This information provides insights into the inhibition mechanism of antibiotics and could also improve and accelerate new antibiotic developments.
FDAAs reflect the anti-cell wall ability of antibiotics in vivo
Antibiotics can be generally separated into three different classes based on the biological targets they inhibit. The first class of antibiotics inhibits nucleotide synthesis. These antibiotics result in failed gene replication and thus lead to inhibited cell division or lysis. Examples include Rifamycin derivatives that inhibit RNA synthesis. The second class of antibiotics inhibits protein formation. This type of antibiotics often targets ribosome's function, such as Chloramphenicol. The third class of antibiotics inhibits bacterial cell wall synthesis, mostly peptidoglycan formation. Since the cell wall is essential for bacterial growth, disrupting its synthesis causes cell lysis or retarded growth/division. The very first antibiotic, penicillin, isolated in 1928, belongs to this class. Antibiotics targeting the cell wall synthesis are believed to have milder side effects compared to the other two classes because human cells do not contain the cell wall homologs.
FDAA labeling can be utilized to study whether an antibiotic has peptidoglycan inhibition ability. The proposed protocol includes the pre-incubation of bacterial cells with the antibiotic for 3-10 minutes, followed by a short pulse labeling of 10-50% doubling time of the species. The effects of antibiotic toward peptidoglycan synthesis then could be quantified using microscopy. An example conducted using well-studied antibiotics is shown below, where peptidoglycan inhibitors (vancomycin and ampicillin) led to a significant reduction of FDAA intensity; while off-targets (PC190723, kanamycin, chloramphenicol) had no significant impact on FDAA intensity. [1]
Optimizing antibiotic dose and treating time using FDAAs
The higher the antibiotic dose, the stronger the antibacterial effects are. However, the side effects of antibiotics also become more obvious at high concentration, so as longer administration period. FDAA labeling provides a new method to optimize the dose and treatment time required for destroying bacterial cell wall synthesis activity. This can be done by using the protocol abovementioned with varied antibiotic dose or incubation time. One example is shown below using ampicillin whose concentration was varied from 0 to 5X MIC (minimum inhibition concentration) in a 12-minutes incubation window. [1]
References
[1] 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.