Visualizing bacterial cell wall structures
Peptidoglycan, the main content of bacterial cell wall, defines cell boundaries and dictates bacterial growth throughout their life cycle. Direct visualization of the cell wall structures provides substantial information about bacterial growth dynamics and thus has become an indispensable approach for developing antibiotics, as well as improving bacteria applications, such as fermentation. Since bacteria were first described in the late 1600s, scientists have made great efforts to investigate peptidoglycan structures. The development of fluorescence microscopy and molecular probes have greatly improved our ability to study peptidoglycan and allowed time-lapse monitoring of its growth process.
Bacterial cell wall labeling using FDAAs
D-amino acid-based probes, such as FDAAs, are powerful tools to label and visualize bacterial peptidoglycan structures. First of all, these probes are highly biocompatible to the cells and also eco-friendly. FDAAs are made of a D-amino acid backbone and a fluorophore. They can be prepared using "green chemistry" without environmentally harmful materials, such as antibiotics. Second, FDAA labeling is highly specific to the cell wall structures and thus can generate precise and low-noise signals in microscopy. This is because nearly all biological reactions use L-amino acids exclusively. Bacterial cell wall, however, is an exception that contains a high level of D-amino acids in its structure. The D-amino acid-directed metabolic labeling, therefore, makes FDAAs highly specific to the cell wall and minimizes the non-specific labeling to other biomolecules. Examples of FDAA labeling, HADA in this case, are shown in the images below.
FDAA, HADA, labeling in various bacterial species [1]
Third, FDAA labeling is straightforward and low-cost. To visualize the whole structure of the cell wall, one simply incubates bacterial cells with FDAAs. This can be done by simply adding FDAAs (0.5 to 1 mM) into cell culture and allowing the cells to grow for 3 generations. Then the cells can be imaged using microscopy either with or without fixation. A washing step is required for removing the excess background signal when FDAAs are used, but not for RfDAAs. A simplified scheme of the labeling process is shown below.
There are alternative tools for bacterial cell wall labeling. Fluorescent wheat germ agglutinin (FWGA) is a very efficient probe that labels the glycan backbone of peptidoglycan in the cell wall. FWGA is made of a lectin (WGA) and a fluorophore. However, the giant size of the probe makes it difficult to penetrate the outer membranes, resulting in poor applicability to label the cell wall of Gram-negative species. FDAAs, on the other hand, have been successfully applied to near all Gram-negative and Gram-positive species. Another alternative of the cell wall labeling tool is fluorescently-labeled antibiotics that can bind to peptidoglycan specifically. These probes have better membrane permeability because they are small molecules. However, their antibiotic moiety might cause toxicity effects to the cells and result in artificial responses, which makes data interpretation more difficult.
Bacterial cell wall labeling using D-Ala-D-Ala dipeptides
Some bacterial species have high carboxypeptidase (CPases) activities which can remove the incorporated FDAAs from peptidoglycan, resulting in failed labeling. These enzymes serve to remodel the cell wall structures for its maturation by removing the terminal residue of the peptidoglycan stem peptide, where FDAA is incorporated into. To address this issue, one can use dipeptide probes that contain D-Ala-D-Ala backbone, such as clickable D-Ala-D-Ala dipeptide: EDA-DA. These probes are small enough to penetrate the inner membrane and incorporated into peptidoglycan precursors. Since the incorporation is C-/N-terminal orientated, we can ensure that the clickable tag remains on the 4th-position of the stem peptide, not at the terminal (5th) position. Therefore, carboxypeptidases can not remove the tag from the cell wall. These probes have been used to visualize the cell wall structure of infective Chlamydia trachomatis that possesses a ring-shaped peptidoglycan structure in the host cells.
Peptidoglycan structures revealed by EDA-DA in Chlamydia trachomatis [2]
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.
[2] Liechti et al. A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis. Nature. 2013, 506, 507-510.