Monitoring bacterial cell wall turnover
Peptidoglycan is the main content of bacterial cell wall. The activities of peptidoglycan synthesis and degradation are highly dynamic and carefully controlled by bacteria. Peptidoglycan fragments are not only a valuable resource for re-building the wall during their life cycle but also key materials for bacterial infection. The DAA-probe-labeled peptidoglycan fragments are, therefore, powerful tools to study such a reconstruction process as well as cell-to-cell interaction during infection.
Studying the turnover rate of bacterial peptidoglycan
In some species, such as E. coli, peptidoglycan (PG) is rapidly turned over and the breakdown components are recycled for new PG formation. [1] The turnover and recycling pathways are mediated by specific gene sets, including PG hydrolases, transferases, and synthases. However, our knowledge about the mechanism is still very limited. How fast the PG structure is degraded and where the degraded components go remain unresolved in most of the species. D-amino acid-based probes, such as FDAAs or clickable DAAs, turn out to be wonderful tools to investigate the turnover process and the components trafficking because the labeled PG structures and degraded fragments can be tracked continuously using microscopy and also down-stream chromatography analyses. With long-pulse labeling, these probes enable homogeneous labeling of whole PG structures. Also, their mall molecular weight can minimize artificial effects during the recycling and trafficking processes.
A good example of investigating PG turnover using FDAAs is provided by Malcolm Winkler's laboratory at Indiana University. In this study, S. pneumoniae cells were first labeled with FDAA and the signal was monitored over several generations. They found that, unlike B. subtilis cells that have more than 50% PG structures being turned over between generations, wide-type S. pneumoniae has minimized PG turnover activity. The labeled old PG remained strongly fluorescent after several generations of the cell growth as shown below. [2]
S. pneumoniae has minimized PG turnover activity. [2]
Cell-cell interactions through degraded PG fragments
In addition to recycling the PG fragments, the release of PG fragments to the culture medium has been known in certain species. These breakdown components play important roles in signaling both to bacterial cells and eukaryotic cell hosts. In some species, the release PG fragments can induce the expression of β-lactamases, which increases the antibiotic resistance of the cells. [3] Some species also utilize PG fragments as signals to induce spore germination. [4]
Another special case of PG component trafficking was found in predatory Bdellovibrio bacteriovorus. Predatory B. bacteriovorus. are naturally antibacterial. It conducts infections by traversing, modifying and finally destroying the cell walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Elucidating these multi-enzymatic processes on two similar PG structures, the predator and the prey, have been challenging. Kuru et al. recently utilized FDAA labeling to visualize the dynamic interactions between the predator PG and prey PG between the invader and host cells, providing new insights into the infection mechanism of B. bacteriovorus. [5]
Fluorescence microscopy of B. bacteriovorus (invader, red) structures and E. coli (host, blue) PG structures. [5]
References
[1] Park et al. How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiol Mol Biol Rev. 2008. 2, 211-27
[2] Boersma et al. Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms. J. Bacteriology, 2015.
[3] Fisher et al. The sentinel role of peptidoglycan recycling in the beta-lactam resistance of the Gram-negative Enterobacteriaceae and Pseudomonas aeruginosa. Bioorg Chem. 2014, 56, 41-8.
[4] Shah et al. A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments. Cell. 2008, 135, 486-96.
[5] Kuru et al. Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation. Nature Microbiology, 2017, 2, 1648–1657