Decorating Bacterial Surfaces By Designer Molecules Advances the Fundamental Knowledge about Bacterial Growth

In addition to the usual protein synthesis and unlike other domains of life, bacteria can and must make a second type of proteinaceous structure, namely their peptidoglycan (PG) cell walls. PG significantly differs from natural proteins by the presence of D-centered amino acids, it defines bacterial shape, growth and division and it is therefore an essential surface element. These properties in turn make PG also the best target for antibiotic development. Due to the lack of tools for spatiotemporal tracking of PG biosynthesis, we have developed a set of simple, yet versatile, ways to tag PG growth of virtually all bacterial species. Exploiting the surprisingly promiscuous nature of the PG metabolic enzymes and following a modular approach, we have designed and synthesized unnatural substrate analogs, such as so-called fluorescent D-amino acids (FDAAs)[1], that can hijack PG biosynthesis at different and mostly essential points of the pathway. Because they are non-toxic D-amino acid derivatives, these molecules specifically and efficiently label bacterial growth, opening the way to specifically engineer surfaces of live bacteria with any functionality conceivable even in complex environments. These tools have already had a profound impact on the field of bacterial cell biology and helped us to address crucial questions in the field. Case in point, we recently settled a 50 years long controversy and revealed presence of PG in sexually transmitted pathogen Chlamydia, which are sensitive to anti-PG drugs despite the failure of all previous attempts to show the presence of a functional PG wall in this bacterium (commonly referred to as the “Chlamydial anomaly”)[2]. More recently, we have improved upon the current probes and introduced a third class of labeling method, opening the way to three distinct fluorogenic PG labeling strategies.

In the near future, we believe that these novel approaches for PG labeling will find a common use as a diagnostic tool for infections, will expand the knowledge base required for new antibiotic discovery, and will represent the core designs for new antibacterials themselves. More generally, we believe that our success in hijacking PG biosynthesis with a library of distinct molecules suggests that PG pathway may be an inherently promiscuous scaffold for metabolic engineering of various polypeptide compounds. Furthermore, our work on fluorogenic D-amino acids, i.e. molecules that ‘turn on’ once incorporated into the PG, may eventually lay the groundwork for the design of genetically encodable fluorogenic L-amino acids as the preferred protein tags as opposed to much larger and fainter fluorescent proteins. 

REFERENCES

1.              Kuru, E., et al., In Situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angewandte Chemie, 2012. 51(50): p. 12519-23.

2.              Liechti, G.W., et al., A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis. Nature, 2013.