d -Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis

The bacterial cell wall is composed of membrane layers and a rigid yet flexible scaffold called peptidoglycan (PG). PG provides mechanical strength to enable bacteria to resist damage from the environment and lysis due to high internal turgor. PG also has a critical role in dictating bacterial cell morphology. The essential nature of PG for bacterial propagation, as well as its value as an antibiotic target, has led to renewed interest in the study of peptidoglycan biosynthesis. However, significant knowledge gaps remain that must be addressed before a clear understanding of peptidoglycan synthesis and dynamics is realized. For example, the enzymes involved in the PG biosynthesis pathway have not been fully characterized. Our understanding of PG biosynthesis has been frequently revamped by the discovery of novel enzymes or newly characterized functions of known enzymes. In addition, we do not clearly know how the respective activities of these enzymes are coordinated with each other and how they control the spatial and temporal dynamics of PG synthesis. The emergence of molecular probes and imaging techniques has significantly advanced the study PG synthesis and modification. Prior efforts utilized the specificity of PG-targeting antibiotics and proteins to develop PG-specific probes, such as fluorescent vancomycin and fluorescent wheat germ agglutinin. However, these probes suffer from limitations due to toxic effects toward bacterial cells and poor membrane permeability. To address these issues, we designed and introduced a family of novel molecular probes, fluorescent d-amino acids (FDAAs), which are covalently incorporated into PG through the activities of endogenous bacterial transpeptidases. Their high biocompatibility and PG specificity have made them powerful tools for labeling peptidoglycan. In addition, their enzyme-mediated incorporation faithfully reflects the activity of PG synthases, providing a direct in situ method for studying PG formation during the bacterial life cycle. In this Account, we describe our efforts directed at the development of FDAAs and their derivatives. These probes have enabled for the first time the ability to visualize PG synthesis in live bacterial cells and in real time. We summarize experimental evidence for FDAA incorporation into PG and the enzyme-mediated incorporation pathway. We demonstrate various applications of FDAAs, including bacterial morphology analyses, PG growth model studies, investigation of PG-enzyme correlation, in vitro PG synthase activity assays, and antibiotic inhibition tests. Finally, we discuss the current limitations of the probes and our ongoing efforts to improve them. We are confident that these probes will prove to be valuable tools that will enable the discovery of new antibiotic targets and expand the available arsenal directed at the public health threat posed by antibiotic resistance.