Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis

Abstract

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

Establishing rod shape from spherical, peptidoglycan-deficient bacterial spores

“…PG growth in many bacteria (22,23). B. subtilis (Gram-positive) and E. coil (Gram-negative) incorporate single D-amino acidbased fluorescent probes into PG using transpeptidases that catalyze the last steps of PG polymerization (24). We postulate that M. xanthus incorporates FDAAs in a similar manner.…”

The versatility of carbohydrates in antimicrobial applications

“…For example, VanNieuwenhze et al studied the peptidoglycan biosynthesis with fluorescent d ‐amino acids. [ 14 ] However, only a handful of reports directly take advantage of the carbohydrate‐based main chain, mainly because of difficulty and complexity in the synthesis of substrates. [ 15–16 ] To address this, our group attempted to design and synthesize such substrates from polymeric material by top‐down rather than bottom‐up strategy.…”

An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy

“…Fluorescent d -amino acids (FDAAs) are molecular probes that are widely used to label the PG layer. They are integrated by D,D- and L,D-transpeptidases, which exchange free d -amino acids and FDAAs into position 4 ( Escherichia coli ) or 5 ( B. subtilus ) of the stem peptide [8]. Once integrated, they can be visualized by fluorescence microscopy.…”