Fluorogenic d-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays

Abstract

Peptidoglycan (PG) is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions which construct peptide cross-linkages between polysaccharide chains to endow mechanical strength. However, tracking transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis, and application of rotor-fluorogenic D-amino acids (RfDAAs) enabling real-time, continuous tracking of transpeptidation reactions. These probes enable monitoring PG biosynthesis in real time through visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D-, L,D-transpeptidases, and sortases in vitro . The unique ability of RfDAAs to become fluorescent when incorporated into PG provides a powerful new tool to study PG biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of PG biosynthesis.

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

“…Given the significant inhibition observed for both DCS and β-lactams—and the well-characterized inhibitory activity of the β-lactams toward the bacterial transpeptidases—we postulated that DCS, a cyclized D -serine analogue with a free amino group (Figure 1b), might behave as a competitive inhibitor of periplasmic L , D - and/or D , D -TPases. In support of this hypothesis, both LdtA Vc , a representative L,D-TPase from Vibrio cholerae , 56 and PBP4 Sa , a soluble version of recombinant PBP4 from Staphylococcus aureus , 17 incorporated DCS into appropriate soluble substrate analogues in vitro : 13 LdtA Vc incorporated DCS into tetrapeptides comparably to D -methionine, a DAA that is naturally produced by Vibrio cholerae cells 2 (Figure 2b); the product of each reaction was confirmed by MS (Figure SI2a). PBP4 Sa incorporated DCS into a synthetic N α , N ε -diacetyl- L -Lys- D -Ala- D -Ala tripeptide (3P) albeit to a lower extent when compared to identical experiments utilizing D -methionine (Figure 2c).…”

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

“…This is most likely because the molecular weight of OGDA (498 Da) is close to the molecular weight cut-off for porins in the outer membrane in E. coli (500-600 Da) [22]. This problem has been noted for most FDAAs [11,12].…”

“…It has previously been documented that most FDAAs do not work in Gram-negative bacteria because they cannot cross the outer membrane [11, 12]. This membrane acts as a permeability barrier to most chemical compounds, in particular those that are larger than 500–600 Da [22].…”

“…Once integrated, they can be visualized by fluorescence microscopy. The VanNieuwenhze laboratory has developed more than 10 different types of FDAAs [9, 10], as well as rotor-fluorogenic versions (RFDAAs) [11]. This collection includes fluorophores that span the entire visible spectrum, providing options for multi-colour labelling and imaging of the PG by sequential addition of different FDAAs.…”

An RGB-emitting molecular cocktail for the detection of bacterial fingerprints

“…[16][17][18] Peptidoglycan, one of the major components of bacterial cell walls, is a multi-layer network structure composed of glycans and short peptides, and it has been labeled to study the synthesis of bacterial cell walls. [19][20][21][22][23] Modied monosaccharides and amino acids have been shown to be incorporated into the peptidoglycan construction without affecting the normal growth, metabolism, or multiplication of bacteria, and have been used to efficiently label bacteria for the purpose of distinguishing them. Specically, uorophore-modied D-amino acids have been developed for the metabolic labeling of bacteria.…”