A Time‐Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW

Abstract

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time‐resolved Förster resonance energy transfer (TR‐FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non‐polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.

Advances and prospects of analytic methods for bacterial transglycosylation and inhibitor discovery

“…8B). 87 They prepared lipid II analogues, namely biotin-lipid II and DNP-lipid II, with lysine residues carrying biotin and 2,4-dinitrophenyl (DNP) tags, respectively. The lipid II analogues copolymerized by the catalysis of S. aureus FtsWI complex in the presence of Mn 2+ ion.…”

“…FRET occurred in the polymeric products because the donor and acceptor were in close proximity. The TR-FRET assay was adapted for high-throughput screening (HTS) of S. aureus-lethal compounds 87,88 that may function as TGase inhibitors. 84 Upon the TGase-catalyzed reaction, the dabsyl-containing lipid chain is cleaved.…”

“…(B) Biotin-lipid II and DNPlipid II copolymerize by the catalysis of S. aureus FtsWI complex in the presence of Mn 2+ ion. 87 Treatment of the polymeric products with anti-DNP Eu-cryptate antibody and Streptavidin-XL665 renders FRET.…”

“…8B) for high-throughput screening of a 900-compound library of S. aureus growth inhibitors. 87,88 Based on the result of microscale thermophoresis (MST), compound 18 selectively competes lipid II for the binding to S. aureus FtsW with a K d value of about 0.8 μM. The MIC value of compound 18 against S. aureus was 1 μM mL −1 , which is comparable to that of vancomycin.…”

Real-Time Biosynthetic Reaction Monitoring Informs the Mechanism of Action of Antibiotics

“…Synthesis of peptidoglycan, a highly conserved component of the bacterial cell wall and the target of many successful antibiotics, begins in the cytosol with the synthesis of disaccharide pentapeptide precursors which are then attached to C 55 -P by MraY to form lipid I . Lipid I is subsequently glycosylated into lipid II by MurG, and then transported across the membrane by flippases such as MurJ and Amj. − The carrier lipid is finally released as undecaprenyl diphosphate (C 55 -PP) after the glycopeptide moieties are incorporated into the nascent peptidoglycan polymer by the penicillin-binding proteins and other machineries (Figure A). − C 55 -PP is also synthesized de novo but must first be dephosphorylated before it can be used for translocating cell wall precursors . Carrier lipid molecules are dephosphorylated by phosphatases of the UppP or PAP2 families, , and are transported back across the plasma membrane by DedA and DUF368 family proteins ,, before re-entering the lipid II cycle .…”