B. subtilis LytR-CpsA-Psr Enzymes Transfer Wall Teichoic Acids from Authentic Lipid-Linked Substrates to Mature Peptidoglycan In Vitro

Abstract

Gram-positive bacteria endow their peptidoglycan with glycopolymers that are crucial for viability and pathogenesis. However, the cellular machinery that executes this function is not well understood. While decades of genetic and phenotypic work have highlighted the LytR-CpsA-Psr (LCP) family of enzymes as cell-wall glycopolymer transferases, their in vitro characterization has been elusive, largely due to a paucity of tools for functional assays. In this report, we synthesized authentic undecaprenyl diphosphate-linked wall teichoic acid (WTA) intermediates and built an assay system capable of monitoring LCP-mediated glycopolymer transfer. We report that all Bacillus subtilis LCP enzymes anchor WTAs to peptidoglycan in vitro. Furthermore, we probed the catalytic requirements and substrate preferences for these LCP enzymes and elaborated in vitro conditions for facile tests of enzyme function. This work sheds light on the molecular features of glycopolymer transfer and aims to aid drug discovery and development programs exploiting this promising antibacterial target.

Structure and Mechanism of LcpA, a Phosphotransferase That Mediates Glycosylation of a Gram-Positive Bacterial Cell Wall-Anchored Protein

“…Consistent with this notion, it has previously been demonstrated that S. pneumoniae CpsA2 and Corynebacterium glutamicum LcpA possess pyrophosphatase activity, and this is likely a characteristic of most LCP enzymes (7, 9–13). Recently, the cell wall ligase activity of the Bacillus subtilis and Staphylococcus aureus LCP enzymes has been reconstituted in vitro (11, 12).…”

Substrate Preferences Establish the Order of Cell Wall Assembly in Staphylococcus aureus

“…Most features of the structures are similar, but the orientations of the saccharide moieties and pyrophosphate are different, and only the structure with the disaccharide (LII A WTA ) contains a divalent cation in the active site (Figure S6). This finding is notable because LCPs are metal ion-dependent transferases (Figure S7) 12b,18–19 and we have previously shown that WTA precursors must contain at least a disaccharide to serve as substrates for transfer. 12b Although the N-acetyl glucosamine sugar of the LI WTA substrate makes several contacts with TagT, the corresponding GlcNAc of LII A WTA is oriented differently due to changes in the glycosidic linkage and pyrophosphate bonds (Figure S6).…”

Structure and mechanism of TagA, a novel membrane-associated glycosyltransferase that produces wall teichoic acids in pathogenic bacteria

“…The membrane fraction was diluted ten-fold for comparison with the supernatant and samples were run on an SDS-PAGE gel for 50 minutes at 170 V and transferred to a PVDF membrane using an iBlot transfer device (ThermoFisher Scientific). The membrane was fixed in methanol for five minutes, briefly washed in water, and then blocked over-day in TBST Blocking Buffer (20 mM Tris-HCl, pH 7.5; 500 mM NaCl; 0.05% Tween; and 5% w/v nonfat milk). The membrane was washed, incubated with primary antibody (Invitrogen #MA-21215, mouse anti-6 X His, 1:1000 dilution in TBST Blocking Buffer) overnight at 4°C, washed again, and incubated with secondary antibody (Sigma-Aldrich #A9044, anti-mouse horseradish peroxidase).…”