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

Abstract

In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.

Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes

“…To assess the role of the GVN tripeptide and Y131 in SrtA functionality, we generated recombinant plasmids that express SrtA mutants with Y131A substitution, deletion of GVN (ΔGVN), replacement of GVN by alanine (3A), or combination of both mutations (Y131A/ ΔGVN). For functional studies here, we chose to test the individual plasmids in an srtA suppressor mutant, Δlcp/ΔsrtA, which was selected to disable the glycosylation step of GspA and hence prevent its toxicity (34,48). An analysis of membrane fractions of the transformed strains by immunoblotting established that none of the mutations affected SrtA expression and instability (Fig.…”

A Cell-based Screen in Actinomyces oris to Identify Sortase Inhibitors

“…Our previous studies have shown that reducing srtA gene expression in A. oris leads to cell death and that this phenotype is conditionally dependent on gspA expression 45,46 . To exploit this unique dependence, we developed a cell-based assay to screen for sortase inhibitors that compares the growth-inhibitory effects of small molecules cultured with A. oris MG-1 (wild-type) versus a ΔsrtA/ΔgspA strain.…”

“…The MIC and IC 50 data suggest that at least three of the hit molecules are capable of inhibiting Ao SrtA either within the context of the cell or as an isolated (2020) 10:8520 | https://doi.org/10.1038/s41598-020-65256-x www.nature.com/scientificreports www.nature.com/scientificreports/ transpeptidase. To understand the physiological effects that these molecules have on the display of pili and surface proteins on the surface of A. oris, we grew cells with 10 µM of each hit molecule and assessed sortase activity by western blot and electron microscopy as previously reported 45 . To avoid a confounding problem that inhibiting Ao SrtA causes cell arrest, we performed the experiments in a mutant devoid of gspA; the aforementioned genetic suppressor of sortase lethality.…”

“…Recently, Wu et al made the surprising discovery that the viability of the oral bacterium Actinomyces oris MG-1 in cell culture depends on the activity of its sortase ( A°S rtA) 45,46 . Ao SrtA anchors the glycosylated surface protein A (GspA) to the cell wall.…”

LytR-CpsA-Psr Glycopolymer Transferases: Essential Bricks in Gram-Positive Bacterial Cell Wall Assembly

“…The LCP homolog LcpA of A. oris provide a so far unique example of an LCP enzyme that is involved in a protein glycosylation process, i.e., the transfer of a saccharide moiety to an amino acid acceptor sequence [ 174 ] instead of a PGN backbone sugar. LcpA is genetically linked to GspA, a glycoprotein that is attached to A. oris PGN by the house-keeping sortase SrtA, which, in turn, recognizes the cell wall sorting signal of the glycoprotein [ 175 ].…”