Discovery and characterization of a new class of O-linking oligosaccharyltransferases from the Moraxellaceae family

Abstract

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered E. coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii

“…Since the identification of bacterial glycosylation more than two decades ago, − a range of discrete bacterial glycosylation systems have been identified responsible for the modification of specific proteins, such as flagella or pilin, − as well as general glycosylation systems responsible for the modification of multiple proteins. , Within known glycosylation pathways, several classes of oligosaccharyltransferases have been identified which enable the en bloc transfer of complex glycans onto proteins from lipid-linked oligosaccharides within the periplasm of Gram-negative species. ,, Of the identified oligosaccharyltransferases, the Pilin-glycosylation ligases, also known as the PglL family of enzymes, , are now recognized to be widely distributed within Gram-negative genera and responsible for O -linked glycosylation across a range of species. ,,− Across the known PglL glycosylation systems, differences in the proteins ,,− and amino acid residues , were able to be modified as well as the compositions , and lengths ,, of glycans were able to be effectively transferred have now been observed supporting extensive diversity in PglL functionality. Consistent with this across the Moraxellaceae , which contains the Acinetobacter genera, divergent Moraxellaceae PglL homologues are now known to possess unique glycosylation targeting specificities . Due to the diversity within the PglL enzymes of the Moraxellaceae understanding the substrates of glycosylation is critical to dissecting the roles of these systems especially within poorly understood species such as the human pathogen Acinetobacter baumannii .…”

“…Consistent with this across the Moraxellaceae, which contains the Acinetobacter genera, divergent Moraxellaceae PglL homologues are now known to possess unique glycosylation targeting specificities. 37 Due to the diversity within the PglL enzymes of the Moraxellaceae understanding the substrates of glycosylation is critical to dissecting the roles of these systems especially within poorly understood species such as the human pathogen Acinetobacter baumannii. A. baumannii is an opportunistic pathogen associated with nosocomial infections including ventilator-associated pneumonia, urinary tract, and wound infections.…”

“…By reducing the triggering of ETD/EThcD events to only potential glycopeptide precursors optimal ETD reaction times and/or extended ion filling times can be undertaken. However, while this approach has been widely utilized for the study of bacterial glycopeptides ,, we recently noted two examples within Moraxella osloensis and N. gonorrheae , wherein alterations within EThcD triggering was essential to allow site localization due to the lack of HexNAc residues within glycopeptides derived from these species. The diversity within the glycan structures utilized for O -linked glycosylation by A. baumannii strains supports the potential benefit of alternative ETD-triggering approaches to further improve glycoproteomic analysis of A. baumannii strains.…”

CRISPRi-Mediated Silencing of Burkholderia O-Linked Glycosylation Systems Enables the Depletion of Glycosylation Yet Results in Modest Proteome Impacts

“…24−26 While early studies sought to confirm the activity of PglL enzymes using heterogeneous expression systems 27−29 or proteomic approaches, 15,24,30,31 recent studies have focused on expanding our understanding of their enzymatic capacities. These recent studies have demonstrated that PglL oligosacchar-yltransferases can possess unique targeting ranges 32−34 as well as diverse glycan tolerances, 35,36 yet little insights have been gained about the functional roles of these glycosylation systems within the bacterial species that possess them or the conserved properties of these enzymes across genera.…”

“…The PglL enzymes represent one of the largest families of oligosaccharyltransferases and have been confirmed to mediate O-linked glycosylation across a range of bacteria including Acinetobacter, , Francisella, Pseudomonas, − Neisseria, − Ralstonia, and Burkholderia species. − While early studies sought to confirm the activity of PglL enzymes using heterogeneous expression systems − or proteomic approaches, ,,, recent studies have focused on expanding our understanding of their enzymatic capacities. These recent studies have demonstrated that PglL oligosaccharyltransferases can possess unique targeting ranges − as well as diverse glycan tolerances, , yet little insights have been gained about the functional roles of these glycosylation systems within the bacterial species that possess them or the conserved properties of these enzymes across genera.…”