The N -linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering

Abstract

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.

Potential targets for next generation antimicrobial glycoconjugate vaccines

“…pleuropneumoniae (Cuccui et al. 2017 ) could also give access to a broader range of hexose ending polysaccharides.…”

Synthetic Glycobiology: Parts, Systems, and Applications

“… 168 Other studies have developed short, optimized GlycTag sequences for NGT 80 and have shown that the modification of a target protein with these GlycTags (such as GGNWTT) can successfully direct efficient NGT glycosylation of diverse recombinant proteins in vivo and in vitro . 80 , 160 , 173 Later studies have found that the single glucose residue installed by NGT can be elaborated to a dextran polymer 160 (which could be useful for vaccines against pathogenic bacteria that use NGTs to adhere to human cells), polysialic acids 131 (which may prolong the serum-half-life of small therapeutic proteins), N -acetyllactosamine (LacNAc), 174 , 175 and other fucosylated and sialylated forms of lactose 174 , 175 by overexpression of elaborating GTs within the cell. 131 This sequential elaboration technique may also allow an NGT-based system to circumvent the limits on glycan structure found in OST systems.…”

How Sweet Are Our Gut Beneficial Bacteria? A Focus on Protein Glycosylation in Lactobacillus

“…Although HMW1 glycosylates proteins preferably at the consensus sequon Asn -Xxx-Thr/Ser, different amino acids can be tolerated in the third position [ 11 , 14 , 15 ]. Recently, the A. pleuropneumoniae glycosylation operon was used to add N -linked glycan consisting of 1–29 hexose units onto an acceptor protein in E. coli , illustrating the potential biotechnological application of this glycosylation system for glycoengineering [ 16 ].…”