Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity

Abstract

SummaryThe structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the Oantigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

“…This study in combination with detailed microscopic investigations in our laboratory 4 revealed that the cell surface architecture of T. forsythia is so far unique, with a typical Gramnegative cell envelope profile being overlaid with an ϳ22-nmthick, closed, monolayer that is formed by co-assembly of the two T. forsythia S-layer glycoproteins. Whereas the TfsA and TsfB S-layer protein portions are antigenically diverse according to immunoblot evidence, 4 identical oligosaccharides are displayed at multiple sites on either S-layer protein via O-glycosidic linkage units between ␣-D-Galp and Thr and Ser residues, respectively, that are scattered over the whole S-layer proteins.…”

“…Whereas the TfsA and TsfB S-layer protein portions are antigenically diverse according to immunoblot evidence, 4 identical oligosaccharides are displayed at multiple sites on either S-layer protein via O-glycosidic linkage units between ␣-D-Galp and Thr and Ser residues, respectively, that are scattered over the whole S-layer proteins. According to combined 600.13-MHz 1 H NMR/LC-ESI-MS approaches, the T. forsythia S-layer oligosaccharide is an overall highly diverse and branched structure containing several modified glycose residues (compare with Fig.…”

“…Interestingly, in several of the investigated bacteria, a scenario of general glycosylation systems and of overlapping roles for distinct carbohydrate-active enzymes is evolving. For instance, the gastrointestinal pathogen Campylobacter jejuni targets multiple proteins at asparagine residues (2); the intestinal symbiont Bacteroides fragilis (3) and Neisseria species (1) possess general O-glycosylation systems; and in Pseudomonas aeruginosa, distinct stages of LPS, exopolysaccharide, and pilin glycoprotein biosynthesis share common enzymes (4,5).…”

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

“…This relaxed specificity is in contrast to the typical eukaryotic OST, where a terminal ␣1-2-linked glucose residue is of central importance for efficient glycosylation (28). Interestingly, it has been reported that the sugars transferred during pilin O glycosylation in P. aeruginosa are products of the O antigen biosynthetic pathway and that the process occurs with low glycan substrate specificity (22). Because O antigen polymerization takes place at the periplasmic side of the inner membrane (17), the efficient transfer of O polysaccharide to proteins by PglB confirmed that N-glycosylation in bacteria takes place in the periplasm.…”

“…Pseudomonas aeruginosa O11 (6,22,23) and the corresponding empty vector control pLAFR1 were kindly provided by Peter Castric (Duquesne University, Pittsburgh). The strains and plasmids used are listed in Table 1.…”