Structural Characterization of the Pseudomonas aeruginosa 1244 Pilin Glycan

Abstract

An antigenic similarity between lipopolysaccharide (LPS) and glycosylated pilin of Pseudomonas aeruginosa 1244 was noted. We purified a glycan-containing molecule from proteolytically digested pili and showed it to be composed of three sugars and serine. This glycan competed with pure pili and LPS for reaction with an LPS-specific monoclonal antibody, which also inhibited twitching motility by P. aeruginosa bearing glycosylated pili. One-dimensional NMR analysis of the glycan indicated the sugars to be 5N␤OHC 4 7NfmPse, Xyl, and FucNAc. The complete proton assignments of these sugars as well as the serine residue were determined by COSY and TOCSY. Electrospray ionization mass spectrometry (MS) determined the mass of this molecule to be 771.5. The ROESY NMR spectrum, tandem MS/MS analysis, and methylation analysis provided information on linkage and the sequence of oligosaccharide components. These data indicated that the molecule had the following structure: ␣-5N␤OHC 4 7NFmPse-(234)-␤-Xyl-(133)-␤-FucNAc-(133)-␤-Ser.Pseudomonas aeruginosa is an opportunistic pathogen capable of causing severe infections in individuals with compromised defense mechanisms (1). The somatic pili, protein filaments that extend as bundles from one or both of the cell poles, are considered to be a major virulence factor, promoting adherence and invasiveness (2, 3). These fibers are composed of a monomeric subunit, pilin, which has a strain-dependent molecular weight of ϳ16,000. The mature form of this protein is produced by the removal of a six-residue leader sequence, a process that is accompanied by methylation of the nascent amino-terminal phenylalanine (4). Although this had initially been considered the only post-translational modification of this protein, evidence has been generated indicating that P. aeruginosa strain 1244 pilin is also glycosylated (5). This process is dependent on pilO, a gene present as part of an operon that also contains the pilin structural gene, pilA.Although archeal and eubacterial S-layers commonly contain covalently bound glycan (6 -8), other examples of glycosylated prokaryotic surface proteins are rare. Cell wall-associated glycoproteins have been demonstrated in Streptococcus sanguis (9) and Mycobacterium tuberculosis (10). Among Gram-negative bacteria, Campylobacter species have been shown to contain a general protein glycosylation system that modifies a number of surface proteins including flagellin (11-13). Evidence has been presented that a glycan is associated with P. aeruginosa flagellin (14). As with P. aeruginosa (5), the pili of Neisseria meningitidis and Neisseria gonorrhoeae have been shown to be glycosylated. X-ray diffraction studies indicate that the N. gonorrhoeae pilin glycan is a disaccharide (15). In addition to glycerol phosphate (16), N. meningitidis pilin contains covalently bound trisaccharide (17).Although the detection of glycosylated bacterial surface proteins is uncommon, the comprehensive determination of their carbohydrate structure has been even more rare. Results present...

Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source

“…Given the similarity of the cj1321 to cj1326 genes to those involved in carbohydrate biosynthesis or sugar modifications, and the genes' location within the O-linked glycosylation locus, this result strongly suggests that these genes are involved in carbohydrate modification of the flagellum. Glycosylation of flagellin is increasingly being recognized in a number of Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa, Helicobacter pylori, and Aeromonas spp (22)(23)(24). The modifications increase the hydrophilicity of flagellin and often influence the cells' immunogenicity and their interaction with eukaryotic cells (22,23).…”

“…Glycosylation of flagellin is increasingly being recognized in a number of Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa, Helicobacter pylori, and Aeromonas spp (22)(23)(24). The modifications increase the hydrophilicity of flagellin and often influence the cells' immunogenicity and their interaction with eukaryotic cells (22,23). The biological significance of the glycosylation island in C. jejuni remains to be determined, but different glycoforms appear to be expressed in different hosts or environments and may provide them with a specific survival advantage (25).…”

Structural and Functional Analysis of Campylobacter jejuni PseG

“…Similar sequences have also been identified in the O-antigen biosynthetic loci of Pseudomonas aeruginosa serotype O7 and O9, ORF11 and ORF9, respectively (48). As these O-antigen biosyn-thetic loci also possess pseudaminic acid-like biosynthetic genes, and the fact that this sugar is known to decorate the lipopolysaccharide and pili of P. aeruginosa (49,50), we propose that these too are PseG members. Importantly, when performing a multiple sequence alignment using MUSCLE (51) with the PseG sequences described, a minimum consensus sequence (DX 5 GXGHX 2 R) for this family of proteins was identified (supplemental Fig.…”

Bacterial glycoproteomics

“…Over the last decade, the prevalence of reported glycoproteins has been best demonstrated in the cell-surface appendages of bacteria, such as pili and flagella. The pilin proteins of the pathogenic bacteria Neisseria meningitidis, Neisseria gonorrhoeae and Pseudomonas aeruginosa have all been demonstrated to be Oglycosylated (Castric et al, 2001;Hegge et al, 2004;Stimson et al, 1995). The pilin glycans from Neisseria species share a common structure, in particular with respect to the unusual O-linked sugar residue 2,4-diacetamido-2,4,6-trideoxyhexose (DATDH) (Hegge et al, 2004).…”

“…Data from chemical degradations are usually required to supplement molecular and fragment ion information to characterize structural features such as sugar type and branching. Another technique that has proved invaluable in carbohydrate structural determination is nuclear magnetic resonance (NMR), and this is often essential in the determination of novel or unusual sugars that are a common feature of bacterial glycoproteins (Castric et al, 2001;Thibault et al, 2001). The increased sample amounts required for NMR analysis can prove problematic but the introduction of cryoprobe technology for increased sensitivity in NMR analysis has shown the potential for detailed structural information from limited amounts of material (Voisin et al, 2005).…”