Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4‐diacetamido‐2,4,6‐trideoxyhexose

Abstract

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N-linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O-linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1-4-linked digalactose moiety covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper-adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.

Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence

“…GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. Besides its function as a substrate for glucosyltransferases resulting in glucosylated surface structures, UDP-glucose plays a well-established biochemical role as a glycosyl donor in the enzymic biosynthesis of carbohydrates (Csonka & Epstein, 1996;Stimson et al, 1995). The galU gene was found to be important for pathogenesis of infections due to a number of Gram-negative pathogens, including Actinobacillus pleuropneumoniae (Rioux et al, 1999), Escherichia coli (Komeda et al, 1977), Klebsiella pneumoniae (Chang et al, 1996), Pseudomonas aeruginosa (Evans et al, 2002;Priebe et al, 2004), Shigella flexneri (Sandlin et al, 1995) and Vibrio cholerae (Nesper et al, 2001).…”

BfpU, a soluble protein essential for type IV pilus biogenesis in enteropathogenic Escherichia coli

“…This protein makes up most, if not all, of the subunits of the pilus, where it is arranged in a helical array (Paranchych & Frost, 1988). The pilin proteins of several bacterial species are known to be modified by glycosylation and\or phosphorylation (Virji et al, 1993 ;Stimson et al, 1995 ;Castric, 1995 ;Forest et al, 1999). The crystal structure of the complete pilin from Neisseria gonorrhoeae has been solved and has been shown to resemble a ladle, with a long, amino-terminal handle composed of an uninterrupted hydrophobic α-helix and a head composed predominantly of anti-parallel β-sheets (Parge et al, 1995).…”

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).…”

“…Both the C. jejuni N-linked glycan and the O-linked glycan of N. meningitidis and N. gonorrhoeae pilin contain the sugar residue DATDH (Hegge et al, 2004;Stimson et al, 1995;Wacker et al, 2002). MS/MS experiments on the C. jejuni variant N-linked glycan structures were supportive of the involvement of PglE and PglF in DATDH biosynthesis (Linton et al, 2005), with these enzymes subsequently determined as an aminotransferase and dehydratase involved in the bacillosamine biosynthetic pathway (Schoenhofen et al, 2006).…”