Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.

Abstract

We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids.These receptors, lactosylceramide Glc(J81-1)Cerj, gangliotriosylceramide [GalNAc(Pl14)Gal(P14)Glc(P81-1)Cer], and gangliotetraosylceramide [Gal(f31-3)GalNAc(131-4)Gal(P14)Glc(,81-1)CerJ, were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MSl1 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesin is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertiohs were required to abolish expression ofthe gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus.The binding of a pathogen to host cells is an important step in most infectious processes. The host cell receptors for several bacterial pathogens have been identified. For example, Escherichia coli strains causing urinary tract infections bind to a digalactoside moiety present in glycolipids (1, 2), and type 1 fimbriated E. coli, also implicated in urinary tract infections, bind to mannose residues in glycoproteins (3). In E. coli strains binding digalactoside, attachment to eukaryotic cells is independent of pilin, the major subunit of the pilus (4). In contrast, attachment of type 1 fimbriated E. coli is dependent on piliation (5). Recent work on digalactosidebinding E. coli demonstrated the presence of a minor protein at the tip of the pilus that binds to the receptor (6). The mannose-binding adhesin on type 1 fimbriae was shown to be present at the tip and along the sides of the pilus fiber (7,8). In addition to these pilus-associated adhesins, a nonpilus-associated surface adhesin responsible for mannoseresistant hemagglutination has been reported in uropathogenic E. coli KS52 (9).Pili have been shown to be important in the attachment of Neisseria gonorrhoeae to human epithelial cells and erythrocytes (10-12). However, recent in vitro studies indicated that both piliated (P+) and nonpiliated (P-) gonococci adhered to and were internalized by tissue culture cells with equal efficiency (13). We recently identified a set ofglycolipid receptors on epithelial cells that are recognized by both P+ and P-N. gonorrhoeae. They share lactose as their core sugar. Two of these, lactosylceramide [LacCer; Gal(, bound to LacCer with a lower affinity in vitro (14). Binding to these receptors was observed to be independent of pilin and protein II expression.A detailed characterization of the gonococcal surface adhesin, which recognizes the glycolipid receptor on host cells, is necessary for an understanding of the attachment process. For this purpose, we developed a thin-layer plate overlay assay for isolating the N. gonorrhoeae gene encoding the GgO4-binding adhesin from a pool of E. coli recombinants. Express...

Identification of glycolipid receptors for Helicobacter pylori by TLC‐immunostaining

“…Using a TLC-immunostaining method, Lingwood et al, found that H, pylori specifically bound to a sulfated alkylacylglycerolipid from pig stomach [9], though the precise structure of the lipid was not elucidated, In this study, we also applied TLC-immunostaining to elucidate the glycosphiagolipids specially recognized by H. pylori and successfully demonstrated direct binding of H. pylori to II3NeuAc-LacCer(GM,0 and sulfatide, Our preliminary results have also shown that freezethawing or sonication differentially influenced H, pylori binding to GM3 and sulfatide, indicating the possible involvement of different proteins in binding of each glycosphingolipid. In fact, the glycolipid-binding adhesins locating on either the pilus fiber or cell surface have been demonstrated in N, gonorrhoea and E. co// [16], On the other hand, our studies showed that sulfatldes were the most predominam acidic $1ycosphingolipids in the human stomach. Taken together, the results indicate that sulfatides might more likely serve as a receptor for H. p21ori in situ.…”

Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains

“…gonorrhoeae MS11 carries a single copy of the pilD gene. The gonococcal chromosome is known to carry several copies of certain genes (e.g., the genes encoding PilE pilin, the pilin accessory protein PilCN, the glycolipid-binding adhesin, and the Opa proteins [21,35,42,57]). Previous studies using the pulO-specific probe suggested that N. gonorrhoeae might also carry several copies of the pilDNg gene (11).…”

Comparative Genome Biology of a Serogroup B Carriage and Disease Strain Supports a Polygenic Nature of Meningococcal Virulence

“…The induction of the meningococcal outer membrane proteins upon contact with host cells, however, occurs in a time-dependent manner (25), and it is plausible that their induction takes place at earlier time points that were not investigated in this study. Finally, the high variability in the genetic architecture of the RTX, TPS, and maf loci together with the high sequence diversity of the encoded orthologous proteins supports an important role for these classes of genes in meningococcal infection biology; however, this remains to be fully resolved (53,55,63,81).…”