Globoseries Of Glycolipids Research Articles

Glycolipid binding epitopes involved in adherence of the periodontitis-associated bacterium Porphyromonas gingivalis

The ability of the periodontal pathogen Porphyromonas gingivalis to use different glycolipid structures as receptors has previously been demonstrated. The bacterium adhered to acid and nonacid glycolipids originating from human organs and to nonacid glycolipids of porcine origin. The aim of the present study was to analyze these binding epitopes by structural characterization. Glycolipid fractions with positive bacterial binding from e.g. human and porcine origin, were purified by the high performance liquid chromatography technique and thereafter used in bacterial overlay assays with (35)S-labeled P. gingivalis. Purified fractions with positive binding were structurally characterized by proton nuclear magnetic resonance spectroscopy. Complementing thin-layer chromatograms and bacterial overlay assays with pure reference glycolipid fractions and competition experiments with lactose were performed to define potential receptors. The P. gingivalis binding epitopes, including cerebrosides with nonhydroxy fatty acids, lactosylceramide with hydroxy fatty acids, sulfatides, lacto-, neolacto- and gangliotetraosylceramides, are in several instances similar to those found for other bacteria, e.g. H. pylori, H. influenzae and N. meningitidis. In addition P. gingivalis also bound to the Galalpha4Gal epitope of the globo series of glycolipids. In the future these results may be valuable for development of new treatment strategies, such as anti-adhesion therapies and vaccines specifically directed against P. gingivalis infection.

Quantitative studies of the binding of the class II PapG adhesin from uropathogenic Escherichia coli to oligosaccharides

Binding of the class II PapG adhesin, found at the tip of filamentous pili on Escherichia coli, to the carbohydrate moiety of globoseries glycolipids in the human kidney is a key step in development of pyelonephritis, a severe form of urinary tract infection. An assay based on surface plasmon resonance for quantification of the binding of the class II PapG adhesin to oligosaccharides has been developed. Using this assay dissociation constants ranging from 80 to 540 μM were determined for binding of the PapG adhesin to di-pentasaccharide fragments from the globoseries of glycolipids. A series of galabiose derivatives, modified at the anomeric position, O-2′ or O-3′, was also investigated. The anomeric position appeared to be the most promising for development of improved inhibitors of PapG-mediated adhesion of E. coli. p-Methoxyphenyl galabioside was found to be most potent ( K d=140 μM), and binds to PapG almost as well as the Forssman pentasaccharide.

Binding of N-trifluoroacetyl-derivatized natural glycosphingolipids by uropathogenic Escherichia coli and Neisseria subflava.

Several neutral glycosphingolipids were hydrogenated and subjected to trifluoroacetylation in trifluoroacetic acid/trifluoroacetic acid anhydride under conditions leading to complete exchange of the N-acetyl groups of GalNAc for N-trifluoroacetyl. The derivatized glycosphingolipids were analyzed for binding by P-fimbriated uropathogenic Escherichia coli, recognizing the globo series of glycolipids (carrying Galalpha1-4Gal). Using E. coli it was shown that a GalNCO-CF3 next to the minimum binding epitope Galalpha1-4Gal did not substantially influence the binding, as did not a trifluoro acetyl group on the ceramide. Exchange of N-acetyl of GalNAc in the receptor active gangliotetraosylceramide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1-1Cer, for N-trifluoroacetyl, did not change the binding of two out of the three strains tested of the bacterium Neisseria subflava. Discussion concerning the binding epitopes of the bacterial adhesins to carbohydrates is based on these results.

P Fimbriae of Uropathogenic Escherichia coli as Multifunctional Adherence Organelles

P fimbriae are the major single virulence factor of uropathogenic Escherichia coli strains. Recent analyses have shown that P fimbriae possess two distinct binding specificities mediated by different fimbrial subunits. P fimbriae bind to Gal alpha (1-4)Gal-containing globoseries of glycolipids of epithelial cells; this binding is mediated by the lectin-like minor protein G of the filament. In vitro mapping of the human urinary tract for binding sites of P fimbriae has revealed that they bind in a Gal alpha (1-4)Gal-inhibitable manner to epithelia of kidney and bladder. On the other hand, P fimbriae bind to immobilized fibronectin and its amino- and carboxyterminal fragments; this binding is dependent on the E and the F minor proteins of the P-fimbrial filament and seems to be based on a protein-protein interaction. The P fimbriae-fibronectin interaction has been demonstrated also on frozen sections of kidney. P fimbriae thus possess two tissue-adherence properties: one specific for epithelial glycoconjugates and the other for fibronectin of subepithelial extracellular matrices. P-fimbrial binding to epithelial glycoconjugates seems to be important in determining the host tropism and enabling the ascent of E. coli urinary tract infections. Binding to fibronectin may be important in secondary phases of the infection, e.g. after epithelial injury.

Bacterial adherence as a virulence factor in urinary tract infection

Escherichia coli (E. coli) causes greater than 90% of urinary tract infections, UTI, in childhood. The capacity to adhere to urinary tract epithelial cells characterizes E. coli strains that cause acute pyelonephritis. Adherence of uropathogenic E. coli is the result of a specific interaction between bacterial adhesins and glycolipid receptors on the host cells, especially the globoseries of glycolipids which share the Galactose alpha 1-greater than 4Galactose beta disaccharide (Gal alpha 1-greater than 4Gal beta). In childhood UTI, Gal alpha 1-greater than 4Gal beta-binding bacteria caused significantly higher body temperature, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and pyuria, and lower renal concentrating capacity, than E. coli lacking this specificity. The Gal alpha 1-greater than 4Gal beta-binding bacteria thus appeared to be more potent inducers of inflammation than other strains. Since inflammation may lead to tissue damage we examined the relationship of infection with Gal alpha 1-greater than 4Gal beta-positive bacteria to renal scarring. The frequency of renal scarring was 5% in boys with Gal alpha 1-greater than 4Gal beta-positive and 40% in boys with Gal alpha 1-greater than 4Gal beta-negative E. coli. Bacterial binding to Gal alpha 1-greater than 4Gal beta can be detected with a commercially available test reagent. This reagent can thus be used as an effective predictor of risk for renal scarring. Interleukin-6 (IL-6) is a pyrogen and inducer of the acute phase reactants. It was shown to be produced locally in the urinary tract, in response to UTI, and to spread systemically. Mucosal challenge with dead bacteria was sufficient to induce the IL-6 response. Circulating IL-6, and/or IL-1 and tumor necrosis factor could explain the fever, as well as increased ESR and CRP found in association with acute symptomatic UTI.

P blood group and proneness to urinary tract infection in Japanese children.

The globoseries of glycolipids are antigens in the P blood group system as well as epithelial cell receptors for uropathogenic Escherichia coli. The P1 blood group is overrepresented in Swedish girls with recurrent pyelonephritis. In this study, Japanese children with urinary tract infection (UTI) were analyzed for P blood group phenotype. Out of 26 children with recurrent UTI, 50% were of the P1 blood group compared to the 31% of P1 individuals in the Japanese population at large (p less than 0.05). Of children defined as having febrile UTI 62% were P1. The P1 blood group was thus significantly enriched (3.5 times) in the children with febrile UTI. These results support the hypothesis that individuals of blood group P1 run an increased risk for recurrent pyelonephritis.

Bacterial and host determinants of renal scarring.

This review summarizes recent work examining the interaction between host and parasite in recurrent urinary tract infection (UTI) and renal scarring. Virulence in uropathogenic E. coli has been defined by the severity of acute disease. Isolates from patients with acute pyelonephritic strains differ from those causing asymptomatic bacteriuria by multiple traits which contribute to virulence, and which are coexpressed in a non-random manner. The single marker most characteristic for the pyelonephritogenic clones is bacterial adherence to uroepithelial cells binding specifically to the disaccaride Gal alpha 1-4 Gal beta within the globoseries of glycolipids. The notion that the most severe consequence of acute pyelonephritis, i.e. renal scarring, was caused by the most virulent clones, was contradicted by comparison of pyelonephritic strains isolated from children with and without scarring. The virulent clones were significantly less frequent in patients with renal scarring (22%) than in patients with recurrent pyelonephritis not developing renal scars (62%). In view of the unexpected inverse association of bacterial virulence with renal scarring lack of Gal alpha 1-4 Gal beta binding capacity of E. coli strains was found to predict the risk for renal scarring among boys with first-time acute pyelonephritis. Vesicoureteric reflux (VUR) is widely accepted as a host determinant of susceptibility to pyelonephritis and renal scarring. In our study the frequency of renal scarring was 57% among girls with VUR as compared to 8% of those without. The reflux alone did however, not explain the selection of bacteria of low virulence. Individuals prone to UTI and renal scarring were found to be a genetically selected subgroup of the general population. A correlation between P1 blood group phenotype and susceptibility to UTI and between blood group non-secretor state and renal scarring was found. The mechanisms behind these relationships need to be defined. The bacterial and host parameters combined indicate that host parameters are essential for the tendency to develop renal scarring after acute pyelonephritis.

Non-antibiotic prevention of urinary tract infection.

The identification of several glycoconjugates as receptors for attaching bacteria has resulted in optimism regarding the use of receptor analogues in preventing infection. The structure of receptor-active oligosaccharides and the patient groups which might benefit from such treatment are most thoroughly understood for urinary tract infections. Prevention of adhesion and delay or decrease of infection was achieved using oligosaccharides from the globoseries of glycolipids containing Gal alpha 1----4Gal. This minimal receptor structure as a free saccharide was, however, not sufficient. It was the intact P, P1 and pk determinants which showed optimal activity in vitro. In addition, protection against experimental UTI results from previous exposure to whole bacteria or isolated antigens. The mechanism(s) of protection and their relevance in clinical UTI remain to be defined.

Non-antibiotic prevention of urinary tract infection.

The identification of several glycoconjugates as receptors for attaching bacteria has resulted in optimism regarding the use of receptor analogues in preventing infection. The structure of receptor-active oligosaccharides and the patient groups which might benefit from such treatment are most thoroughly understood for urinary tract infections. Prevention of adhesion and delay or decrease of infection was achieved using oligosaccharides from the globoseries of glycolipids containing Gal alpha 1----4Gal. This minimal receptor structure as a free saccharide was, however, not sufficient. It was the intact P, P1 and pk determinants which showed optimal activity in vitro. In addition, protection against experimental UTI results from previous exposure to whole bacteria or isolated antigens. The mechanism(s) of protection and their relevance in clinical UTI remain to be defined.