globotriaosylceramide and Urinary-Tract-Infections

E. coli cause 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. Galactose alpha 1-4Galactose beta is the minimal receptor for adhering uropathogenic E. coli. Gal alpha 1-4Gal beta-binding bacteria caused significantly higher body temperature, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), pyuria, and lower renal concentrating capacity than E. coli lacking this specificity. The binding bacteria thus appeared to be more potent inducers of acute inflammation. Since inflammation may lead to tissue damage, we examined the relationship of infection with Gal alpha 1-4Gal beta-positive bacteria to renal scarring. The frequency of renal scarring was 5% in boys with Gal alpha 1-4Gal beta-positive and 40% in boys with Gal alpha 1-4Gal beta-negative E. coli. Analysis of binding capacity with the help of a newly developed latex agglutination assay can thus be used as an effective predictor of risk for renal scarring.

Topics: Bacterial Adhesion; Child; Child, Preschool; Cicatrix; Escherichia coli Infections; Female; Humans; Male; Pyelonephritis; Trihexosylceramides; Urinary Tract Infections

Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases.

Topics: Antibodies, Bacterial; Bacterial Adhesion; Chromatography, Thin Layer; Combinatorial Chemistry Techniques; Erythrocytes; Fimbriae, Bacterial; Globosides; Glycosphingolipids; Humans; Immunoenzyme Techniques; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trihexosylceramides; Urinary Tract Infections; Uropathogenic Escherichia coli

Bacterial adherence to mucosal cells is a key virulence trait of pathogenic bacteria. The type 1 fimbriae and the P-fimbriae of Escherichia coli have both been described to be important for the establishment of urinary tract infections. While P-fimbriae recognize kidney glycosphingolipids carrying the Galalpha4Gal determinant, type 1 fimbriae bind to the urothelial mannosylated glycoproteins uroplakin Ia and Ib. The F1C fimbriae are one additional type of fimbria correlated with uropathogenicity. Although it was identified 20 years ago its receptor has remained unidentified. Here we report that F1C-fimbriated bacteria selectively interact with two minor glycosphingolipids isolated from rat, canine, and human urinary tract. Binding-active compounds were isolated and characterized as galactosylceramide, and globotriaosylceramide, both with phytosphingosine and hydroxy fatty acids. Comparison with reference glycosphingolipids revealed that the receptor specificity is dependent on the ceramide composition. Galactosylceramide was present in the bladder, urethers, and kidney while globotriaosylceramide was present only in the kidney. Using a functional assay, we demonstrate that binding of F1C-fimbriated Escherichia coli to renal cells induces interleukin-8 production, thus suggesting a role for F1C-mediated attachment in mucosal defense against bacterial infections.

Topics: Animals; Bacterial Adhesion; Chromatography, Thin Layer; Dogs; Escherichia coli; Escherichia coli Infections; Galactosylceramides; Glycosphingolipids; Humans; Interleukin-8; Magnetic Resonance Spectroscopy; Mucous Membrane; Rats; Receptors, Immunologic; Sphingosine; Trihexosylceramides; Urinary Tract; Urinary Tract Infections