Thin-layer Chromatography Overlay Assay Research Articles

Quorum Sensing in Halorubrum saccharovorum Facilitates Cross-Domain Signaling between Archaea and Bacteria.

Quorum Sensing (QS) is a well-studied intercellular communication mechanism in bacteria, regulating collective behaviors such as biofilm formation, virulence, and antibiotic resistance. However, cell-cell signaling in haloarchaea remains largely unexplored. The coexistence of bacteria and archaea in various environments, coupled with the known cell-cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms and the presence of cell-cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms, suggests a possibility for haloarchaea to possess analogous cell-cell signaling or QS systems. Recently, N-acylhomoserine lactone (AHL)-like compounds were identified in haloarchaea; yet, their precise role-for example, persister cell formation-remains ambiguous. This study investigated the capacity of crude supernatant extract from the haloarchaeon Halorubrum saccharovorum CSM52 to stimulate bacterial AHL-dependent QS phenotypes using bioreporter strains. Our findings reveal that these crude extracts induced several AHL-dependent bioreporters and modulated pyocyanin and pyoverdine production in Pseudomonas aeruginosa. Importantly, our study suggests cross-domain communication between archaea and bacterial pathogens, providing evidence for archaea potentially influencing bacterial virulence. Using Thin Layer Chromatography overlay assays, lactonolysis, and colorimetric quantification, the bioactive compound was inferred to be a chemically modified AHL-like compound or a diketopiperazine-like molecule, potentially involved in biofilm formation in H. saccharovorum CSM52. This study offers new insights into putative QS mechanisms in haloarchaea and their potential role in interspecies communication and coordination, thereby enriching our understanding of microbial interactions in diverse environments.

Pectin-derived neoglycolipids: Tools for differentiation of Shiga toxin subtypes and inhibitors of Shiga toxin-mediated cellular injury

Gut pathogenic enterohemorrhagic Escherichia coli (EHEC) release Shiga toxins (Stxs) as major virulence factors, which bind to globotriaosylceramide (Gb3Cer, Galα1-4 Galβ1-4Glcβ1-1Cer) on human target cells. The aim of this study was the production of neoglycolipids (neoGLs) using citrus pectin-derived oligosaccharides and their application as potential inhibitors of Stxs. The preparation of neoGLs starts with the reduction of the carboxylic acid group of the pectic poly(α1-4)GalUA core structure to the corresponding alcohol, followed by hydrolytic cleavage of resulting poly(α1-4)Gal into (α1-4)Galn oligosaccharides and their linkage to phosphatidylethanolamine (PE). Thin-layer chromatography overlay assays of the produced (α1-4)Galn-PE and corresponding Amadori (α1-4)Galn=PE neoGLs revealed distinguishable binding patterns for Stx1a, Stx2a, and Stx2e. Furthermore, prepared neoGLs protected Vero cells against the cytotoxic action of Stxs when applied as multivalent glycovesicles. The produced neoGLs are applicable for differentiation of Stx subtypes and represent a promising approach to combat infections of EHEC by blocking their major toxins.

Shiga toxin glycosphingolipid receptor expression and toxin susceptibility of human pancreatic ductal adenocarcinomas of differing origin and differentiation

Shiga toxins (Stxs) are composed of an enzymatically active A subunit (StxA) and a pentameric B subunit (StxB) that preferentially binds to the glycosphingolipid (GSL) globo­triaosylceramide (Gb3Cer/CD77) and to a reduced extent to globotetraosylceramide (Gb4Cer). The identification of Gb3Cer as a tumor-associated GSL in human pancreatic cancer prompted us to investigate the expression of Gb3Cer and Gb4Cer in 15 human pancreatic ductal adenocarcinoma cell lines derived from primary tumors and liver, ascites, and lymph node metastases. Thin-layer chromatography overlay assays revealed the occurrence of Gb3Cer in all and of Gb4Cer in the majority of cell lines, which largely correlated with transcriptional expression analysis of Gb3Cer and Gb4Cer synthases. Prominent Gb3Cer and Gb4Cer lipoform heterogeneity was based on ceramides carrying predominantly C16:0 and C24:0/C24:1 fatty acids. Stx2-mediated cell injury ranged from extremely high sensitivity (CD(50) of 0.94 pg/ml) to high refractiveness (CD(50) of 5.8 μg/ml) and to virtual resistance portrayed by non-determinable CD(50) values even at the highest Stx2 concentration (10 μg/ml) applied. Importantly, Stx2-mediated cytotoxicity did not correlate with Gb3Cer expression (the preferential Stx receptor), suggesting that the GSL receptor content does not primarily determine cell sensitivity and that other, yet to be delineated, cellular factors might influence the responsiveness of cancer cells.

The human H3N2 influenza viruses A/Victoria/3/75 and A/Hiroshima/52/2005 preferentially bind to α2-3-sialylated monosialogangliosides with fucosylated poly-N-acetyllactosaminyl chains

Among influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annually half million deaths worldwide. Influenza A virus infection is initiated via hemagglutinin that binds to terminally sialylated glycoconjugates exposed on the surface of target cells. Gangliosides from human granulocytes were probed using thin-layer chromatography overlay assays for their binding potential to H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. Highly polar gangliosides with poly-N-acetyllactosaminyl chains showing low chromatographic mobility exhibited strong virus adhesion which was entirely abolished by sialidase treatment. Auxiliary overlay assays using anti-sialyl Lewis(x) (sLe(x)) monoclonal antibodies showed identical binding patterns compared with those performed with the viruses. A comprehensive structural analysis of fractionated gangliosides by electrospray ionization quadrupole time-of-flight mass spectrometry revealed sLe(x) gangliosides with terminal Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc epitope and extended neolacto (nLc)-series core structures as the preferential virus binding gangliosides. More precisely, sLe(x) gangliosides with nLc8, nLc10 and nLc12Cer cores, carrying sphingosine (d18:1) and a fatty acid with variable chain length (mostly C24:0, C24:1 or C16:0) in the ceramide moiety and one or two additional internal fucose residues in the oligosaccharide portion, were identified as the preferred receptors recognized by H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. This study describes glycan-binding requirements of hemagglutinin beyond binding to glycans with a specific sialic acid linkage of as yet undefined neutrophil receptors acting as ligands for H3N2 viruses. In addition, our results pose new questions on the biological and clinical relevance of this unexpected specificity of a subtype of influenza A viruses.

Glycosphingolipid receptors for pathogenic vibrios in intestines of mariculture fish

Vibrios are highly motile, facultatively anaerobic bacteria that are ubiquitous in aquatic environments and part of the normal intestinal microflora of healthy fish, but some species can cause vibriosis. The adherence of vibrios to host fish intestines is a significant event not only for their survival and growth, but also in terms of pathogenicity. However, the molecular mechanism underlying the adhesion of vibrios to the intestinal tract of fish is not fully understood. We report here the identification of intestinal glycosphingolipid (GSL) receptors to which pathogenic vibrios attach in typical mariculture fish. Thin-layer chromatography overlay assays using five species of 35S-labeled vibrios and intestinal glycosphingolipids of seven species of mariculture fish revealed that all of the fish tested possessed GM3 (NeuAcα2-3Galβ1-4Glcβ1-1′Cer) and/or GM4 (NeuAcα2-3Galβ1-1′Cer) as major acidic intestinal GSLs and that all of the vibrios tested specifically adhered to GM3 and/or GM4. Our results demonstrate that these GSLs were able to function as a receptor for the various vibrios tested. Analysis of the relationship between sugar structure and receptor activity for vibrios revealed that ‘NeuAcα2-3Galβ1-’ is required at the non-reducing end of glycosphingolipids for the bacteria to attach.

Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

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.

Neutral glycosphingolipid content of ovine milk

Milk glycosphingolipids (GSL) have been reported to participate in the newborn's defense against pathogens. Taking this into account, in this study we determined the neutral GSL content of ovine milk, including its fatty acid profile. Its role in bacterial adhesion was also addressed by immunodetection of separate GSL in a high-performance thin-layer chromatography overlay assay. Ovine milk has a neutral GSL pattern similar to human milk and includes lactosylceramide (LacCer; 45.7%), monohexosylceramide (glucosylceramide and galactosylceramide, 31.2%), globotriaosylceramide (Gb3; 19.1%), and globotetraosylceramide (Gb4; 3.5%). Globotriaosylceramide and Gb4 are present in human but not bovine milk. Neutral GSL contained C23:0 and C24:0 as the most abundant fatty acids, a finding consistent with its high content of very long chain fatty acids (longer than C20). Most fatty acids were saturated and had a low content of polyunsaturated fatty acids. Bovine enterotoxigenic Escherichia coli strains bound strongly to LacCer and showed a weak binding to monohexosylceramide. The K99 strain also bound strongly to Gb3, and F41 to Gb4. Lactosylceramide, monohexosylceramide, and Gb3 were also observed to bind to human uropathogenic E. coli strains. The results reported here show the ability of neutral GSL in ovine milk to bind to E. coli strains. These compounds could be used as an alternative and available source to supplement infant or bovine formulas with a view to preventing bacterial infections.

Shiga toxin receptor Gb3Cer/CD77: tumor-association and promising therapeutic target in pancreas and colon cancer.

BackgroundDespite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.Methodology/Principal FindingsTissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.Conclusions/SignificanceEnhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

Surface plasmon resonance analysis of botulinum neurotoxin binding for lipid receptors

Botulinum neurotoxins (BoNTs) are the most potent protein toxins. There are seven serotypes of BoNTs, termed A to G (BoNT/A to BoNT/G). The carboxyl-terminal half of the heavy chain (Hc) of the neurotoxin recognizes its specific receptor on the plasma membrane. We have previously demonstrated that BoNT/C binds to gangliosides GD1b and GT1b under physiological conditions, while BoNT/D interacts with phosphatidylethanolamine (PE). In this study, the binding specificities of BoNT-Hc for GT1b or PE were determined by surface plasmon resonance (SPR) using a receptor-including liposome. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real-time analysis of toxin binding under conditions that mimic the natural cell surface of these interactions and without any requirement for labeling of toxin or receptor. BoNT/C-Hc showed the binding specificity to GT1b liposome but not PE liposome. On the other hand, BoNT/D-Hc bound to only PE liposome. The SPR analysis also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants. The affinities of BoNTs for liposome-anchored gangliosides or PE were attributable largely to very fast dissociation rate constants. The SPR/liposome analysis should have general applicability in the study of lipid–protein interactions.

Binding of Clostridium botulinum Type C and D Neurotoxins to Ganglioside and Phospholipid: NOVEL INSIGHTS INTO THE RECEPTOR FOR CLOSTRIDIAL NEUROTOXINS

Clostridium botulinum neurotoxins (BoNTs) act on nerve endings to block acetylcholine release. Their potency is due to their enzymatic activity and selective high affinity binding to neurons. Although there are many pieces of data available on the receptor for BoNT, little attempt has been made to characterize the receptors for BoNT/C and BoNT/D. For this purpose, we prepared the recombinant carboxyl-terminal domain of the heavy chain (H(C)) and then examined its binding capability to rat brain synaptosomes treated with enzymes and heating. Synaptosomes treated with proteinase K or heating retained binding capability to both H(C)/C and H(C)/D, suggesting that a proteinaceous substance does not constitute the receptor component. We next performed a thin layer chromatography overlay assay of H(C) with a lipid extract of synaptosomes. Under physiological or higher ionic strengths, H(C)/C bound to gangliosides GD1b and GT1b. These data are in accord with results showing that neuraminidase and endoglycoceramidase treatment decreased H(C)/C binding to synaptosomes. On the other hand, H(C)/D interacted with phosphatidylethanolamine but not with any ganglioside. Using cerebellar granule cells obtained from GM3 synthase knock-out mice, we found that BoNT/C did not elicit a toxic effect but that BoNT/D still inhibited glutamate release to the same extent as in granule cells from wild type mice. These observations suggested that BoNT/C recognized GD1b and GT1b as functional receptors, whereas BoNT/D induced toxicity in a ganglioside-independent manner, possibly through binding to phosphatidylethanolamine. Our results provide novel insights into the receptor for clostridial neurotoxin.

Direct analysis of silica gel extracts from immunostained glycosphingolipids by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry.

A combined strategy of preparative high-performance thin-layer chromatography overlay assay and mass spectrometry was established for the structural characterization of immunostained glycosphingolipids (GSLs) in silica gel extracts. Crude chloroform/methanol/water (30/60/8, v/v/v) extracts of immunostained TLC bands were analyzed by nanoelectrospray low-energy CID mass spectrometry without further purification. The GSL species investigated were isomeric monosialogangliosides of the neolacto series from a ganglioside preparation of human granulocytes, the disialoganglioside GD3 from a human melanoma lipid extract, and ganglio series Gg3Cer of a neutral GSL preparation from murine lymphoreticular MDAY-D2 cells. For the specific detection of lipid-bound oligosaccharides, polyclonal chicken IgY, murine monoclonal IgG3, and IgM antibodies were used. The resulting mass spectra show that only analytical quantities of approximately 1 microg of a single GSL within a complex mixture are required for the structure determination of immunostained GSLs by MS and MS/MS. All species investigated were detected as singly charged deprotonated molecular ions, and neither buffer-derived salt adducts nor coextracted contaminants from the immunostaining procedure or the silica gel layer were observed. This effective HPTLC-MS-joined procedure offers a wide range of applications for any carbohydrate binding agents such as bacterial toxins, plant lectins, and others.

The pH 6 antigen of Yersinia pestis binds to beta1-linked galactosyl residues in glycosphingolipids.

The Yersinia pestis pH 6 antigen was expressed by, and purified from, Escherichia coli containing cloned psa genes. By an enzyme-linked immunosorbence-based assay, purified pH 6 antigen bound to gangliotetraosylceramide (GM1A), gangliotriaosylceramide (GM2A), and lactosylceramide (LC) (designations follow the nomenclature of L. Svennerholm [J. Neurochem. 10:613-623, 1963]). Binding to GM1A, GM2A, and LC was saturable, with 50% maximal binding occurring at 498 +/- 4, 390, and 196 +/- 3 nM, respectively. Thin-layer chromatography (TLC) overlay binding confirmed that purified pH 6 antigen bound to GM1A, GM2A, and LC and also revealed binding to hydroxylated galactosylceramide. Intact E. coli cells which expressed the pH 6 antigen had a specificity similar to that of purified pH 6 in the TLC overlay assay except that nonhydroxylated galactosylceramide was also bound. The binding patterns observed indicate that the presence of beta1-linked galactosyl residues in glycosphingolipids is the minimum determinant required for binding of the pH 6 antigen.

Haemagglutination and glycolipid‐binding activities of Lactobacillus reuteri

The carbohydrate-binding activity of Lactobacillus reuteri was studied by haemagglutination (HA), HA inhibition and thin layer chromatography (TLC) overlay assays. Three of the six Lact. reuteri strains examined showed HA activity. Two strains (JCM1081 and JCM1112T) agglutinated neuraminidase-treated, but not untreated, erythrocytes. Strain JCM2762 agglutinated both treated and untreated erythrocytes. The HA activity of JCM 1081 was inhibited by galactose, lactose, methyl beta-galactoside and asialoglycophorin A. Among 12 glycosphingolipids, TLC overlay assay showed that JCM1081 strongly bound to asialo-GM1. These results indicated that JCM1081 bound to the beta-galactosyl residues of the non-reducing terminal of sugar chains of glycoconjugates. The carbohydrate-binding ability of JCM1081 may be responsible for the adhesion of this strain to the mucosal surface of the intestine.

Glycosphingolipid-binding protein of Borrelia burgdorferi sensu lato.

The binding of Borrelia burgdorferi, the causative agent of Lyme disease, to glycosphingolipids present in various types of cells was examined. B. burgdorferi bound specifically to galactosylceramide (GalCer) and glucosylceramide (GlcCer) but not to other glycosphingolipids, as determined by a thin-layer chromatography (TLC) overlay assay. The binding specificity of B. burgdorferi to various glycosphingolipids suggested that the binding receptor in this species is ceramide monohexoside. The levels of binding of B. burgdorferi virulent strain 297 to GlcCer, sulfatide, lactosylceramide, and galactosylgloboside were 56.2, 1.6, 15.9, and 9.7%, respectively, relative to that to GalCer. Virulent low-passage strains of B. burgdorferi were serially subcultured in BSK II medium, and the resultant high-passage strains were not capable of infecting mice and did not induce footpad swelling. The levels of binding of the low-passage strains to GalCer on TLC plates and to CHO-K1 cells in vitro were threefold higher than those of high-passage strains. Binding was not affected by pretreatment of Borrelia with monospecific anti-outer surface protein C (OspC) antiserum. These results indicated that the binding of Borrelia to glycosphingolipid expressed on the cell surface plays an essential role in infection of mammalian hosts. However, OspC was not associated with binding. The necessity of the sugar and N-acyl moieties in GalCer for the binding of Borrelia was shown by a TLC overlay assay using chemically modified GalCer. Furthermore, three proteins, 67-kDa protein, 62-kDa Hsp60, and 41-kDa flagellin, were involved in binding of B. burgdorferi to GalCer, as shown by blotting assay using biotinylated GalCer as a probe.

Quantitative Analysis of Bacterial Toxin Affinity and Specificity for Glycolipid Receptors by Surface Plasmon Resonance

The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in GM1) with tolerance for substitution with a second internal sialic acid (as in GD1b). Escherichia coli heat-labile enterotoxin bound to GM1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-GM1 and GD1b, respectively) but not substitution of the terminal galactose of GM1. Tetanus toxin showed a requirement for two internal sialic acid residues as in GD1b. Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.

Binding Specificity ofLactobacillusto Glycolipids

Lactobacillus,a representative useful bacterium, in the intestinal tract was found to bind to some specific glycosphingolipids, like the pathogenic intestinal bacteria. Thin layer chromatography overlay assays using rabbit antiserum againstLactobacillus caseirevealed that the bacteria bound to GA1and trihexosylceramide strongly, but not to any gangliosides. The bacteria generally bound to glycosphingolipids having short sugar chains and galactosyl moiety in the non-reducing terminal.L.caseidid not bind to GM1, but bound to the product after sialidase treatment, GA1. This indicated that sialic acid inhibited the adhesion ofL.caseito tissues.L.caseiactually bound nonacid glycosphingolipids but not acid glycosphingolipids extracted from the small intestinal mucosa of rats.

Adherence to lipids and intestinal mucin by a recently recognized human pathogen, Campylobacter upsaliensis.

Campylobacter upsaliensis is a recently recognized human enteric pathogen associated with enteritis, colitis, bacteremia, and sepsis. Very little is known about the mechanisms of pathogenesis of this organism. The goals of this study were to determine whether C. upsaliensis binds to epithelial cells and whether there are specific lipid molecules that might serve as cell membrane receptors. In addition, we also explored C. upsaliensis binding to purified human small-intestinal mucin, since the mucus gel overlying the epithelium provides an initial contact surface for the bacteria and must be penetrated for the organisms to reach their cell receptors. Binding of C. upsaliensis to model epithelial cells was shown by microscopy adhesion assays, and binding to lipids was detected by thin-layer chromatography-overlay assays. Bacteria bound to phosphatidylethanolamine (PE), gangliotetraosylceramide (Gg4), and, more weakly, to phosphatidylserine (PS). There was no binding to ceramide, cholesterol, phosphatidylcholine, and globosides. Using receptor-based microtiter well immunoassays, we observed binding to be equal, specific, and saturable for PE and Gg 4 but low and nonspecific for PS. At least five bacterial surface proteins (50 to 90 kDa) capable of PE binding were identified by a lipid-silica affinity column technique. In slot blot overlay assays, biotin-labeled C. upsaliensis also bound in a concentration-dependent fashion to purified human small-intestinal mucin, implying that these microorganisms also express an adhesin(s) recognizing a specific mucin epitope(s). We speculate that binding to mucins may influence access of the bacteria to cell membrane receptors and thereby influence host resistance to infection.

Porcine 987P glycolipid receptors on intestinal brush borders and their cognate bacterial ligands.

Certain strains of enterotoxigenic Escherichia coli adhere to piglet intestinal epithelial cells by means of the 987P fimbriae. The 987P fimbrial structure consists of a helical arrangement of three fimbrial proteins, namely, the major subunit FasA and two minor subunits, FasF and FasG. FasG, which is located at the fimbrial tip and at various positions along the fimbriae, mediates 987P binding to glycoprotein receptors. In this study, we isolated and analyzed the structure of piglet glycolipid brush border receptors and characterized their cognate ligands on the 987P fimbriae. Two major glycolipid bands recognized by 987P fimbrial probes in thin-layer chromatography overlay assays were further purified by high-performance thin-layer chromatography and shown to comigrate with control galactosylceramide containing hydroxylated fatty acids and with sulfatide. Their structures were confirmed by fast atom bombardment mass spectrometry, which detected homologous series of ceramide monohexoside and sulfatide with hydroxylated fatty acyl chains ranging from h16:0 to h24:0. Assembled 987P fimbriae, pre- and postassembly dissociated fimbrial subunits, and Fab fragments of specific anti-FasG, -FasF, and -FasA were used to inhibit 987P-mediated bacterial binding to the two identified piglet glycolipids and corresponding isoreceptor controls. Only assembled fimbriae and anti-FasG Fab fragments were significantly able to inhibit bacterial binding to sulfatide, indicating that in addition to glycoproteins, FasG recognizes a specific glycolipid of piglet brush borders. In contrast, only anti-FasA Fab fragments were significantly able to inhibit bacterial binding to galactosylceramide with hydroxylated fatty acids and piglet hydroxylated ceramide monohexoside, indicating that FasA may determine a third type of ligand-receptor interaction in the piglet intestines. Since these bacterial adhesins recognize their respective glycolipid receptors only after being assembled in their final fimbrial quaternary structure, adhesin binding may involve cooperative interactions and the subunits by themselves may have very low binding affinities. Alternatively, conformation-sensitive domains of these subunits present in the assembled fimbriae may be required for glycolipid binding.

Burkholderia (basonym Pseudomonas) cepacia binding to lipid receptors

Piliated Burkholderia (formerly Pseudomonas) cepacia from sputa of cys tic fibrosis patients in Toronto, Canada, were shown earlier to bind to purified mucins and to a protein receptor on epithelial cells via a 22-kDa adhesin located on unique cable pili. However, a second receptor, thought to be lipid in nature, was also identified on cells and appeared to serve as the major cell receptor for poorly piliated or nonpiliated isolates. In the present study in vitro approaches were used to identify putative lipid receptors for B. cepacia and to explore the nature of the binding interaction. As judged by thin-layer chromatography overlay assays, the best receptors were digalactosylceramide and globotriosylceramide (Gb(3)). Both contain and unsubstituted terminal Gal alpha 1-4Gal sequence. B cepacia also bound moderately to galactosylceramide, gangliotriosylceramide, and gangliotetraosylceramide. Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking adhesin for GB(3). Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the carbohydrate epitope to the bacteria. Gb(3) was also the major receptor in lipid extracts of human erythrocytes, human buccal epithelial cells and HEp-2 laryngeal epithelial cells. In a receptor-based enzyme-linked immunosorbent assay, binding to Gb(3) within a phospholipid-cholesterol mixture (a membrane-like environment) increased and then approached saturation as a direct function of increasing bacterial concentration. The calculated value of K(a) (3.06 X 10(-8) ml/CFU), the affinity constant, was almost identical to the K(a) calculated earlier for B. cepacia binding to a set of lipid receptors in buccal epithelial cells (1.5 X 10(-8) to 2.0 X 10(-8) ml/CFU). Our findings suggest that within cell membranes, galactose-containing glycolipids, particularly Gb(3) are good candidates for receptors for B. cepacia, particularly for isolates in which cable pili are poorly expressed.

Adhesion of S-fimbriated Escherichia coli to brain glycolipids mediated by sfaA gene-encoded protein of S-fimbriae

In an attempt to further assess the role of S-fimbriae in the pathogenesis of Escherichia coli meningitis, the adherence of E. coli strains with or without S-fimbriae were examined for this study to purified glycolipids using thin layer chromatography overlay assays. Only S-fimbriated E. coli strains bound to sulfatide, seminolipid, galactosyl ceramide, and lactosyl ceramide but not to gangliosides including sialyl neolacto-series and other neutral glycolipids. The binding of S-fimbriated E. coli to sulfatide was temperature dependent (i.e. maximal at 37 degrees C) and inhibited by S-fimbriae, anti-S-fimbriae, and anti-S-adhesin antibodies as well as by sulfatide, galactosyl ceramide, and lactosyl ceramide. E. coli transformants which lack the sfaA gene from the Sfa gene cluster showed no binding to the glycolipids, while other transformants lacking the adhesin gene sfaS or sfaG or H and mutants obtained by site-directed mutagenesis in the sfaS gene exhibited a similar binding to the glycolipids compared to the parent S-fimbriated strain. A large amount of sulfated glycolipids was demonstrated on brain endothelial cells and the binding of S-fimbriated E. coli to brain endothelial cells was inhibited by these glycolipids. These findings suggest that the binding of S-fimbriated E. coli to brain endothelial cells occurs in part via glycolipids containing terminal Gal(3SO4)beta-1 residues and in part by S-fimbriae protein SfaA. S-adhesin was not involved in the binding of S-fimbriae to these glycolipids.