Glycobiology Advance Access originally published online on February 16, 2005
Glycobiology 2005 15(7):700-708; doi:10.1093/glycob/cwi049
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Identification and characterization of binding properties of Helicobacter pylori by glycoconjugate arrays
2 Department of Operative Dentistry and Periodontology, Dental School, University of Regensburg, D-93053 Regensburg, Germany; 3 Max von Pettenkofer Institute, Ludwig-Maximilians University, D-80336 München, Germany; 4 School of Health Sciences, University College of Borås, SE-501 90 Borås, Sweden; and 5 Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden
1 To whom correspondence should be addressed; e-mail: stefan.ruhl{at}klinik.uni-regensburg.de
Received on December 28, 2004; revised on February 11, 2005; accepted on February 14, 2005
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
The microaerophilic bacterium Helicobacter pylori is well established for its role in development of different gastric diseases. Bacterial adhesins and corresponding binding sites on the epithelial surface allow H. pylori to colonize the gastric tissue. In this investigation, the adhesion of H. pylori to dot blot arrays of natural glycoproteins and neoglycoproteins was studied. Adhesion was detected by overlay with fluorescence-labeled bacteria on immobilized (neo)glycoproteins. The results confirmed the interaction between the adhesin BabA and the H-1-, Lewis b-, and related fucose-containing antigens. In addition, H. pylori bound to terminal
2-3-linked sialic acids as previously described. The use of a sabA mutant and sialidase treatment of glycoconjugate arrays showed that the adherence of H. pylori to laminin is mediated by the sialic acid-binding adhesin, SabA. The adhesion to salivary mucin MUC5B is mainly associated with the BabA adhesin and to a lesser extent with the SabA adhesin. This agrees with reports, that MUC5B carries both fucosylated blood group antigens and 2-3-linked sialic acids. The adhesion of H. pylori to fibronectin and lactoferrin persisted in the babA/sabA double mutant. Because binding to these molecules was abolished by denaturation rather than by deglycosylation, it was suggested to depend on the recognition of unknown receptor moieties by an additional unknown bacterial surface component. The results demonstrate that the bacterial overlay method on glycoconjugate arrays is a useful tool for exploration and the characterization of unknown adhesin specificities of H. pylori and other bacteria. Key words: adhesins / glycoproteins / Helicobacter pylori / lectins / neoglycoproteins
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
The spiral, gram-negative bacterium Helicobacter pylori persistently colonizes the gastric mucosa of 50% of the human population and is associated with the induction of chronic type B gastritis, peptic ulceration, and the development of gastric carcinoma and MALT (mucosa-associated lymphoid tissue)-lymphoma (for a review see [Ernst and Gold, 2000
]). Close association of H. pylori with gastric epithelial cells (Hessey et al., 1990) and the mucin layer covering the epithelial surface (Schreiber et al., 1999; Schreiber et al., 2004) is believed to facilitate the permanent colonization of the stomach by this bacterium. An intimate association with the epithelial cells allows the bacteria to encroach upon signal transduction processes, in particular, by the action of the so-called Cag type IV secretion system (Odenbreit et al., 2000; Fischer et al., 2001), resulting in rearrangement of the cytoskeleton (Segal et al., 1996) and the induction of proinflammatory cytokines (Censini et al., 1996).
In the past, biochemical as well as genetic approaches have been chosen to identify bacterial factors involved in the adherence process of H. pylori to gastric cells. By using affinity purification and overlay assays, a subset of putative receptors could be identified, including fucosylated, sialylated, or sulphated oligosaccharides, glycolipids, glycoproteins, mucins, and lipid-like compounds (Evans et al., 1988; Lelwala-Guruge et al., 1992; Lingwood et al., 1992; Ascencio et al., 1993; Borén et al., 1993; Hirmo et al., 1996; Namavar et al., 1998; Hirmo et al., 1999). Recently, two major receptor structures and their corresponding adhesins were characterized in more detail. One is a fucosylated oligosaccharide structure present both in the H-1 and Lewis b blood group antigens (blood group O phenotype) (Borén et al., 1993; Aspholm-Hurtig et al., 2004) that was identified as a receptor motif for the H. pylori outer membrane protein (OMP) BabA, the blood group antigen binding adhesin (Ilver et al., 1998). Another is the sialyl-Lewis x antigen, that is recognized by the OMP SabA, the sialic acid-binding adhesin (Mahdavi et al., 2002). The fucosylated blood group antigens are highly expressed in gastrointestinal epithelium which favors the colonization of the gastric mucosa. The sialyl-Lewis x antigen is described to be predominantly expressed in inflamed tissues and might promote the chronicity of the infection process once gastritis is established (Mahdavi et al., 2002).
Beside these well-characterized interactions, other eucaryotic receptors and bacterial adhesin candidates have been described, suggesting the enrolment of additional interactions that are not fully explored yet. In this regard, extracellular matrix (ECM) proteins, such as laminin and collagen type IV, have been proposed as receptors for H. pylori (Trust et al., 1991; Valkonen et al., 1997). In addition, surface-exposed components of H. pylori such as the OMPs AlpAB (Odenbreit et al., 1999; Odenbreit et al., 2002a) and HopZ (Peck et al., 1999) as well as Lewis x structures in the O-antigen side chain of the lipopolysaccharide (Edwards et al., 2000) have been shown to be involved in adherence to gastric cells.
Recently, arrays of immobilized purified glycoproteins and neoglycoproteins have been successfully used to explore the adhesin specificities of oral actinomyces and viridans streptococci (Ruhl et al., 1996; Ruhl et al., 2004). These arrays are well suited to assess bacterial adhesin specificities because both the naturally occurring glycoproteins and related oligosaccharide structures, presented on neoglycoproteins, can be compared for receptor activity and for the exploration of the minimal oligosaccharide motifs necessary for binding. This method has been now adapted by using a set of sabA- and babA-deficient fluorescence-labeled mutants of H. pylori to investigate a wider range of natural and synthetic glycoproteins for receptor activity.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Characterization of adhesin expression in H. pylori mutants
By using specific antisera directed against BabA and SabA, respectively, the expression of these OMPs was verified on H. pylori J99 wild type (wt) (Figure 1). The BabA and SabA proteins could be detected with apparent molecular weights of
80 and 70 kDa, respectively. The BabA protein was expressed in the J99 sabA mutant but not in the J99 babA and J99 babA/sabA mutants. The expression of the SabA protein could be detected in the J99 babA but not in J99 sabA or J99 babA/sabA mutants. These data confirm the correct mutagenesis of the desired genes in each mutant strain.
|
Validation of glycoconjugate array
To verify that proteins were properly immobilized on the nitrocellulose membrane and are glycosylated, the incubation of blots with 10 mM sodium periodate for the oxidation of carbohydrates and subsequent incubation with biotin-LC-hydrazide was performed. The result confirmed that each protein spot on the array carries sugar chains with the exception of bovine serum albumin (BSA) and human serum albumin (HSA), that were included as internal negative controls (Figure 2A).
|
A further characterization of carbohydrates was performed by lectin blotting with Ulex europaeus agglutinin (UEA-I) and Lotus tetragonolobus agglutinin (LTA) that both bind to -L-fucose and recognize the H-2 trisaccharide epitope (Du et al., 1994; Mollicone et al., 1996) (Figure 2B and C). In previous studies, UEA-I was used for the detection of receptor motifs recognized by H. pylori (Falk et al., 1993). In the present study, both lectins bound strongly to H-2-, Lewis y-, and 2'-fucosyllactose-carrying neoglycoproteins. LTA but not UEA-I recognized also the Lewis x antigen (dot C2). Weaker binding was noted to fucosylated blood group type 1 chains that were recognized by UEA-I and LTA to different extents. Strong binding of both lectins was found to MUC5B (dot A6), indicating the presence of -L-fucose on this glycoprotein as expected from the presence of type 2 human blood group determinants on this molecule (Thomsson et al., 2002).
To further validate the glycoconjugate overlay method, Streptococcus gordonii DL1, expressing an adhesin (Hsa) specific for a2-3-linked sialic acids (Takahashi et al., 1997; Takahashi et al., 2002), was used as a probe. This strain bound strongly to fetuin, glycophorin A, laminin, and 3'-sialyllactose (Figure 3A). Strain D102, which lacks the sialic acid-binding adhesin, failed to bind any component on the glycoconjugate array (Figure 3B).
|
Lectin-dependent H. pylori binding to natural and synthetic glycoproteins
Binding characteristics of H. pylori adhesins were determined by comparing the binding of wt strain J99 with babA- and sabA-deficient mutant strains (Figure 4). J99 wt bound to fetuin, glycophorin A, laminin, MUC5B, sialyl-Lewis a, sialyl-Lewis x, 3'-sialyllactose, 3'-sialyl-3-fucosyllactose, and 3'-sialyl-N-acetyllactosamine (Figure 4A). These glycoproteins were recognized also by the J99 babA mutant strain (Figure 4B). No binding of any H. pylori strain to 6'-sialyllactose (dot D2) could be detected. Strong binding of J99 wt was noted to H-1- and Lewis b-containing neoglycoproteins (Figure 4A). These two determinants were recognized also by the J99 sabA mutant (Figure 4C). Binding to MUC5B (dot A6) was still detected with both, the babA and the sabA mutant, the latter, however, showing a stronger signal. The J99 babA/sabA double mutant failed to bind to any component on the glycoconjugate array (Figure 4D).
|
To further confirm the sialic acid-dependency of SabA-mediated binding, blots were incubated with sialidase before overlay with bacteria (Figure 5). Sialidase treatment abolished binding of J99 wt and the babA mutant to fetuin, laminin, and sialic acid-containing neoglycoproteins (Figure 5A and B), resulting in a binding pattern analogous to the sabA mutant (Figure 4C). Residual binding of the babA mutant to MUC5B (Figure 5C) was completely abolished by pretreatment with sialidase (Figure 5D).
|
BabA- and SabA-independent binding of H. pylori to fibronectin and lactoferrin
In the course of testing additional glycoproteins as putative receptor candidates for H. pylori, adhesion to fibronectin and lactoferrin was noticed (Figure 6). Remarkably, this was observed not only with the wt strain but also with the babA/sabA double mutant, suggesting that a different unknown bacterial surface component on H. pylori might be involved in this interaction. To map the receptor motif in these glycosylated proteins, the protein structure was denatured by sodium dodecyl sulfate (SDS) and heat treatment or the sugar residues were removed by treatment with N-glycosidase F. Following only denaturation, the adhesion to fibronectin and lactoferrin disappeared in both J99 wt and the babA/sabA double mutant. However, denaturation did not influence the SabA-dependent binding of J99 wt to laminin. Binding of J99 wt to laminin only disappeared, when denatured membranes were treated with N-glycosidase F, confirming lectin-like interaction in this recognition process. Aminogroup detection was used to control that denaturation did not result in the loss of immobilized proteins.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Numerous adhesive properties of H. pylori have been described, including hemagglutination, attachment to epithelial cells, and binding to distinct receptors, such as oligosaccarides or proteins of the basement membrane (Trust et al., 1991
; Gerhard et al., 2001). The aim of this study was to establish a screening system for the exploration of novel receptor–adhesin interactions in H. pylori. The results of this study confirmed the binding specificity of H. pylori adhesin BabA to H-1-, Lewis b-, and related oligosaccharide determinants as well as the specificity of adhesin SabA to sialyl-Lewis x and sialyl-Lewis a containing oligosaccharides (Borén et al., 1993; Mahdavi et al., 2002). Preferential binding of H. pylori to 2-3-linked sialic acid (Hirmo et al., 1996) was attributed to the SabA adhesin. In addition, the SabA adhesin was found sufficient to explain binding to laminin that had been previously observed (Trust et al., 1991). Binding of H. pylori to salivary mucin MUC5B that had been also described (Namavar et al., 1998) was now shown to be predominantly mediated by the BabA adhesin and to a lesser degree also by the SabA adhesin. Only the binding to fibronectin and lactoferrin could not be explained by the activities of the SabA or BabA adhesins. Because binding of H. pylori to these glycoproteins was abolished by denaturation rather than by deglycosylation, it is proposed to depend on the recognition of unknown protein moieties by an additional adhesive surface structure on H. pylori.
For the validation of the glycoconjugate array, lectins (UEA-I and LTA) were chosen that exhibit binding specificity for -L-fucose (Falk et al., 1993) and in this respect are similar to the H. pylori BabA adhesin that is known to bind to the Lewis b antigen, an oligosaccharide structure containing terminal 1-2-linked-fucose (Ilver et al., 1998). The minimal structure in the glycoconjugate array for recognition by UEA-I was shown to be Fuc1-2Gal, present in both type 1 and type 2 chains of blood groups, but a preference for the H type 2 trisaccharide epitope (Fuc1-2 Galß1-4GlcNAc) was noticed that is in agreement with earlier reports (Du et al., 1994; Mollicone et al., 1996). Strong binding to 2'-fucosyllactose confirmed that the N-acetyl group of type 2 chains is not necessary for binding (Mollicone et al., 1996). Analogous to UEA-I, the binding specificity of LTA for H-2 antigen (Mollicone et al., 1996) could be confirmed. A significant difference between UEA-I and LTA was the additional recognition of the Lewis x antigen (Galß1-4[Fuc1-3]GlcNAc) by LTA but not by UEA-I, that is also in agreement with previous reports (Yan et al., 1997). So far, UEA-I had been used as a probe to identify potential receptors for H. pylori (Falk et al., 1993). However, the results of this investigation demonstrate, that UEA-I recognizes a broader range of fucose-containing receptors than the H. pylori BabA adhesin. This becomes particularly evident from the finding that, in contrast to UEA-I which strongly bound 2'-fucosyllactose, H. pylori J99 wt and the sabA mutant showed only weak binding. Thus, although terminal Fuc1-2Gal might be sufficient for binding of H. pylori, significantly stronger binding occurs to the Fuc1-2Galß1–3GlcNAc motif found in H-1 and Lewis b antigens.
In the past, two possible explanations for the binding of H. pylori to laminin were proposed. First, a lectin-like interaction of the bacterium with terminal sialic acids on laminin (Valkonen et al., 1993) and second, an interaction of H. pylori lipopolysaccharides with laminin (Valkonen et al., 1994). Our results clearly show that a lectin-like interaction of the SabA adhesin with terminal sialic acid is responsible for binding to laminin. Thus, strong binding to laminin that was detectable with the J99 wt strain disappeared in the sabA-deficient mutant (Figure 4) as well as after the preincubation of the membranes with sialidase (Figure 5). These findings expand the binding activities of the SabA adhesin beyond the previously reported recognition of sialyl-Lewis blood group antigens (Mahdavi et al., 2002). A broader recognition of terminal sialic acid-containing oligosaccharides is supported by the finding that the sabA mutant failed to bind to fetuin, glycophorin, 3'-sialyllactose-HSA, 3'-sialyl-3-fucosyllactose-BSA, and 3'-sialyl-N-acetyllactosamine-BSA. Because 6'-sialyllactose-HSA was not recognized as a receptor, it is proposed that the previously found binding activity of H. pylori to terminal 2-3-linked sialic acid (Hirmo et al., 1996) can be attributed to the SabA adhesin. In this respect, the binding specificity of the SabA adhesin to laminin appears similar to the sialic acid-binding adhesin of S. gordonii DL1 that also exhibits a preference for 2-3-linked sialic acids (Takahashi et al., 1997). Interestingly, the elucidation of the oligosaccharides on laminin had revealed only the presence of terminal 2-3- but not 2-6-linked N-acetyl neuraminic acid (Knibbs et al., 1989). This might explain stronger binding of H. pylori to laminin than to fetuin, the latter carrying both 2-3- and 2-6-linked N-acetyl neuraminic acids (Spiro and Bhoyroo, 1974; Takasaki and Kobata, 1986).
Binding of H. pylori to salivary mucin MUC5B, that had been previously reported (Namavar et al., 1998), could now be confirmed by binding of wt strain J99. Notably, both the sabA and the babA mutants still bound to MUC5B whereas the sabA/babA double mutant failed to bind. Strong binding of the sabA-deficient mutant to MUC5B indicates the importance of H-1-, Lewis b-, and related oligosaccharide epitopes for the recognition of this mucin by the BabA adhesin. Analysis of glycosylation had shown the presence of these oligosaccharide determinants on MUC5B (Thomsson et al., 2002). Binding of the babA-deficient mutant to MUC5B was weaker and suggests an additional involvement of the SabA adhesin with corresponding terminal sialic acids on this molecule. This was further confirmed by pretreatment of the membranes with sialidase that removed the residual binding of the babA-deficient mutant but not of the wt strain J99 to MUC5B (Figure 5). These data suggest that binding to MUC5B can solely be explained on the basis of both the BabA and the SabA adhesin. The requirement of an additional adhesin recognizing sulfated oligosaccharide structures, as previously proposed (Namavar et al., 1998), could not be supported based on the present data. Because the recognition of various sialic acid-containing oligosaccharides differs between the SabA adhesin of H. pylori and the Hsa adhesin of S. gordonii DL1 (Figures 3 and 4), sub-terminal sugars to 2-3-linked sialic acid seem to be involved in recognition. This becomes particularly apparent for MUC5B that is bound by H. pylori but not by S. gordonii DL1. Binding of H. pylori to MUC5B, a human salivary mucin, may enable H. pylori to colonize the oral cavity (Namavar et al., 1998) which may in turn have implications for oral transmission of this pathogen (Dowsett and Kowolik, 2003).
Binding of H. pylori to lactoferrin had been previously described and was attributed to either a 60 kDa heat shock protein (Amini et al., 1996) or a 70 kDa lactoferrin-binding OMP of H. pylori (Dhaenens et al., 1997). For the 60 kDa heat shock protein, it was suggested that carbohydrate moieties of lactoferrin were involved in binding (Amini et al., 1996). In this investigation, however, it could be demonstrated that binding of H. pylori to lactoferrin is not dependent on BabA or SabA activities because the babA/sabA double mutant still bound to this protein. Thus, the presence of an additional binding activity on H. pylori has to be hypothesized. Analogous to lactoferrin, binding of H. pylori to fibronectin, an ECM component, was also independent of BabA or SabA activities, clearly distinguishing it from the SabA-dependent binding to laminin, another protein of the ECM. The fact that denaturation rather than deglycosylation of both lactoferrin and fibronectin abolished binding of H. pylori to these components, suggested that protein moieties rather than carbohydrates might play a role in receptor recognition. ECM proteins such as fibronectin, laminin, or vitronectin are involved in integrin-mediated signal transduction pathways that regulate cellular processes including actin rearrangements, cell cycle regulation, or survival of cells (Schwartz and Shattil, 2000). Several pathogenic bacteria, such as Staphylococcus aureus or Neisseria gonorrhoeae, have learned to exploit this signaling network to invade epithelial cells by bridging fibronectin-binding proteins to ß1-integrins on the epithelial surface (van Putten et al., 1998; Sinha et al., 1999). Because evidence for H. pylori invasiveness has been described (Su et al., 1999; Amieva et al., 2002) but the mechanism of entry is not known, it will be interesting to identify the fibronectin-binding component on H. pylori.
The high specificity and reliability of the current overlay method as well as the simple handling, in combination with well-defined bacterial mutants, may allow future analysis of complex mixtures of, for example, gastric epithelial cell membranes or salivary proteins for the identification of natural receptors for H. pylori adhesion.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Bacteria and growth conditions
The wt H. pylori strain J99 (Alm et al., 1999
) and its isogenic mutants in the sabA and/or babA genes (Mahdavi et al., 2002) were grown for 48–72 h at 37°C in a microaerophilic atmosphere on Wilkins-Chalgren agar (Oxoid, Wesel, Germany) containing 10% horse blood, Dent supplement (Oxoid) and 0.4 g KNO3 per liter. Streptococcus gordonii strain DL1 (Challis) and the sialic acid-binding deficient mutant strain D102, kindly provided by Yukihiro Takahashi (The Nippon Dental University School of Dentistry, Tokyo, Japan) were grown in complex medium as previously described (Takahashi et al., 1997).
SDS–polyacrylamide gel electrophoresis and immunoblot
For immunoblot analysis the bacteria were collected from agar plates and suspended in 300 µL sample solution (Laemmli, 1970). Boiled aliquots were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) in a 10% acrylamide gel by using a mini-gel apparatus (Biorad, München, Germany) and blotted onto nitrocellulose membranes at 1 mA/cm2 by using a semidry blot system (Biotec Fischer, Reiskirchen, Germany). The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) (50 mM Tris–HCl, pH 7.5; 150 mM NaCl) and incubated with antisera AK277 (anti-BabA) (Odenbreit et al., 2002b) or AK278 (anti-SabA) for at least 2 h (1:10,000 dilution). Alkaline phosphatase-coupled protein A was used to visualize the bound antibody by the decomposition of nitroblue tetrazolium.
Glycoproteins and neoglycoproteins
Dry nitrocellulose membranes (Invitrogen, Carlsbad, CA, pore size 0.4 µm) were spotted with 1 µL volumes containing 1 µg of glycoproteins or neoglycoproteins. The glycoproteins used were fetuin (Calbiochem, Bad Soden, Germany), asialofetuin (Sigma), glycophorin A (Sigma), asialoglycophorin (Sigma), laminin (from human placenta, Sigma), MUC5B (kindly provided by M.J. Levine, Department of Oral Biology, SUNY, Buffalo, NY), transferrin (Sigma), fibronectin (from human plasma, Sigma), and lactoferrin (from human milk, Sigma). The neoglycoproteins used are listed in Table I. HSA (fraction V, Sigma) and BSA (fraction V, immunoglobulin-free, protease-free, Sigma) were included as negative (nonglycosylated) controls.
|
Pretreatment of dot blot arrays
For sialidase treatment, membranes were incubated with 0.1 U/mL of sialidase (from Clostridium perfringens type X, Sigma) in TBS containing 5% BSA (fraction V, Sigma), 1 mM CaCl2, 1 mM MgCl2, and 0.1% sodium azide at 37°C before overlay. For the denaturation of spotted proteins, membranes were treated with 0.1% SDS (Merck, Darmstadt, Germany) in 20 mM sodium phosphate buffer (pH 7.2) containing 50 mM ß-mercaptoethanol (Merck) at 100°C for 5 min. For N-glycosidase F digestion, 0.05 U/mL of recombinant Glyko® N-glycanase from Chryseobacterium meningosepticum (PROzyme, San Leandro, CA) and 0.75% NP-40 (PROzyme) were added after denaturation and further incubation was carried out overnight at 37°C. All enzymatic pretreatments of membranes were performed in sealed plastic bags.
Bacterial overlay
The method was performed as previously described (Ruhl et al., 2000) except that fluorescence-labeled bacteria were used as probes. Bacteria at 1 x 108/mL in PBS were fluorescein labeled by incubation with fluorescein-5-isothiocyanate (FITC) (Molecular Probes, Eugene, OR) at 100 µg/mL for 30 min at room temperature. Untreated and pretreated membranes were blocked in TBS containing 5% BSA (fraction V, Sigma), 1mM CaCl2, 1mM MgCl2 for 2 h at room temperature. Labeled bacteria were recovered by centrifugation at 2700 x g for 7 min, resuspended in 10 mL blocking buffer and added to a final concentration of 2.5 x 107 bacteria in a total volume of 40 mL (about 1 mL of bacterial suspension per cm2 of nitrocellulose membrane). The overlays were incubated for 30 min at 4°C in the dark without mixing and were washed three times at room temperature for 5 min on a rotary shaker in TBS containing 0.05% Tween-20, 1 mM CaCl2, and 1 mM MgCl2. The fluorescence of adherent bacteria was detected by a Typhoon imaging system (Typhoon 9200, Amersham Biosciences, Freiburg, Germany).
Chemical labeling of glycoconjugates
For the oxidation of carbohydrates, nitrocellulose membranes with (neo)glycoproteins immobilized were incubated for 30 min in acetate buffer (0.1 M, pH 5.5) with 10 mM sodium periodate (ICN Biomedicals, Aurora, OH) at room temperature in the dark. After washing in PBS, membranes were incubated for 1 h at room temperature in acetate buffer containing 100 µg/mL biotin-LC-hydrazide (Pierce, Rockford, IL). The membranes were washed three times with TBS and were then blocked in TBS containing 3% BSA (Sigma) for 1 h at room temperature. The membranes were subsequently incubated for 30 min in the dark with 5 mg fluorescein avidin-D (Vector Laboratories, Burlingame, CA) per mL in blocking buffer. Membranes were washed three times in TBS containing 0.1% Tween-20 and once in TBS. Fluorescent signals were detected by a Typhoon imaging system (Typhoon 9200, Amersham Biosciences).
Lectin blotting
Membranes were blocked for 1 h at room temperature with TBS containing 2% polyvinyl alcohol (average molecular weight 30,000–70,000, Sigma), 0.1% Tween-20, 1 mM CaCl2, and 1 mM MgCl2. Membranes were subsequently incubated for 1 h at room temperature in the dark with fluorescein-labeled UEA-I (Vector Laboratories) and fluorescein-labeled LTA (Sigma) at concentrations of 5 µg per mL in blocking buffer (Ruhl et al., 2000). The blots were washed three times in TBS containing 0.1% Tween-20 and the fluorescence of bound lectins was detected by a Typhoon imaging system (Typhoon 9200, Amersham Biosciences).
Aminogroup detection
Untreated and pretreated membranes were washed three times in borat buffer pH 9.7 containing 0.05 M Na2B4O7 x 10 H2O (Merck) and 0.2% Tween-20 and then incubated for 1 h with 100 µg/mL of sulfo-NHS-LC-biotin (Pierce, Rodeford, IL). After washing two times in borat buffer and two times in TBS containing 0.1% Tween-20, the membranes were incubated for 30 min in the dark with 5 mg/mL fluorescein avidin-D (Vector Laboratories) in TBS containing 0.1% Tween-20. Membranes were washed again three times in TBS containing 0.1% Tween-20 and fluorescent signals were detected by a Typhoon imaging system (Typhoon 9200, Amersham Biosciences).
|
Acknowledgements |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
We thank Andreas Eidt for excellent technical assistance. We are grateful to Prof. Dr. N. Lehn, Institute of Medical Microbiology, University of Regensburg for his advice and help with bacterial culture. We thank Dr. Wolfgang Fischer for critical reading of the manuscript. This investigation was supported by grants SFB 585/B5 (S.R.), OD 21/1 (S.O.) from the Deutsche Forschungsgemeinschaft (DFG), the Swedish Research Council, the Swedish Cancer Society, and the Kempestiftelserna (T.B.).
|
Abbreviations |
|---|
BSA, bovine serum albumin; ECM, extracellular matrix; FITC, fluorescein-5-isothiocyanate; HSA, human serum albumin; LTA, Lotus tetragonolobus agglutinin; OMP, outer membrane protein; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; UEA-I, Ulex europaeus agglutinin; wt, wild type
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Alm, R.A., Ling, L.S., Moir, D.T., King, B.L., Brown, E.D., Doig, P.C., Smith, D.R., Noonan, B., Guild, B.C., deJonge, B.L., and others. (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature, 397, 176–180.[CrossRef][Medline]
Amieva, M.R., Salama, N.R., Tompkins, L.S., and Falkow, S. (2002) Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells. Cell Microbiol., 4, 677–690.[CrossRef][ISI][Medline]
Amini, H.R., Ascencio, F., Ruiz-Bustos, E., Romero, M.J., and Wadström, T. (1996) Cryptic domains of a, 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin. FEMS Immunol. Med. Microbiol., 16, 247–255.[CrossRef][ISI][Medline]
Ascencio, F., Fransson, L.Å., and Wadström, T. (1993) Affinity of the gastric pathogen Helicobacter pylori for the N-sulphated glycosaminoglycan heparan sulphate. J. Med. Microbiol., 38, 240–244.[Abstract]
Aspholm-Hurtig, M., Dailide, G., Lahmann, M., Kalia, A., Ilver, D., Roche, N., Vikström, S., Sjöström, R., Lindén, S., Bäckström, A., and others. (2004) Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin. Science, 305, 519–522.
Borén, T., Falk, P., Roth, K.A., Larson, G., and Normark, S. (1993) Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. Science, 262, 1892–1895.
Censini, S., Lange, C., Xiang, Z., Crabtree, J.E., Ghiara, P., Borodovsky, M., Rappuoli, R., and Covacci, A. (1996) Cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc. Natl. Acad. Sci. U. S. A., 93, 14648–14653.
Dhaenens, L., Szczebara, F., and Husson, M.O. (1997) Identification, characterization, and immunogenicity of the lactoferrin-binding protein from Helicobacter pylori. Infect. Immun., 65, 514–518.[Abstract]
Dowsett, S.A. and Kowolik, M.J. (2003) Oral Helicobacter pylori: can we stomach it? Crit. Rev. Oral Biol. Med., 14, 226–233.
Du, M.H., Spohr, U., and Lemieux, R.U. (1994) The recognition of three different epitopes for the H-type, 2 human blood group determinant by lectins of Ulex europaeus, Galactia tenuiflora and Psophocarpus tetragonolobus (winged bean). Glycoconj. J. 11, 443–461.[CrossRef][ISI][Medline]
Edwards, N.J., Monteiro, M.A., Faller, G., Walsh, E.J., Moran, A.P., Roberts, I.S., and High, N.J. (2000) Lewis X structures in the O antigen side-chain promote adhesion of Helicobacter pylori to the gastric epithelium. Mol. Microbiol., 35, 1530–1539.[CrossRef][ISI][Medline]
Ernst, P.B. and Gold, B.D. (2000) The disease spectrum of Helicobacter pylori: the immunopathogenesis of gastroduodenal ulcer and gastric cancer. Annu. Rev. Microbiol., 54, 615–640.[CrossRef][ISI][Medline]
Evans, D.G., Evans, D.J. Jr., Moulds, J.J., and Graham, D.Y. (1988) N-acetylneuraminyllactose-binding fibrillar hemagglutinin of Campylobacter pylori: a putative colonization factor antigen. Infect. Immun., 56, 2896–2906.
Falk, P., Roth, K.A., Borén, T., Westblom, T.U., Gordon, J.I., and Normark, S. (1993) An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium. Proc. Natl. Acad. Sci. U. S. A, 90, 2035–2039.
Fischer, W., Puls, J., Buhrdorf, R., Gebert, B., Odenbreit, S., and Haas, R. (2001) Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8. Mol. Microbiol., 42, 1337–1348.[CrossRef][ISI][Medline]
Gerhard, M., Hirmo, S., Wadström, T., Miller-Podraza, H., Teneberg, S., Karlsson, K.A., Appelmelk, B.J., Odenbreit, S., Haas, R., Arnqvist, A., and Borén, T. (2001) Helicobacter pylori, an adherent pain in the stomach. In Achtman, M., (ed.). Helicobacter pylori: Molecular and Cellular Biology. Horizon Scientific Press, Wymondham, UK, pp. 185–206.
Hessey, S.J., Spencer, J., Wyatt, J.I., Sobala, G., Rathbone, B.J., Axon, A.T., and Dixon, M.F. (1990) Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis. Gut, 31, 134–138.
Hirmo, S., Artursson, E., Puu, G., Wadström, T., and Nilsson, B. (1999) Helicobacter pylori interactions with human gastric mucin studied with a resonant mirror biosensor. J. Microbiol. Methods, 37, 177–182.[CrossRef][ISI][Medline]
Hirmo, S., Kelm, S., Schauer, R., Nilsson, B., and Wadström, T. (1996) Adhesion of Helicobacter pylori strains to -2,3-linked sialic acids. Glycoconj. J., 13, 1005–1011.[CrossRef][ISI][Medline]
Ilver, D., Arnqvist, A., Ögren, J., Frick, I.M., Kersulyte, D., Incecik, E.T., Berg, D.E., Covacci, A., Engstrand, L., and Borén, T. (1998) Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science, 279, 373–377.
Knibbs, R.N., Perini, F., and Goldstein, I.J. (1989) Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin. Biochemistry, 28, 6379–6392.[CrossRef][Medline]
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[CrossRef][Medline]
Lelwala-Guruge, J., Ljungh, A., and Wadström, T. (1992) Haemagglutination patterns of Helicobacter pylori. Frequency of sialic acid-specific and non-sialic acid-specific haemagglutinins. APMIS, 100, 908–913.[ISI][Medline]
Lingwood, C.A., Huesca, M., and Kuksis, A. (1992) The glycerolipid receptor for Helicobacter pylori (and exoenzyme S) is phosphatidylethanolamine. Infect. Immun., 60, 2470–2474.
Mahdavi, J., Sondén, B., Hurtig, M., Olfat, F.O., Forsberg, L., Roche, N., Ångström, J., Larsson, T., Teneberg, S., Karlsson, K.A., and others. (2002) Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation. Science, 297, 573–578.
Mollicone, R., Cailleau, A., Imberty, A., Gane, P., Perez, S., and Oriol, R. (1996) Recognition of the blood group H type, 2 trisaccharide epitope by 28 monoclonal antibodies and three lectins. Glycoconj. J., 13, 263–271.[CrossRef][ISI][Medline]
Namavar, F., Sparrius, M., Veerman, E.C., Appelmelk, B.J., and Vandenbroucke-Grauls, C.M. (1998) Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin. Infect. Immun., 66, 444–447.
Odenbreit, S., Faller, G., and Haas, R. (2002a) Role of the AlpAB proteins and lipopolysaccharide in adhesion of Helicobacter pylori to human gastric tissue. Int. J. Med. Microbiol., 292, 247–256.[CrossRef][ISI][Medline]
Odenbreit, S., Kavermann, H., Püls, J., and Haas, R. (2002b) CagA tyrosine phosphorylation and interleukin-8 induction by Helicobacter pylori are independent from AlpAB, HopZ and Bab-group outer membrane proteins. Int. J. Med. Microbiol., 292, 257–266.[CrossRef][ISI][Medline]
Odenbreit, S., Puls, J., Sedlmaier, B., Gerland, E., Fischer, W., and Haas, R. (2000) Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science, 287, 1497–1500.
Odenbreit, S., Till, M., Hofreuter, D., Faller, G., and Haas, R. (1999) Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue. Mol. Microbiol., 31, 1537–1548.[CrossRef][ISI][Medline]
Peck, B., Ortkamp, M., Diehl, K.D., Hundt, E., and Knapp, B. (1999) Conservation, localization and expression of HopZ, a protein involved in adhesion of Helicobacter pylori. Nucleic Acids Res., 27, 3325–3333.
van Putten, J.P., Duensing, T.D., and Cole, R.L. (1998) Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors. Mol. Microbiol., 29, 369–379.[CrossRef][ISI][Medline]
Ruhl, S., Cisar, J.O., and Sandberg, A.L. (2000) Identification of polymorphonuclear leukocyte and HL-60 cell receptors for adhesins of Streptococcus gordonii and Actinomyces naeslundii. Infect. Immun., 68, 6346–6354.
Ruhl, S., Sandberg, A.L., and Cisar, J.O. (1996) Recognition of immunoglobulin A1 by oral actinomyces and streptococcal lectins. Infect. Immun., 64, 5421–5424.[Abstract]
Ruhl, S., Sandberg, A.L., and Cisar, J.O. (2004) Salivary receptors for the proline-rich protein-binding and lectin-like adhesins of oral actinomyces and streptococci. J. Dent. Res., 83, 505–510.
Schreiber, S., Konradt, M., Groll, C., Scheid, P., Hanauer, G., Werling, H.O., Josenhans, C., and Suerbaum, S. (2004) The spatial orientation of Helicobacter pylori in the gastric mucus. Proc. Natl. Acad. Sci. U. S. A., 101, 5024–5029.
Schreiber, S., Stüben, M., Josenhans, C., Scheid, P., and Suerbaum, S. (1999) In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: experimental method and results. Infect. Immun., 67, 5151–5156.
Schwartz, M.A. and Shattil, S.J. (2000) Signaling networks linking integrins and rho family GTPases. Trends Biochem. Sci., 25, 388–391.[CrossRef][ISI][Medline]
Segal, E.D., Falkow, S., and Tompkins, L.S. (1996) Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins. Proc. Natl. Acad. Sci. U. S. A., 93, 1259–1264.
Sinha, B., Francois, P.P., Nusse, O., Foti, M., Hartford, O.M., Vaudaux, P., Foster, T.J., Lew, D.P., Herrmann, M., and Krause, K.H. (1999) Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin 5ß1. Cell Microbiol., 1, 101–117.[CrossRef][ISI][Medline]
Spiro, R.G. and Bhoyroo, V.D. (1974) Structure of the O-glycosidically linked carbohydrate units of fetuin. J. Biol. Chem., 249, 5704–5717.
Su, B., Johansson, S., Fallman, M., Patarroyo, M., Granstrom, M., and Normark, S. (1999) Signal transduction-mediated adherence and entry of Helicobacter pylori into cultured cells. Gastroenterol., 117, 595–604.[CrossRef][ISI][Medline]
Takahashi, Y., Konishi, K., Cisar, J.O., and Yoshikawa, M. (2002) Identification and characterization of hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1. Infect. Immun., 70, 1209–1218.
Takahashi, Y., Sandberg, A.L., Ruhl, S., Muller, J., and Cisar, J.O. (1997) A specific cell surface antigen of Streptococcus gordonii is associated with bacterial hemagglutination and adhesion to 2–3-linked sialic acid-containing receptors. Infect. Immun., 65, 5042–5051.[Abstract]
Takasaki, S. and Kobata, A. (1986) Asparagine-linked sugar chains of fetuin: occurrence of tetrasialyl triantennary sugar chains containing the Gal beta 1–3GlcNAc sequence. Biochemistry, 25, 5709–5715.[CrossRef][Medline]
Thomsson, K.A., Prakobphol, A., Leffler, H., Reddy, M.S., Levine, M.J., Fisher, S.J., and Hansson, G.C. (2002) The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse. Glycobiology, 12, 1–14.
Trust, T.J., Doig, P., Emödy, L., Kienle, Z., Wadström, T., and O’Toole, P. (1991) High-affinity binding of the basement membrane proteins collagen type IV and laminin to the gastric pathogen Helicobacter pylori. Infect. Immun., 59, 4398–4404.
Valkonen, K.H., Ringner, M., Ljungh, A., and Wadström, T. (1993) High-affinity binding of laminin by Helicobacter pylori: evidence for a lectin–like interaction. FEMS Immunol. Med. Microbiol., 7, 29–37.[CrossRef][ISI][Medline]
Valkonen, K.H., Wadström, T., and Moran, A.P. (1994) Interaction of lipopolysaccharides of Helicobacter pylori with basement membrane protein laminin. Infect. Immun., 62, 3640–3648.
Valkonen, K.H., Wadström, T., and Moran, A.P. (1997) Identification of the N-acetylneuraminyllactose-specific laminin-binding protein of Helicobacter pylori. Infect. Immun., 65, 916–923.
Yan, L., Wilkins, P.P., Alvarez-Manilla, G., Do, S.I., Smith, D.F., and Cummings, R.D. (1997) Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le (x) determinant. Glycoconj. J., 14, 45–55.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Muller, G. Groger, K.-A. Hiller, G. Schmalz, and S. Ruhl Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria Appl. Envir. Microbiol., April 15, 2007; 73(8): 2653 - 2660. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. Kusters, A. H. M. van Vliet, and E. J. Kuipers Pathogenesis of Helicobacter pylori Infection Clin. Microbiol. Rev., July 1, 2006; 19(3): 449 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y Yamaoka, O Ojo, S Fujimoto, S Odenbreit, R Haas, O Gutierrez, H M T El-Zimaity, R Reddy, A Arnqvist, and D Y Graham Helicobacter pylori outer membrane proteins and gastroduodenal disease Gut, June 1, 2006; 55(6): 775 - 781. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||