Carbohydrates act as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis: a study of bacterial binding to glycolipids

Ulrika Hellström1,2, Eva C. Hallberg2,3, Jens Sandros2, Lennart Rydberg3 and Annika E. Bäcker4

2 Department of Oral Pathology, Faculty of Odontology, The Sahlgrenska Academy at Göteborg University, Box 450, SE 405 30 Göteborg, Sweden; 3 Department of Clinical Chemistry and Transfusion Medicine, The Sahlgrenska Academy at Göteborg University, SE 413 45 Göteborg, Sweden; and 4 Department of Endodontics and Oral Diagnostics, Faculty of Odontology, The Sahlgrenska Academy at Göteborg University, Box 450, SE 405 30 Göteborg, Sweden

Received on October 30, 2003; revised on February 19, 2004; accepted on March 4, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this study we show for the first time the use of carbohydrate chains on glycolipids as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis. Previous studies have shown that this bacterium has the ability to adhere to and invade the epithelial lining of the dental pocket. Which receptor(s) the adhesin of P. gingivalis exploit in the adhesion to epithelial cells has not been shown. Therefore, the binding preferences of this specific bacterium to structures of carbohydrate origin from more than 120 different acid and nonacid glycolipid fractions were studied. The bacteria were labeled externally with 35S and used in a chromatogram binding assay. To enable detection of carbohydrate receptor structures for P. gingivalis, the bacterium was exposed to a large number of purified total glycolipid fractions from a variety of organs from different species and different histo-blood groups. P. gingivalis showed a preference for fractions of human and pig origin for adhesion. Both nonacid and acid glycolipids were used by the bacterium, and a preference for shorter sugar chains was noticed. Bacterial binding to human acid glycolipid fractions was mainly obtained in the region of the chromatograms where sulfated carbohydrate chains usually are found. However, the binding pattern to nonacid glycolipid fractions suggests a core chain of lactose bound to the ceramide part as a tentative receptor structure. The carbohydrate binding of the bacterium might act as a first step in the bacterial invasion process of the dental pocket epithelium, subsequently leading to damage to periodontal tissue and tooth loss.

Key words: adhesion / bacterial overlay / chromatogram binding assay / glycolipid / Porphyromonas gingivalis


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Over the years more than 400 different bacterial species have been isolated from the oral cavity. Only a small number of these strains are considered to be periodontally harmful isolates (Moore and Moore, 1994Go). Previous studies have shown that the oral pathogen Porphyromonas gingivalis plays a significant role in the development of periodontitis, a disease that affects the supporting structure of the tooth and subsequently leads to tooth loss if left untreated (Haffajee and Socransky, 1994Go). A large array of potential virulence factors produced by the bacterium, including (among others) production of proteases, fimbriae, polysaccharide capsule, and hemagglutinating factors, enables the induction of periodontal tissue damage (Holt and Bramanti, 1991Go). The adhesive and invasive potential of P. gingivalis that provides the bacterium with a temporary shelter against antibodies and phagocytic cells of the host has been carefully examined in previous studies. However, few of the interactions between this microorganism and the host that lead to periodontal tissue destruction are known. The bacterium uses an adhesin located on the tip of its fimbriae to attach to the surface of the epithelial cells. This binding is inhibited if the fimbriae are blocked by monoclonal antibodies (Isogai et al., 1988Go). It is also known that P. gingivalis strains, which express only a minor arsenal of fimbriae or no fimbriae at all, show a decreased binding capacity to epithelial cells (Lee et al., 1992Go).

It has been shown that a number of bacteria can bind to glycoconjugates on the cell surfaces (Kelm and Schauer, 1997Go; Mouricout, 1997Go), for example, Streptococcus suis (Haataja et al., 1994Go), Escherichia coli (Yang et al., 1994Go), and Helicobacter pylori (Borén et al., 1993Go). Which receptor(s) the adhesin of P. gingivalis exploit in the adhesion to epithelial cells has not been shown. Theories and studies based on various proteins and glycosylated proteins have been published, but none have yet been able to characterize the structure of the bacterial binding epitope. In vitro studies with glycoprotein from oral epithelium suggest the use of glycoconjugates as bacterial receptors (Agnani et al., 2003Go). Adhesion to glycoproteins can occur either to the protein or the carbohydrate part of the molecule. A recently published study showed that the addition of soluble saccharides to the bacterial solution can abolish the binding of P. gingivalis to oral epithelial cells (Agnani et al., 2003Go). This fact suggests that the carbohydrate moiety is somehow involved in the adhesion process. Previous studies have also shown that P. gingivalis can hydrolyze proteins and thereby expose previously hidden epitopes (i.e., cryptitopes) (Kontani et al., 1997Go). This procedure might be adopted by the bacterium when adhering to carbohydrate epitopes on glycolipids.

Glycoproteins exist in different forms in the membranes of eukaryotic cells. A characteristic feature of glycoproteins is the dominating protein moiety of the molecule, compared to proteoglycans, which consist of a dominating carbohydrate part. Carbohydrates existing on glycoproteins can often also be found on glycolipids on the cell surface. The carbohydrate epitopes on glycolipids and glycoproteins are in many cases identical, but the core chains do vary (Oriol, 1995Go). When compared to glycoproteins, the core chains on glycolipids are generally shorter and less branched. They are also quite distinct from the core sequences of glycoproteins. The expression of carbohydrate chains can also vary between different animal species (Bäcker et al., 1997Go; Björk et al., 1987Go; Smith et al., 1975Go; Umesaki et al., 1989Go), between different organs (Bäcker et al., 1999Go; Ulfvin et al., 1989), and also between different cells (Strokan et al., 1998a).

Based on these facts, the aim of the present study was to investigate whether the periodontal pathogen P. gingivalis can use glycolipids as receptors by screening a large array of glycolipid structures with a chromatogram binding assay.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
More than 120 glycolipid fractions representing a wide range of different carbohydrate structures were separated by thin-layer chromatography (TLC). The TLC plate was used for bacterial overlay assay with 35S-labeled P. gingivalis FDC381, and an identical TLC plate was stained with anisaldehyde reagent for chemical detection of the glycolipids. Binding was visualized by autoradiography. In general, more binding sites were detected in the autoradiograms after exposure of P. gingivalis to nonacid (one to four binding sites) compared to exposure to acid glycolipid fractions (one to two binding sites) (Figures 1GoGoGoGo6). When the autoradiograms where applied on the anisaldehyde-stained TLC plates with nonacid glycolipid fractions, binding of the bacteria was mostly to medium moving fractions in the chromatogram. This region of the TLC plate contains glycolipids with three to five saccharides in their carbohydrate chain. In acid fractions bacterial binding mostly occurred to fast moving fractions which is the part of the chromatogram that contains glycolipids with one to three saccharides in their carbohydrate chains.



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Fig. 1. Thin-layer chromatogram of nonacid glycolipid fractions of human origin with positive binding of 35S-labeled P. gingivalis. (A) Detection of glycolipids with anisaldehyde reagent. (B) Glycolipids detected by autoradiography after binding of 35S-labeled P. gingivalis. The binding assay was performed as described in Materials and methods. Approximately 2–10 µg of the glycolipid mixtures were added to the lanes (1–5), depending on their complexity. The solvent system used was chloroform/methanol/water (60:35:8, by volume). The lanes contained kidney A (lane 1, reference), transplantation kidney A1 Le(a–b+) (lane 2), vein A1 Le(a–b+) (lane 3), ureter A1 Le(a–b+) (lane 4), and artery A1 Le(a–b+) (lane 5). The sugar residues in the glycolipid chains are indicated on the left side.

 


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Fig. 2. Thin-layer chromatogram of acid glycolipid fractions of human origin with positive binding of 35S-labeled P. gingivalis. (A) Detection of glycolipids with anisaldehyde reagent. (B) Glycolipids detected by autoradiography after binding of 35S-labeled P. gingivalis. The binding assay was performed as described in Materials and methods. Approximately 2–10 µg of the glycolipid mixtures were added to the lanes (1–11), depending on their complexity. The solvent system used was chloroform/methanol/water with 0.2% CaCl2 (60:40:9, by volume). The lanes contained kidney A (lane 1, reference), intestine B (lane 2), kidney B (lane 3), ureter A1 Le(a–b+) (lane 4), sulfolipid fraction from kidney B (lane 5), sulfatide fraction from pancreas (lane 6), kidney (lane 7), transplantation kidney (lane 8), transplantation kidney (lane 9), transplantation kidney (lane 10), and sulfatide fraction from kidney (lane 11). The sugar residues in the glycolipid chains are indicated on the left side.

 


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Fig. 3. Thin-layer chromatogram of acid glycolipid fractions of human origin with positive binding of 35S-labeled P. gingivalis. (A) Detection of glycolipids with anisaldehyde reagent. (B) Glycolipids detected by autoradiography after binding of 35S-labeled P. gingivalis. The binding assay was performed as described in Materials and methods. Approximately 2–10 µg of the glycolipid mixtures were added to the lanes (1–5), depending on their complexity. The solvent system used was chloroform/methanol/water with 0.2% CaCl2 (60:40:9, by volume). The lanes contained kidney A (lane 1, reference), kidney A (lane 2), ganglioside fraction from kidney (lane 3), ganglioside fraction from transplantation kidney (lane 4), and sulfated sulfolipid fraction from kidney (lane 5). The sugar residues in the glycolipid chains are indicated on the left side.

 


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Fig. 4. Thin-layer chromatogram of nonacid glycolipid fractions of pig origin with positive binding of 35S-labeled P. gingivalis. (A) Detection of glycolipids with anisaldehyde reagent. (B) Glycolipids detected by autoradiography after binding of 35S-labeled P. gingivalis. The binding assay was performed as described in Materials and methods. Approximately 2–10 µg of the glycolipid mixtures were added to the lanes (1–4), depending on their complexity. The solvent system used was chloroform/methanol/water (60:35:8, by volume). The lanes contained human acid kidney A (lane 1, reference), pig liver O (lane 2), pig kidney O (lane 3), and pig small intestine O (lane 4). The sugar residues in the glycolipid chains are indicated on the left side.

 


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Fig. 5. Thin-layer chromatogram of nonacid glycolipid fractions of pig origin with positive binding of 35S-labeled P. gingivalis. (A) Detection of glycolipids with anisaldehyde reagent. (B) Glycolipids detected by autoradiography after binding of 35S-labeled P. gingivalis. The binding assay was performed as described in Materials and methods. Approximately 2–10 µg of the glycolipid mixtures were added to the lanes (1–4), depending on their complexity. The solvent system used was chloroform/methanol/water (60:35:8, by volume). The lanes contained nonacid glycolipid fraction from human acid kidney A (lane 1, reference), pig salivary gland O (lane 2), pig heart O (lane 3), and pig spleen O (lane 4). The sugar residues in the glycolipid chains are indicated on the left side.

 


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Fig. 6. Thin-layer chromatogram of acid glycolipid fractions of pig origin with negative binding of 35S-labeled P. gingivalis. (A) Detection of glycolipids with anisaldehyde reagent. (B) Glycolipids detected by autoradiography after binding of 35S-labeled P. gingivalis. The binding assay was performed as described in Materials and methods. Approximately 2–10 µg of the glycolipid mixtures were added to the lanes (1–5), depending on their complexity. The solvent system used was chloroform/methanol/water with 0.2% CaCl2 (60:40:9, by volume). The lanes contained acid glycolipid fractions from kidney A (lane 1, reference), ganglioside fraction from pig heart (lane 2), sulfatide fraction from pig heart (lane 3), ganglioside fraction from pig kidney (lane 4), and sulfatide fraction from pig kidney (lane 5). The sugar residues in the glycolipid chains are indicated on the left side.

 
Binding of P. gingivalis to nonacid glycolipid fractions of human origin was observed in the region of the TLC plates with one to four sugar residues (Table I, Figure 1). Considering human acid glycolipid fractions, bacterial binding was mainly obtained in the short sugar residue region of the TLC plates, a region where sulfated carbohydrate chains usually are found (Table I, Figures 2 and 3). No binding was detected to human acid fractions originating from blood group A plasma, colon, liver, small intestine, and blood group A1 transplantation liver (data not shown). The total acid fraction from ureter showed a weak staining on the autoradiogram, but binding was not always obtained to this fraction (Figure 2, lane 4). Neither could positive binding be confirmed with purified fractions of ganglioside or sulfatide from this organ (data not shown).


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Table I. Glycolipid fractions of human origin with positive binding of 35S-labeled P. gingivalis in the bacterial overlay assay

 
The binding pattern of nonacid glycolipid fractions from pig organs was generally more distinct compared to autoradiograms of bacterial overlay assay with glycolipid fractions from human organs (Table II, Figures 4 and 5). Binding to acid glycolipid fractions of pig origin could not be detected (Figure 6).


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Table II. Glycolipid fractions of pig origin with positive binding of 35S-labeled P.gingivalis in the bacterial overlay assay

 
Organs of sheep origin were also used in the bacterial overlay assay. Total acid glycolipid fractions of blood group A (Strokan et al., 1998b) were screened. All fractions tested negative for binding with radiolabeled bacteria (data not shown).

Binding was also obtained to gangliotriaosylceramide and gangliotetraosylceramide, the pure reference glycolipid fractions used for studying binding analogy with other bacteria (Figure 7).



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Fig. 7. Thin-layer chromatogram of pure reference glycolipid fractions with positive binding of 35S-labeled P. gingivalis. (A) Detection of glycolipids with anisaldehyde reagent. (B) Glycolipids detected by autoradiography after binding of 35S-labeled P. gingivalis. The binding assay was performed as described in Materials and methods. 8 µg of the glycolipid mixtures were added to the lanes (1–2). The solvent system used was chloroform/methanol/water (60:35:8, by volume). The lanes contained gangliotriaosylceramide from guinea pig erythrocytes (lane 1), and gangliotetraosylceramide from mouse feces (lane 2). These reference glycolipid fractions were kindly provided by M.D., Ph.D. Teneberg; for a more detailed description, we refer to Teneberg et al. (2004)Go.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Periodontitis is known to be a multifactorial disease, involving a variety of bacterial species. The complexes of microorganisms involved in the process appear to relate to the severity of the periodontal destruction (Socransky et al., 1988Go). In the present study we focus on P. gingivalis strain FDC381 because previously published results have shown that this particular strain has the ability to adhere to and invade oral epithelial cells and human pocket epithelium in vitro (Sandros et al., 1993Go, 1994Go). P. gingivalis seems to act as a key bacteria found in a majority of the microbial complexes causing severe and aggressive periodontitis with a resulting loss of periodontal attachment. However, it is important to keep in mind the complexity of this disease and the cooperation of the periodontally harmful bacterial species existing in the dental crevice. The binding epitopes used by P. gingivalis for attachment to and invasion of oral epithelium are not fully understood.

The fact that bacteria use short carbohydrate sequences as receptors is well known (Karlsson, 1995Go) and has been studied at different locations, such as in the human intestine (Borén et al., 1993Go; Holgersson et al., 1991aGo) and oral cavity (Strömberg and Borén, 1992Go). The blood group of the individual (e.g., ABO and Lewis blood group systems) is of importance. In fact, in some cases the blood group has been shown to be a crucial factor for the bacterial capacity to adhere to the specific tissue. This is exemplified by the bacterium H. pylori, which binds to stomach mucosa in individuals expressing the Leb structure (a five-sugar-long carbohydrate) in the tissue (Borén et al., 1993Go). Both neutral (nonacid) and acid carbohydrate chains are known to act as bacterial receptors (Ångström et al., 1998Go; Borén et al., 1993Go; Holgersson et al., 1991aGo; Karlsson, 1995Go; Miller-Podraza et al., 1996Go; Teneberg et al., 2002Go, 2004Go). It is not known whether the binding of P. gingivalis to carbohydrates is dependent on the type of blood group or not.

We found that it was adequate to cultivate the bacteria for 3 days prior to the bacterial overlay assays. At this point P. gingivalis seems to express properties necessary for adhesion to glycolipid structures. The bacteria in the study were cultured on agar plates. Other glycolipid binding bacteria, like H. pylori, show the same binding pattern when cultured on agar as in broth (Teneberg et al., 2002Go). It is also known that the use of different coating solutions in the chromatogram binding assay can affect the reproducibility of binding (Teneberg et al., 2002Go). The use of 2% bovine serum albumin (w/v) in phosphate buffered saline (PBS) with 0.1% Tween 20 (v/v) and 0.01% sodium azid (w/v) was found to be useful in our bacterial overlays.

The bacterial overlay assay showed binding of P. gingivalis to human nonacid and acid fractions and to pig nonacid fractions. Acid fractions of pig origin tested negative despite the close similarity of glycolipid expression in human and pig organs. Binding was found in the four-sugar region but not to longer sugar chains in the glycolipid fractions. The bacterial binding sites are presumably not located on the terminal sugars of the glycolipid carbohydrate chain but on the core chain. This is an important observation because previous studies have shown that P. gingivalis has the ability to enzymatically cleave proteins (Kontani et al., 1997Go) and thereby reveal cryptitopes of protein origin. Enzymatic cleavage might also be used by the bacterium on structures of carbohydrate origin, thus enabling adhesion of the bacterium to cell surfaces. If we expose P. gingivalis solely to carbohydrate epitopes that normally are expressed only in oral mucosa, there would in fact be a reduced chance of finding the actual binding epitopes used by the bacterium in vivo. The glycolipid expression in oral epithelium varies between different individuals because of, among other things, genetic conditions such as the type of blood group (Ravn and Dabelsteen, 2000Go). Therefore, it is of great importance to use a variety of species (in this case human, pig, and sheep) to find structures of carbohydrate origin that are normally not exposed in human tissue. These structures can be exposed as cryptitopes, for example, as previously mentioned. Consideration must also be taken of the fact that it is difficult to purify glycolipids from human pocket epithelium because it is very hard to gain access to the large amount of tissue necessary for glycolipid purification. Studies on glycolipid expression in gingival tissue only consists of assays with monoclonal antibodies on surfaces of gingival epithelial cells (Mackenzie et al., 1989Go). The glycolipids constitute only a small part of the total weight of the tissue which results in approximately only 20 mg total glycolipid fraction from 200 g tissue. Another important reason for using glycolipids from different species and different organs is that information can be determined about the possible influence and/or involvement on the binding of the bacterium to the ceramide part of the glycolipid chain. Studies with H. pylori have shown that the ceramide part is involved in the binding of this bacterium to glycolipids (Ångström et al., 1998Go).

The blood group type of the organ that the glycolipid fraction originated from did not seem to be significant. The blood group epitopes are in most cases terminally expressed on precursor chains with four to five sugar residues. One can speculate whether the presence of a blood group epitope might inhibit bacterial binding to the carbohydrate. Instead, binding might occur to the core chain or the intermediate part of the carbohydrate chain depending on the 3D structure of the carbohydrate chain. A core chain similarity is indicated in the bacterial binding region in the non-acid glycolipid fractions from pig. In this region of the TLC plate the globo chain and its precursors are often found (Bäcker et al., 1998Go).

Binding of P. gingivalis to acid glycolipid fractions from organs of human origin was noticed in the upper part of the chromatogram. In this region of the TLC plates sulfatide fractions are usually found, which indicates that this may be a tentative structure for adhesion of this specific bacterium. Sulfatide fractions have also been included in our screening of glycolipid structures and positive binding was obtained (Figure 2, lane 5, 6 and 11; and Figure 3, lane 5), a result that supports our hypothesis. This has recently also been indicated in the case of glycoprotein binding of P. gingivalis (Agnani et al., 2003Go). In sulfatide glycolipid fractions the sugar chain shows a one- or two-sugar residue length with terminal sulfate group (SO3). The core structure tentatively consists of a galactose connected to glucose before connecting to the polar ceramide chain.

Sugar chains consisting of one-, two-, three-, and four-sugar residues are used by P. gingivalis as receptor structures in nonacid fractions from pig organs. A comparison of the binding areas in this study, with earlier published structural characterization studies of the pig organs (Bäcker et al., 1998Go), indicated that the bacterial glycolipid receptor structure might be a so-called type 4 chain, that is, the globo chain and its core structures (Holgersson et al., 1992Go). Considering the binding in the two-sugar region, it is not possible to distinguish between binding to lactosylceramide and/or to galabiosylceramide, because these glycolipids do not separate on the TLC plates due to similar polarities.

Our results also showed that P. gingivalis has the ability to adhere to gangliotriaosylceramide (GgO3) and gangliotetraosylceramide (GgO4). These purified reference glycolipid fractions were used to study analogy of binding patterns to other bacteria. Previous studies has shown that, among others, Actinobacillus pleuropneumoniae (Abul-Milh et al., 1999Go) and E. coli (Teneberg et al., 2004Go) can bind to GgO3 and GgO4. The fact that P. gingivalis also can use these glycolipids as receptor structures shows that this bacterium has a similar binding pattern. What type of other pure glycolipid structures P. gingivalis recognizes will be fully described in our following study.

Our screening of a large library of glycolipid carbohydrate chains with TLC and bacterial overlay assay showed that P. gingivalis can bind to sugar residues on glycolipids. A screening of more than 120 different fractions by TLC where each fraction was tested at least three times is very time-consuming. Therefore, this screening will be followed in a forthcoming study by further purification of the glycolipid fractions with positive binding of the bacterium.

In this screening study we show for the first time that the periodontal pathogen P. gingivalis strain FDC381 can use glycolipid sugar chains as bacterial receptors. Both nonacid and acid glycolipids can be used by the bacterium and shorter carbohydrate chains are mainly preferred. We have also found that sulfated glycolipids probably are favored by the bacterium in acid glycolipid fractions. Glycolipids based on the globo chain with a core chain of lactosylceramide bound to the ceramide part, seem to be preferred in the nonacid glycolipid fractions.

The carbohydrate binding of the bacterium might act as a first step in the bacterial invasion process of the pocket epithelium, subsequently leading to periodontal damage in the tissue. A mapping of the receptor structures that P. gingivalis uses for adhesion and invasion of eukaryotic cells would increase the knowledge of the origin of periodontitis. This knowledge might in turn be used in new treatment strategies, for example receptor analogs, to inhibit bacterial binding and the production of vaccines and other antimicrobial therapies specifically directed against P. gingivalis. The future goal is to reduce the incidence, onset, and spread of periodontal disease.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycolipid preparations
Non-acid glycolipids from different species, organs, and blood groups were isolated according to the previously reported method of Karlsson (Karlsson, 1987Go). These nonacid glycolipid fractions are referred to as total fractions. Fractions with acid properties were separated from the nonacid fractions during the purification procedure and used separately in the screening process. These fractions are referred to as total acid fractions. In some cases the glycolipid fractions have been further purified (e.g., acid fractions from human kidneys, nonacid fractions originating from pig small intestine, salivary gland, kidney, liver, and spleen) by high-performance liquid chromatography (HPLC) (Bäcker et al., 1998Go). The samples have then been pooled and are referred to as, for example, fractions with one, two, or three sugar residues. Some of the longer carbohydrate chain fractions have also undergone structural analysis by masspectrometry and/or nuclear magnetic resonance for the identification of the component(s) (Bäcker et al., 1997Go, 1998Go; Gustavsson et al., 1996Go).

Sample criteria
The aim of the experiments was to perform a screening of a wide range of glycolipid structures with TLC and bacterial overlay assay. In this way P. gingivalis is exposed to a large number of different carbohydrate structures, which enables detection of receptor structures that are used by the bacterium. Different types of organs with different blood groups were analyzed and compared for differences in binding patterns and/or to study special bacterial preferences for any particular blood group epitope. Several fractions from the same kind of organ, but from different organ donors, were used in the experiments as well as organs with different blood group subgroups. Glycolipid fractions of both acid and nonacid origin were used to see if, for example, sialic acid is necessary for adhesion. In the majority of the screening experiments, total glycolipid fractions were used.

Fractions used in TLC and bacterial overlay
The following section is a listing of all fractions that were used in the TLC and bacterial overlay assays. Any subgrouping, such as serological typing (e.g., A2), blood group Lewis (Le(ab)), and Secretor (Se) status, is shown in parentheses. Fractions named transplantation indicate organs that originate from a human transplantation donor.

Fractions of human origin
Total fractions originating from human organs of blood group A used for screening included acid and nonacid kidney (A2), nonacid transplantation kidney (A1), nonacid transplantation kidney (A1 Le(a+b–)), nonacid transplantation kidney (A2 Le(a+b–)), nonacid liver (A1B), nonacid liver (A1 Le(a–b+)), nonacid liver (A1B Le(a–b–)), nonacid liver (A1 Le(a+b–)), nonacid liver (A2B Le(a–b+)), acid transplantation liver (A1), nonacid jejunum, nonacid ileum (A1), nonacid pancreas, acid plasma (A1 Le(a–b–)Se), acid plasma (A2 Le(a–b+)), nonacid thrombocytes (A1), and nonacid thrombocytes (A2).

Total fractions originating from human organs of blood group B type used for screening included acid and nonacid kidney, nonacid transplantation kidney (Le(a–b+)), acid intestine, acid and nonacid ureter (Le(a–b+)), acid and nonacid artery (Le(a–b+)), acid and nonacid vein (Le(a–b+)), nonacid liver, nonacid liver (Le(a–b+)), acid erythrocytes (Le(a–b+)), and acid plasma (A1 Le(a–b+)).

Total fractions originating from human organs of blood group AB type used for screening included nonacid liver (A1B) and nonacid transplantation kidney (AB Le(a–b+)).

Total fractions originating from human organs of blood group O type used for screening included nonacid kidney, nonacid transplantation kidney (Le(a–b+)), nonacid erythrocytes (Le(a–b+)), and nonacid plasma (Le(a–b+)).

Total fractions originating from human organs with pooled and/or mixed nonspecified blood group included acid kidney, acid and nonacid human liver, acid ureter, acid transplantation kidney, acid and nonacid colon, and acid small intestine.

HPLC-purified human fractions used in the screening included ganglioside fraction from colon, brain ganglioside fraction, pancreas ganglioside fraction, pancreas sulfatide fraction, ureter ganglioside fraction, transplantation kidney ganglioside fraction, sulfolipid fraction from ureter (residual stroma) blood group B, sulfolipid fraction from kidney blood group B, kidney ganglioside fraction, sulfolipid fraction from kidney, and a pure globoside fraction.

Fractions of nonhuman origin
Total fractions originating from pig organs of blood group O used for screening included nonacid small intestine, nonacid kidney, nonacid liver, nonacid spleen, and acid erythrocytes.

Total fractions originating from pig organs of blood group A included nonacid salivary gland and nonacid heart. A total fraction originating from about 80 pig blood group A and O aortas was also used.

HPLC-purified pig organ fractions used included ganglioside, globotriaosylceramide and globotetraosylceramide fractions from heart; sulfolipid fractions from heart, salivary gland, kidney, small intestine, liver, and spleen; and ganglioside fractions from intestine, kidney, salivary gland, heart, and liver. Fractions from small intestine, kidney, liver, salivary gland, spleen, and heart have also been purified and pooled into fractions with nonspecified monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and fractions with >= tetrasaccharide glycolipids (e.g., four to eight sugars in the sugar chain), respectively.

Total fractions originating from sheep organs of blood group A type included acid pancreas, acid small intestine, acid colon, acid lung, and acid kidney. Purified reference glycolipid fractions included gangliotriaosylceramide from guinea pig erythrocytes and gangliotetraosylceramide from mouse feces.

Thin Layer Chromatography
TLC was performed as previously described (Hansson et al., 1985Go) on aluminum-backed silica gel 60 high-performance TLC plates (Merck, Darmstadt, Germany and HP-KF, Whatman, Maidstone, U.K.). Glycolipid fractions (2–10 µg/ lane) were added to the TLC plates and separated in a specific mixture of organic solvents and water. The nonacid components were chromatographed in chloroform/methanol/water, 60:35:8 by volume, and the acid components in chloroform/methanol/water with 0.2% CaCl2, 60:40:9 by volume. On all TLC plates, a reference fraction from human kidney blood group A (an acid glycolipid fraction) that was positive for bacterial binding was added in lane 1.

For each chromatogram with bacterial overlay, two sets of identical glycolipid fractions were separated on the same TLC plate. In this manner we received two TLC plates with as identical a separation pattern as possible. The TLC plate was dried at room temperature until the organic solvents had evaporated from the silica gel, and thereafter it was cut in two halves, thus separating the identical TLC sets. The TLC plate that was used in the bacterial overlay experiment was coated in 0.3% (w/v) polyisobutylmethacrylate, P28 (Sigma-Aldrich), in diethyl ether/n-hexane (1:1 by volume), or in 0.5% P28 in diethyl ether/n-hexane (1:4 by volume) and left to polymerize overnight to avoid unspecific binding to nonpolymerized monomers. The identical TLC plate was visualized chemically by anisaldehyde reagent and used for inspection of the separation pattern and for identification of the bacterial binding region in the autoradiogram (Karlsson, 1987Go). All glycolipid-containing lines were visualized by chemical detection with anisaldehyde reagent. This TLC plate was then compared with the identical plate used in the chromatogram binding assay, where only lines with positive binding can be seen.

Bacterial strains and growth conditions
In this study P. gingivalis FDC381 (collection of Forsyth Dental Center, Boston, MA), a strain isolated from a periodontitis patient, was used. The bacteria were grown on Brucella agar plates (BBL Microbiology Systems, Cockeysville, MD) enriched with 5% defibrinated horse blood, 0.5% hemolyzed blood, and 5 µl/ml menadione in jars with 95% H2 and 5% CO2 at 37°C. A suspension of 5 µl L-[35S]methionine and L-[35S]cysteine in PBS with a concentration of 14.3 mCi/ml and t1/2 of 87.4 days (Redivue ProMix L-[35S] in vitro labeling mix, Amersham Biosciences, Uppsala, Sweden) was added to the agar plates. After 3 days of growth, bacterial cultures were collected and washed three times by centrifugation at approximately 5000 rpm in PBS. A spectrophotometer analysis at 550 nm was performed for evaluation of the bacterial concentration and compared to an absorbance standard curve for P. gingivalis. The bacteria were suspended in PBS with 2% bovine serum albumin (w/v), 0.1% Tween 20 (v/v), and 0.01% sodium azid (w/v) and diluted to a final concentration of 1.5–3 x 108 bacteria/ml used in the bacterial overlay assays.

Bacterial overlay and detection of bacterial binding
The method used in the bacterial overlay assays is a modified version of the method by Karlsson and Strömberg (1987)Go. The TLC plates were coated with 2% bovine serum albumin (w/v), 0.1% Tween 20 (v/v), and 0.01% sodium azid (w/v) in PBS for 2 h at room temperature to block nonspecific binding sites. Thereafter, the coating solution was carefully removed from the TLC plates. Approximately 5 ml of the bacterial suspension was sprinkled over the TLC plates with a Pasteur pipette and then incubated at room temperature for 9 h or overnight. The TLC plates were gently washed four times with PBS, dried in room temperature for at least 1 h, and autoradiographed for approximately 7–14 days using a ß-sensitive film (Kodak BioMax MR, Amersham Biosciences). After development of the film (on Agfa Curix HT-530 U equipment), bacterial binding was analyzed by visual inspection. Significant staining/darkening of the film was registered as positive binding and marked with asterisks (one to four, depending on intensity of the binding; one asterisk = weak staining, four asterisks = strong staining). When positive binding was detected in the autoradiogram, the film was placed on top of the identical anisaldehyde-stained TLC plate by means of easily identified landmarks. In this way the specific sugar binding region was identified. A comparison to the TLC plate in the reference article for that specific organ enabled estimation of the glycolipid structure that P. gingivalis can use as a receptor.


    Acknowledgements
 
Stina Olsson is gratefully acknowledged for valuable help with the bacteria. We also thank Lola Svensson for technical assistance with the TLC plates and M.D., Ph.D. Teneberg for discussions and troubleshooting concerning the bacterial overlay method and for lending us the pure reference glycolipid fractions. This work was supported by the Swedish Research Council (grant no. K2002-73X-14018-02B), a research grant from TUA-SAM, the Magn. Bergwall Foundation, Wilhelm and Martina Lundgren's Scientific Fund, the Royal Society of Arts and Sciences in Göteborg, the Swedish Dental Society, and the Dental Society of Göteborg.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: ulrika.hellstrom{at}odontologi.gu.se


    Abbreviations
 
HPLC, high-performance liquid chromatography; PBS, phosphate buffered saline; TLC, thin-layer chromatography


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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