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ATTACHMENT OF TREPONEMA DENTICOLA, IN PARTICULAR STRAIN ATCC 33520, TO EPITHELIAL CELLS AND ERYTHROCYTES. - AN IN VITRO STUDY - L—J Print: Offsetdrukkerij Ridderprint B.V., Ridderkerk ATTACHMENT OF TREPONEMA DENTICOLA, IN PARTICULAR STRAIN ATCC 33520, TO EPITHELIAL CELLS AND ERYTHROCYTES. - AN IN VITRO STUDY -
een wetenschappelijke proeve op het gebied van de Medische Wetenschappen
Proefschrift ter verkrijging van de graad van doctor aan de Katholieke Universiteit Nijmegen, volgens besluit van het College van Decanen in het openbaar te verdedigen op vrijdag 19 mei 1995 des namiddags te 3.30 uur precies door
Robert Antoine Cornelius Keulers geboren op 4 april 1957 te Geertruidenberg Promotor: Prof. Dr. K.G. König. Co-promotores: Dr. J.C. Maltha Dr. F.H.M. Mikx Ouders, Familie, Vrienden
Table of contents Page
Chapter 1: General introduction 9
Chapter 2: Attachment of Treponema denticola strains to monolayers of epithelial cells of different origin 29
Chapter 3: Attachment of Treponema denticola strains ATCC 33520, ATCC 35405, Bll and Ny541 to a morphologically distinct population of rat palatal epithelial cells 35
Chapter 4: Involvement of treponemal surface-located protein and carbohydrate moieties in the attachment of Treponema denticola ATCC 33520 to cultured rat palatal epithelial cells 43
Chapter 5: Hemagglutination activity of Treponema denticola grown in serum-free medium in continuous culture 51
Chapter 6: Development of an in vitro model to study the
invasion of oral spirochetes: A pilot study 59
Chapter 7: General discussion 71
Chapter 8: Summary, Samenvatting, References 85
Appendix: Ultrastructure of Treponema denticola ATCC 33520 113
Dankwoord 121
Curriculum vitae 123
Chapter 1
General introduction Table of contents chapter 1 Page
1.1. Introductory remarks 11 - Association studies - Response to treatment studies - Immunological studies
1.2. Morphology, occurrence and taxonomy of spirochetes 13 - Morphology - Occurrence - Taxonomy
1.3. Potential virulence factors of oral spirochetes 18 - Introduction - Endotoxins, metabolic products and enzymes - Attachment = Attachment of non-oral spirochetes, in particular, T. pallidum spp. pallidum = Attachment of T. denticola - Invasion
1.4. Aim and outline of the investigation 27 1.1. Introductory remarks
Periodontal disease affects the tooth-supporting tissues and may, in advanced stages, destroy alveolar bone, causing loss of teeth. As dental plaque, particularly subgingival plaque, is closely associated with diseased periodontal tissues, it is generally assumed that plaque microorganisms are responsible for periodontal disease (85,127,151,158,169). Up to now it has not been possible to ascribe the aetiology of periodontal disease to specific oral microorganisms or groups of microorganisms. This is mainly due to the high complexity of the subgingival microflora in which over 325 distinct bacterial species have been observed (116). Oral spirochetes are members of the subgingival microflora and have been associated with periodontal disease as far back as 1890 (2,42,94,115,146,147). Oral spirochetes were for the first time described between 1683 and 1686 by Antonie van Leeuwenhoek in dental plaque of an old man who never cleaned his teeth (74). Nowadays knowledge of the involvement of oral spirochetes in periodontal disease is mainly based on association studies, on response to treatment studies and, to a lesser extent, on immunological studies.
- Association studies.
Association studies mainly involve microscopic examination of samples of subgingival plaque or oral tissue. In plaque removed from non-diseased sites the mean proportions of oral spirochetes is less than 3% (Table 1.1). Slightly increased proportions of oral spirochetes (7-8%) have been reported in gingivitis and juvenile periodontitis. High proportions of spirochetes have been observed in untreated lesions of acute necrotizing ulcerative gingivitis (ANUG; 40%), adult periodontitis (29-57%) and post-juvenile periodontitis (56%). In localized juvenile periodontitis high proportions of oral spirochetes are an inconsistent observation (150,186). All oral spirochetes identified so far have been assigned to the genus Treponema. Treponema denticola, which is cultivated routinely from diseased
11 periodontal pockets (117-120), is the only cultured oral spirochete species of which a positive correlation has been found between numbers of a particular serotype of this microorganism in the subgingival microflora and severe periodontitis (146,147).
- Response to treatment studies.
Response to treatment studies show that successful treatment of different forms of periodontitis by scaling and root planning, surgery, or other therapy, is accompanied by a significant reduction in the percentage of oral spirochetes in the subgingival microflora (Table 1.1).
Table 1.1: Mean percentage of Spirochetes in Untreated and Treated Subjects*
Disease Mean Percentage of Spirochetes References Category Untreated Treated
Health 1-3 1,79,86,148 Gingivitis 8 8 79,89,90 ANUG 40 12 97 Adult 29-57 4-11 19,27,73,75,77- Periodontitis 79,84,86,96,125, 136,149,153 Juvenile 7 2 75,77 Periodontitis Post-Juvenile 56 75 Periodontitis
Based on M.A. Listgarten (84)
12 - Immunological studies.
Immunological studies on humoral response to oral spirochetal infections are limited in number and not conclusive. Significantly elevated levels of antibodies against oral spirochetes, including T. denticola, were found in patients with moderate to advanced periodontitis (62,71,104,162,163) or ANUG (17). Elevated levels of humoral antibodies to oral spirochetes were not found in patients with gingivitis (17), in cases of severe periodontitis (168), or in patients totally edentulous due to periodontitis (62). Tew et al. (168) observed that patients with localized juvenile periodontitis were more often seropositive for several strains of T. denticola and T. socranskii than healthy persons, whereas Lai et al. (71) did not observe elevated serum antibody levels against T. denticola and T.vincentii in comparable patients. In two studies on patients suffering from either adult or juvenile periodontitis (71,104), no significant correlation was found between serum antibody levels against any of the tested oral spirochetes and their proportional presence in the subgingival microflora.
1.2. Morphology, occurrence and taxonomy of spirochetes.
- Morphology.
Spirochetes are helically shaped gram-negative bacteria with a characteristic morphology and locomotion (13). They possess an outer sheath, endoflagella and a protoplasmic cylinder (56,64,91; Figure 1.1). The outer sheath is the most external layer and envelops the endoflagella and protoplasmic cylinder. It consists of a membrane that in some Treponema species is covered by an external "Ruthenium-red positive" layer (56,123; Appendix). The membrane has, in most cases, a normal unit membrane construction containing simple proteins, glycoproteins, lipoproteins and lipopolysaccharides (190). However, in some species of Spirochaeta and Treponema, including T. denticola, it consists
13 Figure 1.1. Schematic illustrations of helically shaped spirochetes. В and С are enlargements of A and В respectively An outer sheath (OS) envellops the endoflagella (EF) and protoplasmic cylinder (PC) The endoflagella are located in the periplasmic space (PS) and inserted into the cylinder by way of an insertion pore (IP). The periplasmic space is the space between the outer sheath and the cell wall-cell membrane-complex (CWCM- complex). Sometimes spirochetes are covered with a Ruthenium-red positive layer (RR). CC' cell content (Modified from Holt, [56])
of polygonally arranged subunits which morphologically resemble an S- layer(56,57,106, Appendix). The Ruthenium-red positive layer is supposed to consist of acid glycosaminoglycan (99) and appears to be not present on every individual spirochete within one species (123; Appendix). The endoflagella lie in the periplasmatic space, the space between the outer membrane and the protoplasmic cylinder. They are composed almost entirely of protein, with only trace amounts of hexose- and pentose positive material. Endoflagella are at one side inserted into a subterminal depression in the protoplasmic cylinder near the ends of the spirochete. Each endoflagellum winds
14 around the protoplasmic cylinder and overlaps, near the center of the spirochete, other endo flagella that originate from the opposite end of the spirochete. The number of endoflagella per spirochete ranges from two to more than one hundred, depending on the species. Endoflagella are involved in cell motility (76,128). The protoplasmic cylinder is the internal part of the spirochetes. It consists of a cell wall-cytoplasmic membrane complex and encloses the cytoplasmic contents. The cell wall is the outermost layer of the cell wall-cell membrane complex and consists of a peptidoglycan layer containing muramic acid (190). The cytoplasmic membrane is the innermost layer of the cell wall-cytoplasmic membrane complex and is composed of phospholipids, proteins and enzymes.
- Occurrence.
Spirochetes occur in a wide variety of natural habitats (11,53,64). Many species grow and persist as free living forms in marine and fresh water, and in soils. Others are part of the commensal microflora in diverse hosts ranging from protozoa to mammals (53). A relatively small number is pathogenic for animals or humans (14,53). Oral spirochetes do not belong to the pioneers of the oral cavity. The establishment of spirochetes in the human oral cavity probably takes place once the teeth have erupted and dental plaque is formed. Their number and frequency increase with the age of the host (6,113,160). Once established, oral spirochetes are mainly present in the subgingival microflora of the gingival crevice but they are also found at the dorsum of the tongue, the tonsils and in the supragingival plaque (46,65,114,180,187). Oral spirochetes are probably acquired from other humans via oral contact (94,160) and their maintainance in plaque seems to be the result of autogenic succession (113). Electron microscopic investigations have shown that the distribution of oral spirochetes in the subgingival microflora is not at random. They often dominate in the outer zone and the apical portions of the subgingival microflora. Here they form, together with flagellated bacteria, a loose layer which lacks an intermicrobial matrix, and which covers the underlying mass of densely
15 packed bacteria adherent to the tooth surface (64,82,83,95,137). This positions spirochetes in close vicinity of the sulcular or pocket epithelium and the maintainance of these microorgasnisms in the subgingival microflora might be due to attachment to these tissues. Though epithelial cells, as well as polymorphonuclear leukocytes, are frequently found attached to the most apical portion of the subgingival microbial mass (82,83), information concerning the attachment of motile organisms to the sulcular epithelium is lacking.
- Taxonomy.
Spirochetes belong to the order of the Spirochaetales. This order is subdivided in two families, the Spirochaetaceae and the Leptospiraceae. The Spirochaetaceae comprise of five genera: Spirochaeta, Cristispira, Treponema, Serpulina and
Table 1.2: Recognized pathogenic spirochete species .
Treponema pallidum ssp pallidum venerai syphilis ssp pertenue yaws ssp endemicum endemic syphilis Treponema carateum pinta Treponema paraluiscuniculi syphilis (rabbits) Serpulina hyodysenteriae swine dysentery Borrelia burgdorferi Lyme disease Borrelia hermsii relapsing fever (tick-bome) Borrelia recurrentis relapsing fever (mouse-bome) Leptospira interrogans leptospirosis
Based on references 14, 155 and 161.
16 Borrelia. The family of Leptospiraceae comprises the genera Leptospira and Leptonema. The genera Treponema, Borrelia, Serpulina and Leptospira contain species that are recognized as being pathogenic to humans or animals (Table 1.2).
All oral spirochetes have been assigned to the genus Treponema (14,57,64). Within this genus T. denticola, T. scoliodontum, T. vincentii, T. pectinovorum and
Table 1.3: Oral spirochetes.
species size flagella reference
"Not carbohydrate dependent"
Treponema denticola small 4-10 29,16 Treponema scoliodontum small ? 14 Treponema vincentii intermediate 8-12 14,165 'Treponema orale' small 2 14,159
"Carbohydrate dependent"
Treponema pectinovorum small 2-4 156,166 Treponema socranskii small 2 157 'Treponema macrodentium ' small 2 14,159
"To be characterised"
Large spirochetes 93,113,120 Pathogen related oral spirochetes (PROS) 6,133,135
17 T. socranskii are recognized as species, while "T. orale" and "T. macrodentium" have no standing in the nomenclature up to now (14,95,156,157; Table 1.3).T. denticela, T. scoliodontum and T. vincentii are proteolytic species which do not depend on carbohydrates for growth in culture, whereas T. socranskii depends on glucose and other sugars (157) and T. pectinovorum requires pectin or its constituents galacturonic and glucuronic acid (156).
Based on the ultrastructural studies of Listgarten and Socransky (93), the oral spirochetes have been divided into three groups: small-sized, intermediate-sized and large-sized spirochetes (95,159). Most of the cultivable oral spirochetes have been assigned to the small-sized group (52,155,156,157,183) and only the cultivable T. vincentii is assigned to the intermediate group (Table 1.3). Although large-sized oral spirochetes have been observed in dental plaque (17,113,120), they have never been obtained in culture and are poorly characterised. Apparently novel oral spirochetes have been described by Barron et al. (6) and Riviera et al. (133,134). In their immunochemical assays they observed that infected gingival tissue and plaque from patients with periodontitis harbour spirochetes that did not react with monoclonal antibodies against T. denticela but did so with monoclonal antibodies against T. pallidum. These pathogen related oral spirochetes (PROS) are able to penetrate tissue (135) and share structural surface characteristics (20) and several surface cross-reactive antigens with T. pallidum.
1.3. Potential virulence factors of oral spirochetes.
- Introduction.
Present knowledge of the putative pathogenicity of oral spirochetes is limited to the species that have been isolated and obtained in culture. In vitro studies have shown that these oral spirochetes, including T. denticela, may contribute to the pathogenesis of periodontal disease, by suppressing the proliferation of fibroblasts (10,164), epithelial (132,178), or endothelial cells (165). T. denticola or its products
18 are also cytotoxic to epithelial cells (3,4,132,178), induce actin rearrangement in fibroblasts (5), lyse human red blood cells (47), enhance bone resorption (44), and are capable of modulating host responses of neutrophils (9,60,80,81,143,164) and lymphocytes (144,164).
- Endotoxins, metabolic products and enzymes.
Oral spirochetes produce endotoxins (51,80,107,190) and metabolic products (14,29,30,80,95) such as indole, putrescine, hydrogen sulfide, ammonia, and certain low molecular fatty acids which are potentially toxic to gingival tissues (152). They also produce a wide variety of potential tissue degrading enzymes such as proteolytic enzymes (124), including collagenolytic (101,176,179), fibrinolytic (121), chymotrypsin- and trypsinlike enzymes (48,72,98,100,110,122,176,177), esterases, glycosaminoglycidases (8), iminopeptidascs (101,102,110), phospholipase С (108,109,145) and phosphatases (28,29,30,59).
- Attachment.
Attachment of oral spirochetes to epithelial cells is the main issue of this thesis and therefore will be introduced in greater detail. Bacteria can become attached to different surfaces by electrostatic or hydrophobic forces of low specificity. This aspecific attachment to cells or other surfaces has been called adherence (67). On the other hand, many bacteria possess proteinaceous surface structures, called adhesins, which are responsible for attachment by specific binding to complementary molecules, called receptors (38,67). This type of relatively stable, irreversible bacterial attachment is described by the term adhesion (69). Adhesins are often associated with filamentous appendages known as fimbriae, fibrillae or pili (24,66,126), but also non-appendage related forms of adhesins exist (12,25). Many adhesins are non-immune proteins or glycoproteins with selective carbohydrate-binding properties and are called lectins (130,175). Others, however, apparently bind to non-glycosylated proteins (40).
19 Attachment is considered to be the first step in colonization (39,41,63) and infection (141) by many bacteria, including different Treponema species. However, the mechanisms by which oral spirochetes attach are not yet elucidated. Most research concerning the mechanism of attachment of spirochetes is performed on the non-oral species T. pallidum.
= Attachment of non-oral spirochetes, in particular T. pallidum spp. pallidum.
In vitro attachment has been established for different non-oral spirochetes such as Leptospira spp., B. burgdorferi, S. hyodysenteriae, and T. pallidum spp. pallidum (Nichols strain) (68,70,154,182). T. pallidum spp. pallidum (Nichols strain) is able to attach to a wide variety of cell types under culture conditions, including those derived from testis, kidney, spleen, lung, epidermis, cervix, urethra, and nerve tissue of human, rabbit or rat origin (33). In addition, this spirochete attaches to the extracellular matrix components collagen, fibronectin, laminin, and hyaluronic acid (36). The in vitro attachment of T. pallidum to cultured cells depends on inoculum size, incubation time and incubation temperature (31,54). Fitzgerald et al. (35) stated that attachment is an active process and that viability of the bacteria is essential, since spirochetes killed by heat or formalin do no longer attach. Under in vitro conditions, only 50 to 60% of the spirochetes attach to cultured cells (31,33,35). Spirochetes mainly attach with their tip to cells in culture (54). Most T. pallidum attach only at one end, a few (5 to 10%) attach at both ends. Fitzgerald et al. (33) stated that T. pallidum attaches equally well to actively growing and stationary-phase cells in culture. However, Wong et al. (188,189) showed that larger numbers of spirochetes attached to metabolic active cells than to quiescent or slowly growing cells. The mechanisms by which T. pallidum attaches to cultured cells have not yet been elucidated. Although fimbriae have been described on T. pallidum subsp. pertenue (58), the general consensus is that spirochetes do not express specific attachment structures as fimbriae, fibrillae or pili (Appendix). Fitzgerald et al.
20 (32,34) postulated that T. pallidum attaches by the interaction of the enzyme glycosaminoglycanase on the spirochetal surface and its substrate on the surface of the cultured cells. Besides this enzyme-substrate interaction, the interaction of specific adhesins on the surface of T. pallidum and receptors on the mammalian cells might be involved in attachment (7). Biochemical studies (7,129) have identified three spirochetal proteins as putative adhesins. These proteins are designated PI, P2, and P3 and are located on the outer sheath of T. pallidum. The mammalian cell receptor is possibly the glycoprotein fibronectin, to which all three putative spirochetal adhesins can bind. The binding site of PI, P2, and P3 exists of a four amino acid sequence Arg-Gly-Asp-Ser in the cell-binding domain of fibronectin (170-172).
Most of the attachment characteristics described for T. pallidum apply also to the in vitro attachment of Leptospira to cultured cells (61,173,174,182). However, for Leptospira, fibronectin is not the principal mediator of adherence (61) and besides tip-oriented attachment, leptospires also attach along their whole length (173). Although little information is available, leptospiral attachment is believed to be mediated by protein containing moieties on both the leptospires and the host cells (173).
= Attachment o/T. denticola.
At the time the studies described in this thesis were initiated, investigations regarding interactions between oral spirochetes and cultured cells were primarily focussed on the cytotoxic effects of spirochetes on these cells (4,10,47,123,132,143,144,178). Little attention was paid to the underlying mechanism(s) of attachment. Conflicting data were reported on the ability of oral spirochetes, in particular T. denticola, to attach to cultured cells in vitro. Fitzgerald et al. (33,35) were not able to detect attachment of the different oral Treponema strains T. denticola, T. denticola biotypes ambiguum, TD-2 and microdentium, T. vincentii, and T. scoliodontum to cultured cells of different origin. This was in
21 contrast to the results of Olson (123) who showed attachment of two strains of T. denticola (B2 and Tl) to cultured human skin epithelial cells. Reijntjens et al. (132) showed attachment of the oral spirochetes T. denticola LI 2D and T. vincentii RitzA to guinea pig ear epithelial cells. Moreover, Cimasoni and McBride (18) showed abundant adherence of the T. denticola strains 51B2, CD-I, RitzA and LA-1 to small particles of uncoated hydroxyapatite and to hydroxyapatite coated with saliva, serum or crevicular fluid. These attachment studies (123,132) revealed some similarities between oral spirochetes and T. pallidum. The number of attached oral spirochetes is dependent on inoculum-size and incubation time and it varies per epithelial cell. Usually only the tips of the spirochetes are involved in the attachment process. In addition, accumulations of oral spirochetes on epithelial cells immediately after mitosis were observed (123).
More recently, studies focused on the attachment mechanism of T. denticola to different proteins (23,49,50), human gingival fibroblasts (184,185), or human red blood cells (22,47) were published.
Attachment of T. denticola to basement membrane proteins was examined by Dawson and Ellen (23) and Haapasalo et al. (49,50). Dawson and Ellen (23) studied the attachment of the T. denticola strains ATCC 33520, ATCC 35405, ATCC 35404, b, d, e and e' to human Fibronectin (Fn), Fn-fragments (the intergrin recognition sequence RGDS), non-RGDS peptides, type IV collagen, laminin and bovine serum albumin (BSA), absorbed on plastic cover slips. They observed that T. denticola displayed interstrain differences in the degree of attachment to Fn, laminin and the RGDS peptide, whereas type IV collagen, non-RGDS peptides and BSA did not support binding. Strains of T. denticola which bound in high numbers to Fn bind with a greater percentage by tip-orientation, indicating clustering of Fn-specific adhesins at the tips of the tréponèmes. Haapasalo et al. (50) used ELISA to study the attachment of T. denticola
22 ATCC 33405 to laminiti, Fn, collagen types I and IV, gelatin, fibrinogen and RGD peptide with bovine serum albumin (BSA) as a reference. Compared to BSA, T. denticola displayed a high affinity to laminin, Fn, fibrinogen, gelatin, and type I collagen. In contrast to the results obtained by Dawson and Ellen (23), Haapasalo et al. observed that T. denticola ATCC 35405 attachment to collagen type IV is comparable to the attachment to fibronectin. Tests with laminin fragments obtained through elastase digestion showed that the spirochetes attached well to the 140-kDa eukaryotic cell-binding domain of laminin but not to the 50-kDa heparin-binding domain of laminin. Heating of the spirochetes to at least 70°C or treatment of the spirochetes with the sulfhydryl reagents p-chloromercuribenzoic acid and oxidized glutathion, or mixed glycosidases, markedly reduced T. denticola ATCC 35405 attachment to laminin, gelatin and fibrinogen, but not to BSA. Haapasalo et al. concluded that the attachment of T. denticola ATCC 35405 to proteins is mediated by a sulfhydryl-containing, glycoprotein-like adhesin with no affinity for galactose and mannose. Due to the attachment of T. denticola to proteins that do not contain sulfhydryl, for example collagen type I, attachment of this spirochete to proteins is not a sulfhydryl-disulfide exchange reaction. Because T. denticola attachment to the distinct proteins depended on sulfhydryl groups, electrostatic properties, and carbohydrate composition of the receptor molecules, Haapasalo et al. postulated that T. denticola ATCC 35405 has the capacity to bind to different kinds of proteins by utilizing specific attachment mechanisms.
The in vitro attachment of the T. denticola strains ATCC 35404 (TD-4), GM-1 and MS25 to human gingival fibroblasts (HGFs) and purified bovine Fn was studied by Weinberg and Holt (184). They observed that all T. denticola strains attached to the HGFs, although strain differences were noted. Scanning electron microscopic studies showed that the spirochetes attach primarily to the HGF surface villi and membrane trabeculations. T. denticola attachment was not exclusively tip-associated but appeared to be random along the treponemal surface. Pretreatment of the tested T. denticola strains with trypsin did not inhibit attachment to HGFs, whereas
23 proteinase-K pretreatment did. Exposure of spirochetes to different sugars showed that the attachment to HGFs is mediated by mannose and galactose. Addition of purified Fn resulted in reduction of the attachment to HGFs in case of T. denticola GM-1 and MS25, while it increased the attachment of ЛТСС 35404 (TD-4) to these cells. While strain differences were noted in some of the parameters studied, Weinberg and Holt postulated two possibilities for T. denticola-HGF attachment: a lectin-like adhesin(s) on the T. denticola surface with an affinity for galactose and mannose on the HGF surface, and a serum host factor(s) bridging T. denticola to HGFs.
Several studies (20,49,106,180,184,190) have shown that different strains of T. denticola express different serological active protéines on their surface, however the biological activity of these proteins have only been investigated by Haapasalo et al. (49) and Weinberg and Holt (185). Haapasalo et al. (49) investigated the role of T. denticola surface proteins in the attachment to laminin, Fn, gelatin, fibrinogen and BSA by a Western blot binding assay. They isolated a major 53-kDa protein on the T. denticola strains ATCC 35404, ATCC 35405, and ATCC 33520 to which Fn, laminin, and fibrinogen attached, whereas no attachment to gelatin or BSA was observed. The 53-kDa protein is a major antigen of the cell envelope of T. denticola ATCC 33520 (20,180) and constitutes the major component of oligomers with molecular masses ranging from 130 to 300 kDa (49). The oligomeric forms are highly resistant to proteolysis by trypsin and proteinase-K, whereas the monomeric 53-kDa protein is readily digested. The 53-kDa protein may be an adhesin, as it is able to bind Fn, fibrinogen and laminin, and it is located on the T. denticola cell surface. Weinberg and Holt investigated the role of T. denticola surface proteins in the attachment to HGFs (185). They isolated treponemal major outer sheath proteins (MOSP) of 58-kDa and 64-kDa. A significant observation was that the expression of these proteins is strain dependent. For example, T. denticola strain ATCC 35404 (TD-4) and SR-4 did not contain the 64-kDa MOSP but possessed the 58-kDa
24 MOSP, while MS25 had both the 64- and 58-kDa MOSP. Weinberg and Holt concluded that T. denticola strains expressing the 58-kDa MOSP might bind to host cells through the mediation of fibronectin, while those expressing a 64-kDa MOSP might attach directly to carbohydrate receptors on host cell surfaces.
The ability of T. denticola to agglutinate erythrocytes was studied by Grenier (47) and Cowan et al. (22). Grenier (47) investigated the ability of T. denticola ATCC 35405 and Dil to agglutinate and lyse erythrocytes of human and animal origin. They observed that the hemagglutinating activity of T. denticola is cell-associated, heat-labile, and not strain-specific. In addition, hemagglutination activity varied for different erythrocytes, was expressed during the exponential growth phase, and was reduced in the presence of D-glucosamine. Their data indicated that the bacterial cells bind to erythrocytes via a D-glucosamine-like-containing component. A different approach was used by Cowan et al. (22) who used electrophoretic mobility measurements to investigate the surface of T. denticola ATCC 33520 and its participation in hemagglutination of erythrocytes. Their data indicated that a pure culture of spirochetes displays surface heterogeneity with respect to charge, and that a less and a more negatively charged subpopulation exist, and that absorption of T. denticola cultures with an excess of human O-type erythrocytes reduced the more negatively charged subpopulation, but not the less negatively charged subpopulation. Because the unadsorbed fraction showed no hemagglutination activity, Cowan et al conluded that the more negatively charged subpopulation, which is in the minority in all spirochete cultures studied, contains the spirochetes capable of adhesion to erythrocytes. They postulated that the agglutination is mediated by electrostatic interactions between oppositely charged domains in the interacting surfaces.
However, none of the above mentioned attachment studies is focussed on the attachment to cells of epithelial origin, although epithelial tissue is most likely the primary target for spirochetal attachment in the oral cavity.
25 - Invasion.
Penetration of epithelial tissues by oral spirochetes, occasionally extending into the underlying connective tissue and alveolar bone, is reported in humans suffering from necrotizing ulcerative gingivitis (21,55,82,88,92,142,167), advanced periodontitis (37,105,137,140), and localized juvenile periodontitis (15,138,139). Invaded oral spirochetes are mostly identified as being of the intermediate type (82,111) and occasionally of the small (167) or large type (82). Invaded oral spirochetes are primarily located in the intercellular spaces between epithelial cells (21,37,103,111,138). Invaded oral spirochetes are frequently found ahead of other types of invading oral bacteria (82,167). An initiating role of oral spirochetes in the onset of periodontal disease has been indicated by Maltha et al. (103) and Mikx et al. (111,112) who observed that oral spirochetes were able to penetrate normal sulcular epithelium in beagle dogs with experimentally induced necrotizing ulcerative gingivitis. Besides in animal experiments (51,103,111) this is also observed in human studies (82,167). However, the mechanisms involved in epithelial penetration of oral spirochetes are not yet understood and results of studies on the invasive properties of T. denticola (112,135) are still inconclusive.
26 1.4. Aim and outline of the investigation.
T. denticola is a small spirochete which regurlarly has been sampled from pockets associated with periodontal disease. It is the only cultivated oral spirochete of which a positive correlation has been established with periodontitis in humans and which has the potential to mediate tissue destruction by direct action against host cells and matrix proteins. For oral spirochetes, including T. denticola, to be involved in periodontal disease, these microorganisms must be able to colonize, survive and grow in the vicinity of periodontal tissues. In vivo, oral spirochetes are dominantly located in the subgingival microflora of the gingival crevice in intimate contact with the sulcular or pocket epithelium. Attachment to these oral tissues might enable these organisms to maintain in this area. Knowledge about the mechanism of attachment of oral spirochetes might, in the long run, result in methods to prevent or limit disease progression. At present it is not known how oral spirochetes attach to oral epithelial surfaces. The attachment mechanism of oral spirochetes has mainly been studied using extracellular matrix proteins, human gingival fibroblasts and erythrocytes. Studies regarding the interaction of oral spirochetes with epithelial cells have primarily been focussed on the damaging effects of oral spirochetes on these cells and information concerning the mechanism of attachment of oral spirochetes to epithelial cells is still lacking.
The aim of the present study is the development of an in vitro assay for the attachment oí Treponema denticola to cultured cells of epithelial origin and to study the spirochete-associated factors involved in this attachment process.
The development of the in vitro attachment assay was initiated by establishing a microscopic scoring system and screening the attachment often different T. denticola strains to monolayers of four types of keratinocytes (Chapter 2).
27 Fine-tuning of the attachment assay was further pursued by focussing on the attachment of T. denticola ATCC 33520 to monolayers of a rat palatal epithelial cell line {Chapter 3). Subsequently the involvement of spirochetal surface-located protein and carbohydrate moieties in the attachment of T. denticola ATCC 33520 to cultured rat palatal epithelial cells was studied (Chapter 4). Parallel with the attachment studies the haemagglutination activity of T. denticola was investigated as a model for further investigation into its attachment properties (Chapter 5).
In a pilot study the putative invasive activity of different oral- and non-oral spirochetes was investigated. For this purpose the development of an in vitro invasion model with a reconstructed epidermis was initiated (Chapter 6).
Because the attachment of T. denticola can be envisaged as an interaction beween its outermost surface and the substrate to which attachment takes place, an electron microscopic study into the surface of T. denticola ATCC 33520 is included (Appendix).
For references see chapter 8.3.
28 Chapter 2
Attachment of T. denticola strains to monolayers of epithelial cells of different origin.
Keulers, R.A.C., Maltha, J.C., Mïkx, F.H.M., and Wolters-Lutgerhorst, J.M.L.
Oral Microbiol Immunol 1993: 8: 84-88. (Reprint) Oral Uicrohml Immunol 1993 Я 84 -Ufi Copyright Г 4unksguard 1993 Prmitâ in Dinmark All rights re\er\ed Ога/МпоЬюкуу апсікпіпипоіоду /ss\ tmt-mss
R. A. С Keuler·1, J. С. Maltha1, F. H. M. МІкя2, Attachment of Treponema 1 J. M. L. Wóltara-Lutgarhortt 'Laboratory of Oral Histology and 'Department of Penodontology and Preventive denticola strains to monolayers of Dentistry TRIKON Research Program Microbiology of Canes and Periodontal Disease University of Nijmegen the epithelial cells of different origin Netherlands
Кеиіегч R4C Maltha J С Mik χ FH M Wolter ч-Lutgerhorst JML Attachment of Treponema denticola strains to monola\ers of epithelial cells of différent origin Oral Uurohio/ Immunol 1993 8 84 88 t Munksgaard, 1993
The attachment of 10 different Treponema denticola strains to monolayers of 4 types of epithelial cells derived from rat palatal epithelium, guinea pig ear, human buccal epithelium and human corneal epithelium was screened microscopi cally Most Τ dentuola strains were able to attach to all four types of epithelial cells The Τ dentuola strains seemed lo attach better to epithelial cells derned from primary cultured material The Τ dentuola strains showed different degrees Key words attachment Treponema denticola of attachment Scanning electron microscopy studies» revealed that the attachment epithelial cell in vitro of Τ denticola was not only tip-associated but occurred also at random points J С Maltha University of Nijmegen Labora in close contact with microvilli of the epithelial cells Attached spirochetes were tory of Oral Histology PO Box 9101 ML 6500 non-uniformly distributed over the monolayers, indicating the presence of receptive HB Ni|megen Ihe Netherlands subpopulaiions of epithelial cells in the monolayers Accepted for publication June 16 1992
Oral spirochetes belong to the genus (19, 20), hydroxyapatite (5) and the gly All strains were stored in skim milk at Treponema and are closely associated coproteins fibronectin and laminin (6) -80 С and subsequently subcultured with periodontal disease The percen In order to gain more general appli in our laboratory They were cultured tage of spirochetes in the subgingival cable information about the attachment anaerobically in modified GM-1 me microflora is increased at periodontal^ of Τ denticola to epithelial cells the de dium (2 20) The Treponema cultures diseased sites (12 15 18) and increases gree of attachment of 10 different Γ were transferred in fresh medium 5 days with periodontal disease severity ( 1 22) denticola strains to monolayers of 4 before the attachment experiments The presence of spirochetes in perio types of epithelial cells was screened dontal tissues has been reported in cases of acute necrotizing ulcerative gingivitis Epithelial cell· Material and methods (11) in localized juvenile periodontitis The monolayers of human cornea epi Oral spirochete· O) and in advanced periodontitis (10, thelial cells (HCE) (23) were a gift from 21) In animal experiments the presence The Τ denticola strains Bil, B12, Dr J Ρ M van der Putten (Department of spirochetes in intact sulcular epithel Ny535, Ny541 Ny572 and Ny573 were of Medical Microbiology, University of ium near subgingival lesions has been isolated from periodontal patients at the Amsterdam, the Netherlands) The demonstrated (13) Spirochetes might Department of Penodontology and Pre monolayers of human buccal epithelial pla> a role in the initiation of the patho ventive Dentistry, University of Nijmeg cells (HBL·) (24) were a gift from Dr R genesis of periodontal disease by pene en the Netherlands (14) The strains M A Hoet (Department of Biochemis tration of intercellular spaces of the sul В11 and В12 originated from one isolate try, University of Nijmegen) The rat cular epithelium (13 16 17) that showed rough ( В11 ) and smooth (Sprague-Dawley strain) palatal cells The mechanisms of tissue invasion by (BI2) colonies of the same size and color (RPE) were a gift of Dr A Arenholt oral bacteria are not yet understood A on a blood agar plate Τ denticola LUD (University of Ârhus, Denmark) The first step m this process probably is the originated from the Department of guinea pig ear cells (lot 23952) (GPE) attachment of bacteria to the oral mu Pathology, School of Dental Medicine, were obtained from Flow Laboratories cosa followed by disruption of the inter University of PennsyKania, Philadel (Irvine, UK) The RPE and GPE cells cellular junctions and penetration into phia Strains ATCC 35404. ATCC 35405 were cultured aerobicall) (9V/U air and the epithelial tissue In utro attachment and ATCC 33520 were purchased from 5% CO-.) in Eagle minimal essential me of Treponema dentuola has been de the American Type Culture Collection dium (MEM) (Gibco Renfrewshire, scribed to fibroblasts (25) epithelial cells and represent 3 different serotypes (4) UK) with Earlc's salts and 25 mM He-
30 Attachment of Treponema to epithelial celts 85 pes buder. supplemented with 6% fetal denticola strains was estimated in at ladium and examined in a Philips SEM calf serum The cells were grown on least 15 randomly chosen microscopic 500 (Philips, Eindhoven, the Nether Thermanox covershps (Flow Labora fields (Carl Zeiss Universal II, magnifi lands) tories) placed in petn dishes (diameter cation χ 1250) For every combination 6 cm), (Gibco, Breda, the Netherlands) of Treponema strain and type of epi Resulta After 4 days the cells had grown to con thelial cell, one sample was studied The fluent monolayers Before the attach results are presented in an arbitrary The results of the estimations for the ment assay, the growth medium was scale ol high, medium, low and no different Τ denticola strains are pre drained and the monolayers were attachment The score high was given if sented in Table 1 Almost all the Τ den washed twice in phosphate-buffered sa each examined microscopic field ticola strains were able to attach to the line (PBS. pH 7 2) and the covershps showed one or more attached spiro 4 types οΓ epithelial cells The only ex were placed in multidish 24-wells plates chetes If not every examined micro ception was Τ denticola strain ATCC (Nunc, Roskilde, Denmark) scopic field showed one or more at 35404, which did not attach to GPE or tached spirochetes, but most (more than HCF cells Strain ATCC 35405, and to 50%) of the examined fields did the a lesser extent strain В12, showed good Attachment assay score medium was given If one or more attachment to the 4 types of epithelial The attachment of the Τ denticola attached spirochetes was observed in cells The strains Ny541, Ny572 Ny573 strains to the different epithelial cells only a minority (less than 50%) of the and Bll displayed poor attachment was tested in separate experiments, but examined microscopic fields the score The 3 different serotspes ATCC 15405 performed according to the same proto low was given The score none was given ATCC 33520, and ATCC 35404 varied col Five-day-old Τ denticola cultures if no attached spirochetes were found in their degree of attachment Τ dentico were washed twice by centnfugation In addition to these estimations, at la strain ATCC 35405 showed a high (I800xg, 10 mm), vortexed and «sus tached spirochetes were actually degree of attachment to all 4 types of pended in PBS (pH 7 2) Finally the counted for the RPE and HBE cells epithelial cells However ATCC 33520 pellet was resuspended in MEM The At least 5 randomly chosen microscopic scored /im to RPE and GPE cells and number of spirochetes per milliliter was fields (magnification χ 1250) were strain ATCC 35404 scored none to GPE counted in a bacterial counting cham counted For each microscopic field, the and HCE cells The strains Bl2 and Bl I ber with a grid of 0 02 mm χ mean number of attached spirochetes scored different degrees of attachment 1/400 mm- (Hawksley, Lancing. UK) per RPF or HBb cell was calculated If For example for the GPE and HBE using phase contrast microscopy One the number of attached spirochetes per cells, strain B12 scored high while strain milliliter of the suspension was added microscopic field was 100 or more, a Bll scored Ion (Table 1) to the wells containing the previously value of 100 was scored The degree of The epithelial cells were derived from washed (PBS, pH 7 2) monolayers of attachment is presented as the median continuous cell lines (RPF and GPE) or epithelial cells The inocula varied be and range of these calculated values from primary cultured biopsy material tween 101 10' spirochetes per milliliter The calculated values were also used (HCE and HBt) The results (Table 1) Incubation was performed under aerob to rank the Τ denticola strains by the indicate that at least 5 Τ denticola ic conditions at 37 С for 6 h Thereafter Wilcoxon scores strains expressed low to no attachment the monolayers were firmly washed 3 Specimens of RPE cells incubated to epithelial cells from continuous cell limes in PBS fixed in 3% glutaraldc- with the difieren! Τ denticela strains lines, whereas to epithelial cells from hyde in Sorensen's buffer (pH 7 4) for were also examined by scanning elec primary cultured biopsy malcnal, only 45 mm at 4 С and stained with a modi tron microscopy (SEM) Fixed mono 2 strains showed low or no attachment fied Warthin Silver staining (13, 20) layers were dehydrated in a series of to HBE and only 3 strains attached For each type of epithelial cell, the ethanol, critical point-dried with carbon poorly to HCE degree of attachment of the different Τ dioxide, sputter-coated with gold pal In addtion to the estimation, the
Table Ì The1 degree ol' attachment of 10 Τ dcnttiola strains to cultured epithelial cells of different origin estimated in randomly chosen microscopic fields Degree of attachment High Medium Low None strain RPh GPh HBF HCE RPE GPL HBF HCL RPE GPE HBF HCF RPF GPb HBF НСГ ATCC 35405 B12 ATCC 33520 Ny535 nl LUD + ATCC 35404 Ny541 N>572 N>573 Bll RPE rat palatal epithelium GPE guinea pig ear epithelium HBE human buccal epithelial cells HCF human cornea epithelial celb nl not tested
31 86 Keulers el al
Table 2. Median and range of the mean number oi attached Τ tient η ola spirochetes per RPF ination. The results showed that most or HBF. cell, calculated per microscopic field and the Wilcoxon (rank sums) stores of these T. denticola strains were able to attach calculated values to all 4 types of epithelial cells The Τ denticola median and (range) median and (range) Wilcoxon only exception was strain ATCC 35404. strain per RPE cell per H BE cell scores* which did not attach to GPE and HCE LUD 1 1 (09 19) 0.4(0.0 2.1) 137 cells ATCC 35405 1.0(0.2 2.0) 04 (0.0 2.5) 135 The attached spirochetes were non Ny535 0.2 (0.0 0.5) 1.4 (0.1 2.7) 1 14 uniform^ distributed over the mono BÍ: 0.0 (0.0 0.2) 0.7 (0.0-2.6) 92 layers. This was not caused by clumping ATCC 33520 I) 1 11)1) 0 Ί ox (00 2111 92 or uneven distribution of the spirochetes ATCC 35404 0.1 (0.0-0.2) 0.3(0.0-2.1) 90 in the inoculum, as was shown by Ny572 1) 1 (Oil 1 4) 0.0(0.0 1.5) 86 microscopical examination prior to in NyS73 0.0 (0.0 0.8) 0 3(0.1 2.3) 85 cubation. The number of spirochetes Ny54l 0.2(0.1 1 1) 0.1 (0.0 0.1) 78 added to the monolayers exceeded the Bll 0.0(0.0 1 1) 0.0 (0.0-0.4) 50 number of epithelial cells at least by a RPF: rat palatal epithelium: HBE: human buccal epithelial cells Kruskal-Wallis Test: factor of 10. making it unlikely that the (RPF and HBE cells) «38.03, df-9, />< 0.001. non-uniform distribution of the spiro chetes is caused by local differences in attachment of the T. denticola strains to treponemal surface and was only oc the density of the inoculum. Haphazard Ihe RPE and HBE cells was quantified casionally tip-associated (Fig. 1). Many trapping of the spirochetes was elimin by counting the epithelial cells and at of the tréponèmes gave the impression ated by firmly washing the monolayers tached spirochetes per microscopic of being attached along its body to the at the end of the incubation period. field. When confluent, the HBE cells microvilli of the RPE cells (Fig. 2). Most likely, the non-uniform distri* were larger than the RPE cells. The Penetration of spirochetes into the RPF bution of the attached spirochetes is due mean number of cells per microscopic surface was not observed. to a epithelial cell-related factor and in field was 105 ±36 for RPE and 47 + 15 dicates a heterogeneity and probably a for HBE. This resulted in an estimated receptive subpopulation of epithelial number of epithelial cells per well of Discussion cells in the monolayers. Heterogeneity in monolayers of epithelial cells and approximately 10'' for RPE and ΗΓ for Attachment of oral tréponèmes has concomitant variation in the number of HBE. The treponemal inoculum size been described for cultured epithelial V attached spirochetes has been noted by varied between 10"-I0 . The difference cells (19. 20) and fibroblasts (25). In others (9. 19. 20) but might have been in inoculum size was not correlated with the present study the degree of attach overlooked in attachment studies using the degree of attachment, and although ment of 10 different Τ denticola strains non-visual methods as conventional the number of spirochetes in the incuba to monolayers of 4 types of epithelial radiolabeling techniques (25). Olson tion well outnumber the number of epi cells derived from rat palatal epithel (19) observed an enhanced attachment thelial cells by at least a factor 10, sub ium, guinea pig ear. human buccal epi of 7.' denticola to epithelial cells with a stantial differences were detected in the thelium or human corneal epithelium more rounded appearance. This could number of attached spirochetes per was screened by light microscopic exam microscopic field. In one sample, the number of attached spirochetes per microscopic field could vary from none to more than 100. although the epi thelial cells were evenly distributed and displayed the same morphology. The medians of the mean numbers of at tached spirochetes per RPE or HBE cell per microscopic field were low with rela tive large ranges (Table 2). The latter are due to the non-uniform distribution of attached spirochetes over the mono layers. Ranking of the Wilcoxon scores per Τ denticola strain revealed a signifi cant heterogeneity with the Kruskal- Wallis test (P< 0.001) (Table 2). The Τ denticola strains LMD. ATCC 35405 and Ny535 showed the highest Wil coxon rank scores. The lowest Wilcoxon rank score was found for the strain Bll. SEM studies of the T. denticola strains incubated with RPE cells con firmed the inter-strain differences as presented in Table I. In genera!, the attachment appeared random on the Fig. I Scanning electron micrograph of Τ denticola ATCC 35405 attached to RPE cells Bar: 40 ftm.
32 Attachment of Treponema to epithelial cells 87
population of cells that is more recep tive for the attachment of Τ denticola.
Acknowledgement The authors would like to thank Gijs de Jongh (Department of Dermatology, University of Nijmegen) for his skillful advice on culture technique.
References
1. Armitage GC. Dickinson WR. Jender- seck RS. Levine SM. Chambers DW. Re lationship between the percentage of sub gingival spirochetes and the severity of periodontal disease. J Periodontol 1982: 53: 550-556. 2. Blakemore RP. Canale-Parola E. Argi nine catabolismi by Treponema denticola Fig 2. Scanning electron micrograph of Τ denticola ЛТСС 35405 attached along its body to J Bacteriol 1976: 128: 616 622. the microvilli of RPE cells (arrows). Bar: 0.5 μτη. 3. Carranza Jr FA, Saghe R. Newman MG. Valetin PL Scanning and transmission electron microscopic study of tissue-in not be confirmed in the present study, cola strains seemed to attach better to vading microorganisms in localized juv probably due to the applied experimen the epithelial cells derived from primary enile periodontitis. J Periodontol 1983: tal techniques. cultured biopsy material. The most 54: 598 617. The different degrees of attachment likely explanation is a difference in the 4. Cheng SL. Siboo R. Chin Quee T. John of the Treponema strains indicate strain- epithelial cell surface, expressing recep son JL. Mayberry WR. Chan ECS. specific factors. For example, the Τ den- tors in different number or nature. For Comparative study of six random oral ticola strains ATCC 35404, ATCC 35405 example, the amount of fibronectin spirochete isolates. Serological heteroge neity of Treponema denticola J Periodont and ATCC 33520 are serologically dis might differ between the primary cul Res 1985: 20: 602 612. tinct (4) and showed differing degrees tured cells and the cultures of the con 5 Cimasoni G. McBride ВС. Adherence of of attachment to RPE. HCE and GPE tinuous cell lines. This could be import Treponema denticola to modified hy- cells. If the attachment of T. denticola ant, as the degree of attachment has droxyapalite. J Dent Res 1987: 66: is mediated by surface-located adhesins been related to the presence of fibro- 1727-1729. that interact with receptors on the epi neclin-specific adhesins on Τ denticola 6. Dawson JR. Ellen RP. Tip-oriented ad thelial cells, than different serotypes (6). herence of Treponema denticola to fib might have different adhesins with im SEM studies with RPE cells showed ronectin. Infect Immun 1990: 58: 3924 3928. munological activity. The immunolog that most Τ denticola spirochetes pri 7. Fitzgerald TJ. Cleveland P. Johnson RC. ical activity of surface located proteins marily attached to the microvilli of the Miller JN. Sykes JA. Scanning electron as mediators of adherence is assumed epithelial cells at random points on the microscopy of Treponema pallidum by Weinberg & Holt (25). treponemal surface, as described by (Nichols strains) attached to cultured The quantification of the degree of Weinberg & Holt for the attachment of mammalian cells. J Bactenol 1977: 130: attachment by actual counting generally Τ denticola to fibroblasts (25). No pref 1333-1344. confirmed the previously estimated dif erence was found for tip-associated 8. Fitzgerald TJ. Johnson RC. Miller JN. ferences between the T. denticola strains. attachment, as is noted for T. denticola Sykes JA Characterization of the attach The mean number of attached spiro (6, 19) and Treponema pallidum (7). Tip- ment of Treponema pallidum (Nichols chetes per cell per microscopic field was associated attachment could be related strain) to cultured mammalian cells and the potential relationship of attachment rather low. with relative wide ranges. to different species, strains, substrates to pathogenicity. Infect Immun 1977: 18: Weinberg & Holt (25) found higher or experimental conditions. 467^*78 numbers of attached Τ denticola strains In conclusion, the attachment of Τ 9. Fitzgerald TJ. Miller JN. Sykes JA. Tre to human gingival fibroblasts. On the denticola is not only tip-associated but ponema pallidum (Nichols strain) in other hand. Fitzgerald et al. (8, 9) did occurs also at random points on the tissue cultures: cellular attachment, entry not find any attachment of Τ denticola treponemal surface in close contact with and survival. Infect Immun 1975: II: biotypes to 19 different cultured cell microvilli of the epithelial cells. The de 1133 1140. types. This supports our findings that gree of attachment of Τ denticola to 10. Frank RM Bacterial penetration in the attachment is related to strain and cell epithelial cells is related to strain specific apical pocket wall of advanced human periodontitis. J Periodont Res 1980: 15: type. differences. Most Τ denticola strains 563 573. tested attach better to primary cultured The epithelial cells used in the present II Listgarten MA. Electron microscopic study were derived from continuous cell epithelial cells than to continuous cell observations on the bacterial flora of lines or from primary cultured biopsy lines. The non-uniform distribution of acute necrotizing ulcerative gingivitis. J material. In general, the tested T. denti attached spirochetes indicates a sub- Periodontol 1965: 36: 68-79.
33 88 Keu/er s er at
12 Listgarten МЛ Hellden L Relative dis 17 Mikx FHM, Ngassapa DM). Reijntjens 22 Simonson LG, Goodman CH, Bial JJ, tribution of bacterid at clinically healthy ί-MJ, Maltha JC Effect of splint place Morton HE Quantitative relationship of and periodontal^ diseased sites m ment on black-pigmented Batieroides Treponema denticola lo seventy of perio humans J Clin Periodont I97S 5 and spirochetes in the dental plaque of dontal disease Infect Immun 1988 56 115 132 beagle dogs J Dent Res 1984 63 726-728 Π Maltha JC Mikx ЩМ. Kuypers FJ 1284-1288 23 Tjai К h van Putten JPM. Pels E. Zanen Necrotizing ulcerative gingis ins in beagle 18 Moore WEC. Holdeman LV, Smibert HC The interaction between Neisseria dogb III Distribution ol spi roche tei in RM. Hash Db, Burmeisler JA. Ranney gonorrhoeae and the human cornea in interdental gingival tissue J Periodont RR Bacteriology of severe periodontitis organ culture An electron microscopic Res 1985 20 522 531 in young adult humans Infect Immun study Graefe's Arch Clin Exp Ophthal 14 Mikx UHM Comparison of peptidase 1982 J8 1137-1148 mol 1988 226 341 345 glycoside se and esterase activities of oral 19 Olson Ï Attachment of Treponema den- 24 Violette SM, King IMS. Sarlorelh AC and non-oral Tre ропота species J Gen titola to cultured human epithelial cells Antagonistic effects ol retinole acid and Microbiol 1991 137 63-68 Scand J Dent Res 1984 92 <5-63 hydrocortisone on terminal differen 15 Mikx FHM van Campen GJ Micro 20 Rcimljens FMJ, Mikx FHM, Wolters- tiation of human squamous carcinoma scopical evaluation of the microflora in LulgerhorM JML, Maltha JC Adher cells J Invest Dermatol 1989 93 relation to necrotizing ulcerative gingi ence of oral tréponèmes and their effect 165 168 vitis in the beagle dog J Periodont Res on morphological damage and detach 25 Weinberg A, Holt SC Interaction of Tre 1982 17 576 584 ment of epithelial cells in utro Infect ponema denticola TD-4. GM-1 and 16 Mikx hHM Maltha JC. %an Campen Immun 1986 $1 642 647 MS25 with human gingival fibroblasts GJ Spirochetes in early lesions ofnccro- 21 Saghe R, Newman MG Carranza Jr FA, Infect Immun 1990 58 1720 1729 ti7ing ulcerative gingivitis experimentally Patlison GL Bacterial invasion of gin induced in beagles Oral Microbiol Im giva in advanced periodontitis in humans munol 1990 5 86 89 JPenodontol 1982 S3 217 222
34 Chapter 3
Attachment of T. denticela strains ATCC 33520, ATCC 35405, Bll and Ny541 to a morphologically distinct population of rat palatal epithelial cells.
Keulers, R.A.C., Maltha, J.C., Mikx, F.H.M., and Wolters-Lutgerhorst, J.M.L.
J Periodont Res 1993: 28: 274-280. (Reprint) J Pin demi Res 1991 * "< 'M) Ctip\right ( MunksgaanJ 199} /Vi' ft/ π Dtninurk 411 rghi re\ei Ы JOl RNAL OF PFRIODONIAL RESEARCH /SSV m"J4S4
R А С Keulers', J С Maltha1, Attachment of T. denticola strains F H M Mlkx2, J M L Wollera-Liilgerhorst' Laboratory ol Oral Hstology department of Penodontology and Preventive Dentistry ATCC 33520, ATCC 35405, B11 and TRIKON Research Program Microbiology of Car es and Periodontal Disease Ny541 to a morphologically University of Nijmegen The Netherlands distinct population of rat palatal epithelial cells
Keulers RAC Maltha JC Mikx FHM Wollen Lulgerhorst JML Attachment ο/τ denticola strains ATCC 33^20 ATCC 3U0S Bll and Nyï4l to a morpho logicali} distim t population of rat palatal ¿pithclial celli J Periodont Res 1993 28 274-280 С Munksgaard 1993
In the present study an assay for the attachment of Τ denticela to epithelial cells is described An indirect tmmunohistochemical staining method using two native polyclonal antisera revealed dark brown coloured spirochetes attached to rat palatal epithelial cell (RPE) monolayers In addition two morphologically distinct populations of RPE cells could be distinguished in the monolayers when using phase contrast microscopy One minor population consisted of isolated rounded RPE cells that were lying on top of a confluent monolayer of flattened RPF cells The rounded RPE cells were more receptive for the attach ment of Τ denticola than the flattened cells The rounded RPE cells were evenly distributed over the monolayer but the attachment of spirochetes to the rounded cells was greater at tht edge than in the centre of the monolayers The percentage of rounded RPt cells with attached spirochetes depended on the incubation time (optimum 6 h) temperature (optimum 37 C) and pH (optimum 7 0) It is speculated that the attachment of Τ denticola is a physical chemical process of yet unknown nature and that differences in the number of microvilli and or the amount of available receptors between the two morphologically Key words attachment - Treponema denti distinct cell types accounts for the differences in the numbers of attached cola - epithelial cells in vitro spirochetes Accepted for publication 23 November 1ΘΘ2
Treponema denticola and other subgingival located contact with the gingival tissue and initiate peri oral spirochetes are closely associated with various odontal infections (19-21) In \ilro attachment of forms of periodontal disease (1 3) Different Τ denticola to hydroxyapatite, fibronectin, lamin- studies have established a positive correlation be ìn, type I and IV collagens, fibrinogen, gelatin, tween the percentage of oral spirochetes in the fibroblasts and epithelial cells has been described subgingival plaque and the severity of periodontal (19-25) Studies on the attachment to cultured epi disease (4- 6) In addition, invasion of the gingival thelial cells (20, 21) revealed a non-uniform distri tissue by oral spirochetes has been reported (7-13) bution and a low mean number of attached trépon In vitro, Τ denticola produces metabolic products èmes per cell The presence of a receptive subpopu- (14) and enzymes (15 18) which can act as poten iation of epithelial cells in monolayers has been tial virulence factors with destructive properties for indicated (24) surrounding tissue The present study describes the development of Attachment to gingival epithelial cells could be an assay for the attachment of Τ denticela to one of the mechanisms by which oral spirochetes monolayers of rat palatal epithelium (RPE) cells, maintain themselves in the gingival crevice in close by studying the attachment of Τ denticela ATCC
36 Treponema attachment to epithelial cells 275
33520 to d morphologically distinct population of mM Na,HP04, 1 8 mM KH,PO„, 1 mM CaCl, RPE cells and application of the assay to three and 1 mM MgCl,) (PBS, pH 7 2) and placed in different Τ ¿tenutola strams multidish 24-well plates (Nunc, Roskilde, Den mark) To estimate the mean number of RPE cells pres Material and methods ent in confluent monolayers, the cells of three con Τ denticola strains fluent coverslips were detached by 0 25% trypsin Τ denticela ATCC 33520 and ATCC 35405 were pH 7 5 (250 USP units/mg, Difco laboratories, purchased from the American Type Culture Collec Detroit, USA) for 10 mm at 37 С and subsequently tion The Τ delineóla strains Bl 1 and Ny541 were counted in a Burker counting chamber (Schreck, isolated from periodontal patients (18) in our own Germany) Confluent monolayers consisted of ap laboratories Strain ATCC 33520 was grown in proximately 10' RPF cells per covershp serum-free Proleose-Tryplicase-yeasl (PTY) me dium containing 10 g of Proteose Peptone no 2 (Difco Detroit, Mi ), 5 g of Trypticase peptone Attachment assay (BBL, Cockeysville, Md ) 2 5 g of KCl and 0 5 g Samples of freshly harvested tréponèmes were of L-cysteine HCl (Merck, Darmstadt, Germany) washed twice by centrifugaron (1800 g 10 min) per litre The medium was titrated to pH 7 0 with and resuspension in PBS supplemented with 25 1 N KOH and heat sterilized After cooling, a mM MgCl, (MBS, pH 7 2) The pellet was finally filter-sterilized mixture of 5 ml of 10% (w/v) resuspended in Dulbecco's Minimal Essential Me NaHCO,, 0 025 g of thiamine PP, (Sigma, St Louis dium (DMEM) supplemented with Ham's FI2 me USA) and 5 ml of volatile fatty acid solution was dium (DMEM/F12 3 1 v/v) (riow laboratories, added The volatile fatty acid solution contained Irvine, UK) Incubation was performed in air con 100 ml οΓ 0 1 N КОН and 0 5 ml of isobutyric taining 7 5% CO, and a relative humidity of 95% acid 0 5 ml of DL-2-methylbutyric acid, 0 5 ml of The number of spirochetes per ml was counted in isovaleric acid and 0 5 ml of valeric acid (all from a bacterial counting chamber (Hawksley, Lancing, Merck) Most experiments were performed with Τ UK) by phase contrast microscopy One ml of the denticela ATCC 33520 grown in serum free PTY suspension, containing approximately 10' spiro medium in continuous culture in a chemostat at chetes, was added to each well containing a washed pH 7 0, at an redox potential of — 532 mV, at 37 C, RPE monolayer, resulting in an estimated spiro a dilution rate of 0 04 h ' and an atmosphere of chete cell ratio of 1000 1 After incubation for 91% N„ 5% H, and 4% CO,, resulting in a density 6 h at 37 C, the monolayers were firmly washed of 5 χ 10s spirochetes per ml and an optical density three times in PBS and finally fixed in 3% glular- of 0 650 at 550 nm The strains Bl 1, Ny541, and aldehyde in Sorensen's buffer (pH 7 4) for 45 mm ATCC 35405 were anaerobically batch grown for at 4 С four days in modified GM-1 medium as previously For staining of the different Τ delincala strains described (21) and tested in the attachment assay we used two native polyclonal antisera These anti- in the same spirochete cell ratio as estimated for sera were a gift of Sjoerd Rijpkcma (National Insti ATCC 33520 (see below) tute of Public Health and Environmental Protec tion Bilthovcn The Netherlands) and had been elicited in New Zealand White rabbits which were Epithelial celle challenged with sonicated extracts of whole spiro The Sprague Dawley rat palatal epithelial cells chetes of Τ denticola strain ATCC 33520 or Τ (RPE) were a gift of Dr A Arenholt (University ι intent и strain LA-1 The staining of the Г dentice of Ârhus, Denmark) The RPL cells were cultured la strains, attached to RPE cells was performed aerobically (95% air and 5% CO,) in Fagle Minimal with two different native polyclonal antisera be Essential Medium (MEM) (Gibco, Breda, The cause pilot studies had revealed that optimal stain Netherlands) with Earlc s salts and 25 mM Hepes ing for all the Τ denticola strains was obtained if buffer supplemented with 6% fetal calf serum The both antisera were used in combination Fixed cells were grown on 13-mm circular Thermanox" RPE monolayers with attached spirochetes were (Nunc, Naperville, USA) or glass coverslips placed first incubated with the antiserum directed against in Petri dishes (diameter 6 cm), (Nunc, Roskilde, Τ denticola ATCC 33520 at a dilution of 1 1000 in Denmark) PBS supplemented with 1% bovine serum albumin Before the attachment assay, the growth medium (BSA) for 1 h at room temperature After three was drained and the confluent monolayers were washing steps in PBS for 10 min each, the mono washed twice in TC-Dulbecco s (27) phosphate layers were incubated in the antiserum directed buffered saline (140 mM NaCI, 2 7 mM KCl, 8 against Τ \mcentu LA-1 diluted 1 500 in PBS
37 276 Keulers et al supplemented with 1% BSA for 1 h followed by Attachment to rounded RPE cells three washing steps in PBS The monolayers were then incubated in peroxidase-conjugated swine In three separate experiments the attachment of Τ immunoglobulins to rabbit immunoglobulins dentinola ATCC 33520 to at least 50 rounded RPE (SWARPO) (Dakopatts, Copenhagen. Denmark) cells in the centre and at the edge of the RPE diluted 1 100 in PBS supplemented with 1% BSA monolayer was scored for 1 h After washing, the immunoreaction was The attachment of 7 denticela ATCC 35405, Bl 1 visualized with diammo-benzidine (DAB 0 5 mg' and Ny541 was scored to at least fifty rounded ml) dissolved in PBS with 0 03% (ν/ν) Η,Ο, RPE cells located only at the edge The attachment The attachment of the Τ denticela strains to the of Τ denticela strain ATCC 35405 was scored in RPE cells was scored in randomly chosen micro three experiments and the attachment of the strams scopic fields (magn 1250 χ ) in the area approxi Bil and Ny541 was scored in five experiments mately 0-2 mm from the edge of the RPE mono layer (edge), unless otherwise indicated In each microscopic field the number of rounded RPE cells Incubation time temperature and PH with zero (score 0), one to five (score 1-5) or more than five (score >5) attached spirochetes was The effects of the incubation time, incubation tem counted Scoring was performed in as many micro perature and pH on the attachment of Τ dentuola scopic fields as needed to count a minimum of 50 ATCC 33520 to the rounded RPE cells were investi rounded RPE cells per monolayer Three mono gated in different experiments layers were examined per experiment The attach The effect of the incubation time was investi ment is presented as the mean percentages (and gated in 4 experiments In each experiment strain standard errors of the mean) of RPE cells with 0, ATCC 33520 was incubated with RPE monolayers 1-5 or >5 attached spirochetes for periods of 2 h or 6 h at 37 С and at pH 7 The effect of the incubation temperature was determined in 8 experiments In each experiment Statistical procedures strain ATCC 33520 was incubated with RPE monolayers at 4 С or 37 С for 6 h at pH 7 Differences between groups were evaluated with The effect of the pH was determined in 2 experi the Students t-test The scores of the different Τ ments In one experiment the attachment assay was dentuola strains were mutually compared using a performed for 6 h at 37 С in PBS at pH values of one-way analysis of variance (One-way ANOVA) 4, 5, 6, 7 and 8 In the other experiment the pH followed up with a Tukey-HSD multiple range test values were 6, 6 5, 7, 7 5 and 8
Attachment to rounded and llattened RPE cells Results Confluent monolayers consisted of flattened and In all the attachment assays using Τ dentuola and rounded RPF cells In two separate experiments RPE cells it was noted that only a small proportion the attachment of Τ demicola ATCC 13520 to the of the added spirochetes attached to the epithelial morphologically distinct cell populations was cells The attachment assay revealed dark-brown scored Storing was carried out to all rounded and coloured spirochetes attached to RPE cell mono flattened RPF cells visible in a microscopic field layers (Figs 1, 2) Two morphologically distinct of magnification 1250 χ and performed in as many populations of RPF cells could be distinguished in microscopic fields as needed to count a minimum the monolayers when using phase contrast micro of 50 rounded RPF cells per monolayer scopy One minor population consisted of isolated rounded RPE cells laying on top of a confluent monolayer of flattened RPE cells (Figs 1 and 2) Distribution of rounded RPE cells In most cases the rounded cells were slightly brown The distribution of the rounded RPE cells over the stained, while the flattened cells were not monolayer was investigated in 10 monolayers In The rounded RPE cells seemed to be more recep each monolayer the number of rounded RPE cells tive to the attachment of Τ denticela ATCC 33520 was counted m 5 microscopic fields (magn 1250 χ ) than the flattened cells (Figs 1 and 2) Quantitative located in the central area (centre) or in the area analysis of the distribution of Τ denticela ATCC approximately 0-2 mm from the edge of the RPE 33520 over the two RPE populations showed that monolayer (edge) The mean numbers of rounded the mean percentage of rounded RPF cells with RPE cells per microscopic field between both loca more than five attached spirochetes (score >5) tions were compared was significantly higher (P<0 05) than the mean
38 Treponema attachment to epithelial cells 277
tached spirochetes was lower. After incubation at 37 С the mean percentage of rounded RPE cells with a score of > 5 attached spirochetes was higher than after incubation at 4 C, while the mean per centage of RPE cells without attached spirochetes was lower. All the differences described above were statistically significant at the level Ρ< 0.05. The effect of the pH of the incubation medium on the attachment of T. denticela ATCC 33520, as scored to the rounded RPE cells located near the edge, is shown in Figure 3. With increasing pH there was a shift in the mean percentage of rounded RPE cells with a score of 1-5 attached spirochetes towards rounded RPE cells with a score of > 5, Fig. I. Phase contrast micrograph of Τ denttcola ATCC 33520 while the mean percentages of rounded RPE cells attached to a monolayer of RPE cells. Notice that the rounded RPE cells are more receptive for the attachment of Τ denticola with one or more attached spirochetes (sum of ATCC 33520 than the flattened cells. Bar: 20 //m. both scores) varied between 68% (at pH 5) and 93% (at pH 7).
Table ¡. Attachment of Τ denticola ATCC 33520 to flattened and rounded RPE cells h Mean number + SEM"1 Mean percentage ±SEM of RPE cells per of RPE cells with score RPE cells microscopic field 0 1-5 >5 flattened *| 45 + 2 67±6 30±7 J 5±1 Rounded 1 li + i 45±3 24±1 1 33±4 1 Mean and standard error of seven different monolayers. b Mean and standard error of two separate experiments, * Significant difference; Students t-test: Ρ < 0.05.
10 pm A 0 Table 2. Location of rounded RPE (rRPE) cells in the mono layer and the attachment of Τ denticola ATCC 33520. Fig. 2. Phase contrast micrograph of rounded RPE cells with Location Mean number+ SEM·1 Mean percentage + SEM scores of I-5 (A) or > 5 (B) attached spirochetes of T. denttcola of rRPE or rRPE cells or rRPE cells wich score ATCC 33520. Bar: 10 //m. cells per microscopic field 0 1-5 >5 edge 11 + 1 , 33 - 2 25±2 , 1 42 ±2 percentage of flattened cells with a score of > 5 centre 10±1 69 + 3 24 + 2 1 7 + 2 (Table l ). л Mean and standard error of 10 different monolayers. The rounded RPE cells were evenly distributed h Mean and standard error of three separate experiments. over the monolayer (Table 2). The distribution of * Significant difference; Students t-test: P<0.05. the attached spirochetes, however, was not. At the edge of the RPE monolayer the mean percentage Table 3. Effect of the incubation time and temperature on the of rounded RPE cells with an score of >5 was attachment of Τ denticola ATCC 33520 to rounded (rRPE) higher, while the percentage of cells without at cells. tached spirochetes (score 0) was lower (Table 2). M ean percentage + SEM of rRPE cells. Based on these results it was decided to score the located near the edge. with score attachment only to rounded RPE cells located near 0 1-5 --5 the edge of the monolayer. Incubation time: In Table 3 the effects of the incubation time and (n=4) incubation temperature on the attachment of T. 2h 27±2 43 + 3 ,1 30 + 2 6 h 13±2 37 ±2 1 50±3 denticela ATCC 33520 to the rounded RPE cells located near the edge is shown. After an incubation Incubation temperature: (n = 8) period of 6 hours the mean percentage of rounded 4C 24 + 2 45 + 2 *| 31+3 RPE cells with a score of > 5 attached spirochetes 37 С 13±1 36±1 1 51 ±2 was higher than after an incubation period of 2 η = number of separate experiments hours, while the percentage of cells without at- * Significant difference: Students t-test: Ρ < 0.05.
39 278 Keulers et al
% rRPE indicated by a significantly higher number of 100 rounded RPb cells with no attached spirochetes in comparison to the other strains (Tukey-HSD multiple range test, P<0 05)
Discussion In the present study an assay for the attachment of Τ denticela to epithelial cells is described An indirect immunohistochemical staining method, using native rabbit polyclonal antisera directed against Τ denticola ATCC 33520 and Τ vincenti! LA-1, was employed Previous pilot studies had revealed that optimal staining for Τ denticola strains, other than the ones used to elicited the antiserum, was obtained if both antisera were used in combination This might be explained by cross reactivity between the different species resulting in a complementary effect of both antisera on the investigated Τ denticola strains Advantages of this staining method are the absence of interfering background colouring of the RPE cells, as was observed with the modified Warthin silver staining used in previous experiments (24), and the possi bility to distinguish two RPE populations in the 3 4 5 6 7 8 9 monolayer, one consisting of flattened and the PH other of rounded RPE cells Fig 3 The effect of the pH on the attachment ol Τ denticola The higher attachment scores to the rounded ATCC 33520 to rounded RPE (rRPE) cells located near the edge of the monolayer Results are expresed
40 Treponema attachment to epithelial cells 279 tors for the attachment of tréponèmes During cell from which bacteria are readily removed with the division, flattened cells relocate cell membrane shed epithelial cells and in which at least some of components to the cell membrane ot the developing the progeny have to colonize new available sur rounded ' daughter" cells (28, 31) This, ds well as faces de no\o synthesis of cell membrane components The m vivo presistence of Τ denticola in the during late interphase (33), could result in more junctional epithelium (38) might, in part, be related available cell membrane receptors on the rounded to the availability of specific receptors in this tissue RPE cells for the attachment of Τ denticola In It is tempting to speculate that the receptors of the this context it is of interest that mitotic cells have junctional epithelium, with its high cell turnover, been shown to contain relative more sialic acid have similarities with the implicated receptors on than do cells in interphase (33-35) Sialic acid has the rounded cells in our in \ilro model Further recently been implicated in the attachment of Τ research is needed to elucidate this issue denticela to erythrocytes (26) and epithelial cells In conclusion, the attachment of Τ denticola (36) ATCC 33520 to RPF cells is optimal after an in Besides a possible influence of the cell cycle of cubation period of 6 h at 37 С and pH 7 RPF cells the RPE cells on the attachment of Τ dentuola with a rounded morphology are more receptive for ATCC 33520, other factors seem to be involved A the attachment of Τ denticola than cells that have significantly higher attachment to the rounded a flattened morphology It is speculated that the RPE cells was noted near the edge of the mono attachment is a physical/chemical process of yet layers The reason for this phenomenon remains unknown nature and that differences in the number unexplained but is not due to an initial non-uni of microvilli and/or the amount of available recep form distribution of the inoculum, because the tors, between the two morphologically distinct cell number of spirochetes in the inoculum outnum types, accounts for the differences in the numbers bered the RPE cells with a estimated factor of 10' of attached spirochetes The increase in the number of tréponèmes at tached to the epithelial cells with increasing contact time is in accordance with the saturation curve Acknowledgements described by Reijntjens et al (21) The influence We gratefully acknowledge Sjoerd Rijpkema (Na of the temperature resulted in an increase in the tional Institute of Public Health and Environmen number of attached spirochetes when incubated tal Protection, Bilthoven, The Netherlands) for at 37 С in comparison to 4 С An influence of preparation of the polyclonal antisera and Birgitte temperature on the attachment of Τ denticola has Hasenack and William Mulder for their technical been previously noted by Dawson et al (23) In assistance addition we found a pH optimum curve for the attachment of Τ denticola ATCC 33520 to the rounded RPE cells Theoretically, shifting of tem References perature and pH in the attachment assay may lead 1 ListgarlenMA Hellden L Relative distribution of bacteria to the formation of rounded RPF cells which are at clinically healthy and pcnodontally diseased sites in distinct from the ones that develop as a result of humans J Clin Periodont 1978 S 115-112 the cell cycle However, no morphologic alterations 2 Moore WFC Holdeman IV Smibert RM Hash DE Bur- were noted at the light microscopic level The ef meister JA Ranney RR Bacteriology of severe peno dontilis in young adult humans Infect Immun 1982 38 fects of the incubation temperature the incubation ΙΠ7 1148 time and the pH on the attachment indicate a 3 Loesche WJ Laughon BB Role ol spirochetes in pen physical/chemical process odonlal disease In Genco RJ Mergenhagen Sb eds Host Applying the attachment assay to three batch- parligli intiraition\ in periodontal diuascs Washington DC Am Soc Microbiol 1982 62 75 cultured Τ denticola strains ATCC 35405, Bl 1 and 4 Listgartcn MA Levin S Positive correlation between Ihe Ny541 revealed strain differences Especially strain proportions of subgingival spirochetes and motile bacteria Ny541 attached poorly, confirming previous results and susceptibility ol human subjects lo periodontal deteno (24) Strain differences in attachment have been ration J С Im Periodont 1981 8 122 138 noted by others (19, 22, 26) and might be due to 5 Armitage GC Dickinson WR Jendcrscck RS Levine SM Chambers DW Relationship between the percentage of difference in expression of adhesin(s) subgingival spirochetes and the seventy of periodontal dis Microscopical observation at the end of the in ease J Periodontal 1982 ЧЗ 550-556 cubation period revealed, in all the tested Τ denti- 6 Simonson LG Goodman CH Bial JJ Morion HE Quanti cola strains, many unattached spirochetes Fluctu tative relationship of Trtponemo dentuola to scvenH of periodontal disease Infect Immun 1988 Sé 726 728 ations in the adherent population have been noted 7 Listgarten MA Flectron microscopic observations on the (26, 37) and might contribute to the persistence of bacterial flora of acute necrotizing ulcerative gingivitis J a species in an environment like the gingival sulcus, Periodontal 1965 36 68-79
41 260 Keulers et al
8 Carranza Jr FA Saghe R Newman MG Valetm PL Scan 25 Haapasalo M, Singh U. McBnde ВС, Uitto V-J Sulf- ning and transmission electron microscopic study of lissuc- hydryldependenl attachment of Treponema denticola to la- imading microorganisms in localized juvenile perio minin and other proteins infect Immun 1991, 59 dontitis J Periodontal 1983 54 598 617 423CM237 9 Prank RM Bacterial penetration in the apical pocket wall 26 Mikx FHM, Keulers RAC Hemagglutination activity of of advanced human periodontitis J Periodont Res 1980, Treponema denticola grown in serum-free medium in con 15 561-571 tinuous culture Infect Immun 1992 60 1761-1766 10 Saghe R, Newman MG, Carranza Jr FA. Pattison GI 27 Dulbecco R & Vogt M Plaque formation and isolation of Bactena! invasion of gingiva in advanced periodontitis in pure lines with poliomyelitis viruses J Exp Sied 1954, 99 humans J Periodontal 1982, 53 217 222 167 11 Maltha JC, Mikx FHM, Kuijpcrs FJ Necrotizing ulcer 28 Porter K, Prescott D, Frye J Changes in surface morph ative gingivitis in beagle dogs III Distribution of spiro ology of chínese hamster ovary cells during the cell cycle chetes in interdental gingival tissue J Periodont Res 1985, J Cell Biol 1973, 57 814 £36 20 522-531 29 Fnckson CA, Trinkaus Ρ Microvilli and blebs as sources 12 Mikx FHM, Maltha JC van Campen GJ Spirochetes in of reserve surface membrane during cell spreading Exp early lesions of necrotizing ulcerative gingivitis experimen Cell Res 1976,99 375-384 tally induced in beagles Oral Microbiol Immunol 1990, 5 30 Wong GHW, Sterner B, Fame S, Graves S Effect of serum 86-89 concentration and metabolic inhibitors on the attachment 13 Saghe FR, Carranza Jr FA, Newman MG, Cheng L, Lewin of Treponema pallidum to rabbit cells J Med Microbiol KJ Identification of tissue-invading bacteria in human 1983, 16 281-293 periodontal disease J Periodont Res 1982, 17 452 455 31 Pasternak CA Surface membranes during the cell cycle 14 Lmdhe J, Socransky SS Chemotaxis and vascular per TIBS 1976, 1 148 151 meability produced by human periodontopathy bacteria 32 Willmgham MC, Pastan I Cyclic AMP modulates J Periodontal Res 1979, 14 138-146 microvillus formation and agglutinabihty in transformed 15 Makinen KK, Syed SA. Makinen P-L, Loesche WJ Dom and normal mouse fibroblasts Proc Natl Acad Sci 1975, inance of iminopeptidase activity in the human oral bac 72 1263 1267 terium Treponema denticola ATCC 35405 Curr Microbiol 33 Graham JM, Sumner MCB, Curtis DH, Pasternak. CA 1987, 14 341-346 Sequence of events m plasma membrane assembly dunng 16 Grenier D. Litto V-J, McBnde ВС Cellular location оГа the cell cycle Nature 1973, 246 291 295 Treponema denticola chymolrypsinlike protease and im 34 Mayhew E Effect of nbonuclease and neuraminidase on portance of the protease in migration through the basement the electrophoretic mobility of tissue culture cells in para- membrane Infect Immun 1990, 58 347 351 synchronous growth J Cell Ph\stoi 1967. 69 305-309 17 Uitto V-J, Grenier D, Chan ECS, McBnde ВС Isolation 35 Ghck MC, Gerner FW, Warren L Changes in the carbo of a chymotr>psinlikc enz>me from Treponema denticola hydrate content of the KB cell during the growth cycle J Infect Immun 1988, 56 2717 2722 Ceil Physiol 1971, 77 1-5 18 Mikx FHM Comparison of peptidase, glycosidasc and 36 Keulers RAC, Maltha JC, Mikx FHM, Wolters-Lutger- esterase activities of oral and non oral Treponema species horst JM L Involvement of treponemal surface located pro J Gen Microbiol 1991, 137 63-68 tein and carbohydrate moieties in the attachment of Tre 19 Weinberg A. Holt SC Interaction of Treponema denticela ponema denticola ATCC 33520 to cultured rat palatal epi TD-4, GM-I and MS25 with human gingival fibroblasts thelial cells Oral Microbiol Immunol 1993 (in press) Infett Immun 1990 58 1720 1729 37 Cowan MM Mikx FHM, Keulers RAC, Busscher HJ 20 Olson I Attachment of Treponema denticola to cultured Electrophoretic mobility, ultrastruclure and hemagglutina human epithelial cells Scand J Dent Res 1984 92 55-63 tion of Treponema denticola ATCC 33520 Infect Immun 21 Rcijntjens FMJ, Mikx HHM Wolters-Lutgerhorst JML, (submitted) Maltha JC Adherence of oral tréponèmes and their efTecl 38 Mikx FHM, Maltha JC, Keulers RAC Are there differ on morphological damage and detachment of epithelial ences between oral treponema in dental plaque and in cells in vitro Infect Immun 1986, 51 642-647 gingival tissue7 J Dent Res 1989, 68 Abstr No 87 22 Cimasom G McBnde ВС Adherence of Treponema denti- cola to modified hydroxy a patite J Dent Res 1987, 66 1727 1729 Address 23 Dawson JR, Ellen RP Tip-orientated adherence of Tre ponema denticola to fibronectm Infect Immun 1990, 58 J С Maltha 3924 3928 Um\erstt\ of Nijmegen 24 Keulers RAC. Maltha JC. Mikx FHM, Woltcrs-Lulger- Laboratory of Oral Histology horsl JML Attachment of Treponema denticola strains to PO Box 9101 keratinocyle-monolayers of different ongin Oral Microbiol NL-6500 HB Nijmegen Immunol 1993, 8 73 77 The Netherlands
42 Chapter 4
Involvement of treponemal surface-located protein and carbohydrate moieties in the attachment of Treponema denticola ATCC 33520 to cultured rat palatal epithelial cells.
Keulers, R.A.C., Maltha, 1С, Mikx, F.H.M., and Wolters-Lutgerhorst, J.M.L.
Oral Microbiol Immunol 1993: 8: 236-241. (Reprint) Oral \1 it mhial Immunol 1993 8 236 241 Copyright % Munksgaard ¡993 Primal m Dirwwrk AH rig/it^ renr\etl OtíM¡aotÉÉKjy~ anätmmrdogy ISSN 0901-OOSS
R. A. C. Keuler·1, J. C. Maltha1, Involvement of treponemal F. H. M. Mlkx', J. M. L. Wolters-Lutgerhortt1 'Laboratory o( Oral Histology end 'Department of Pen odontology and Preventive surface-located protein and Dentistry TRIKON Research Program in Oral Microbiology University of Nijmegen carbohydrate moieties in the the Netherlands attachment of Treponema denticola ATCC 33520 to cultured rat palatal epithelial cells
Keulerч RAC Maltha JC \fikx FHM Uolters-Lutgahorst JML Invohement of treponemal sw face-locatedprotun and tarbofndrate moieties m the attachment of Treponema den.tn.oId ATCC 33520 to cuitwed rat palatal epithelial cells Oral Microbio! Immunol 1993 8 236 241 С Munksgaard 1993
We studied the nature of attachment of Ti eponima denticola ATCC 33520 to a microscopical!) distinct population of rounded rat palatal epithelial cells The motility of the freshly han.est.ed spirochetes appeared not to be a prerequisite for attachment Treatment of Τ denticola ATCC 33520 with proteinase-K heat glutaraldehyde, formaldehyde and periodate oxidation decreased the attachment to the rounded rat palatal epithelial cells indicating the involvement of protein and carbohydrate moieties Trypsin treatment had no effect on the attachment The attachment of Τ denticola ATCC 33520 was decreased after treatment with Key words attachment Treponema denticola name non-immune rabbit serum native polyclonal rabbit serum D-mannose, epithelial cell m vitro Vacetyl-D-galactosamine and sialic acid The results indicate that the attach J С Maltha Laboratory of Oral Histology ment of Τ denticola ATCC 33520 to rounded rat palatal epithelial cells is mediated University of Nijmegen PO Box 9101 by trypsin-rcsistant adhesin(s) of protein and carbohydrate nature, with affinity NL-6500 HB Ni|megen the Netherlands for D-mannose N-acetyl-D-galactosamine and sialic acid Accepted (or publication December 7 1992
Oral spirochetes including Treponema and immune-defense processes has been for D-galactose, D-mannose D-glu- denticola are consistently observed in suggested (2 26 27) cosamine N-acetyl-D-galactosamine increased numbers in periodontal A prerequisite for the involvement of sialic acid or fibronectm have been im pockets with suspected periodontal dis spirochetes in the development of peri plicated (9, 21 29) Also, intra- (4) as ease activity (1 16 28) In addition odontal disease is that these bacteria well as înlerstram differences (5, 12, 29) the presence of oral spirochetes in the can colonize survive and grow in the a nonuniform attachment to cultured gingival tissue has been reported (15 periodontal pockets Attachment is con epithelial cells (12, 11) and a preference 19 22 25) Little is known about the sidered to be the first step in coloniza of attachment to rounded epithelial cells contribution of oral tréponèmes to the tion (8) In \itro studies on the attach (13, 23) has been noted The mechanism onset and development of periodontal ment of Τ dentiiola (3, 5 9-Π. 21, 23 of attachment however remains un diseases In \itro Τ denticola produces 24 29) have revealed that they are able clear This study investigated the in metabolic products (14) and enzymes to attach to human fibroblasts, epi volvement of treponemal surface (10 18 20) that can act as potential thelial cells erythrocytes hydroxyapa- located protein and carbohydrate moi virulence factors with tissue-destructre tite fibronectm laminin fibrinogen, eties in the attachment of Τ dent noia properties Also an indirect influence gelatin and type I and type IV collagens ATCC 33520 to a microscopically dis of oral tréponèmes on periodontal dis The involvement of treponemal ad- tinct population of rounded rat palatal ease bv suppression of host reparative hesins and hemagglutinins with affinity epithelial cells
44 Attachment of Τ denticela to epithelial celh 237
mately 108 spirochetes was added to RPE monolayer In each microscopic Material and methods each well of a multidish 24-well plate field the number of rounded RPE cells Bacterial strain and growth conditions (Nunc) containing covershps with con with 0 (score 0), 1-5 (score 1-5) or > Τ denticela ATCC 33520 was purchased fluent RPE monolayers, resulting in an 5 (score > 5) attached spirochetes was from the American Type Culture Col estimated spirochete epithelial cell ratio counted Scoring was perlormed in as lection The spirochetes were grown in of 1000 1 Incubation was performed in many microscopic fields as needed to continuous culture in serum free PTY air containing 7 5% CO, and a relative count a minimum of 50 rounded RPE medium (21) in a chemostat at pH 7 0, humidity of 95% for 6 h (unless other cells per monolayer In each test the Rh -532 mV, 37 С, a dilution rate of wise indicated) at 37 С Hereafter the number of spirochetes attached to the
0 04 h~' and an atmosphere of 91% N2, monolayers were firmly washed 3 times monolayers was determined in triplicate 5% H, and 4% CO,, resulting in a den in PBS and fixed in 3% glutaraldehyde samples sity of 5 χ 10я spirochetes per milliliter in Sorensen's phosphate buffer (pH 7 4) In an attempt to characterize the na and an optical density of 0 650 at 550 for 45 min at 4 С ture of treponemal surface macromol- nm For the staining of Τ denticela ATCC ccules involved in the attachment, the 33520, we used 2 native polyclonal anti- effect of proteolytic enzymes, alkylation sera These antisera were a gift of Sjoerd and heat pretreatment of Τ denticela on Epithelial cells end culture conditions Rijpkema (National Institute of Public the attachment was examined The Sprague-Dawlcy rat palatal epi Health and Environmental Protection, thelial cells (RPE) were a gift of Dr A Bilthoven, the Netherlands) and had Protease Irealmenl Arenholt (University of Ârhus, Den been elicited in New Zealand White rab mark) The RPE cells were cultured bits challenged with sonicated extracts Samples of the Treponema culture were aerobically (95% air and 5% CO,) in of whole spirochetes of Τ denticela harvested, washed and incubated for I Eagle minimal essential medium (GIB- strain ATCC 33520 or Treponema \in- h at 37 С with trypsin (250 USP units CO Laboratories, Breda, the Nether centii strain LA-1 The staining of 7* mg, Difco Laboratories Detroit, MI) at lands) with Earle's salts and 25 mM denticela ATCC 33520, attached to RPF concentrations of 0 05, 0 1 and 0 5 mg/ HEPES buffer, supplemented with 6% cells, was performed in a standard pro ml MBS or proleinase-K (27 m Anson fetal calf serum The cells were grown cedure that used both native polyclonal units/mg, F Merck, Darmstadt Ger on Π-mm circular glass covershps antisera because previous studies had many) at concentrations of0 05 0 3 and placed in petn dishes (diameter 6 cm) revealed that optimal staining for Τ 0 5 mg/ml MBS The choice of concen (Nunc, Roskilde, Denmark) and denticela strains, other than the one trations was based on the work of Wein reached confluence in approximately 4 used to elicit the antiserum, was ob berg & Holt (29) Control bacterial sus days Before the attachment assay, the tained if both antisera were used in com pensions without the proteases were in growth medium was drained and the bination (13) Tixed RPb monolayers cubated under identical conditons The confluent monolayers were washed with attached spirochetes were first in samples with the serine protease inhibi twice in TC-Dulbecco's (6) phosphate- cubated with the antiserum directed tor phenylmethylsulfonyl fluoride (Sig buffered saline (PBS, pH 7 2) and against Τ denticela ATCC 33520 at a ma Chemical Co ) consisted of I mM placed in multidish 24-well plates dilution of 1 1000 in PBS supplemented phenylmethylsulfonyl fluoride with (Nunc) Confluent monolayers of 3-4 with 1% bovine serum albumin (A7030, either protemase-K (0 5 mg/ml) or tryp days old consisted of an estimated num Sigma Chemical Co, St Louis, MO) sin (0 5 mg/ml) and were preincubated ber of 10s RPE cells per coverslip (13) for 1 h at room temperature After 3 at 37 С for 30 mm prior to addition washing steps in PBS for 10 mm each, of the spirochetes After incubation the the monolayers were incubated in the spirochetes were harvested by centn Attachment assay antiserum directed against Τ іпсепш fugation, washed once in MBS, sus The attachment assay was performed as LA-1 diluted 1 500 in PBS supple pended in DMEM and incubated with previously described (13) Samples of Г mented with 1% bovine serum albumin the RPE cells for 3 hours To eliminate demicola ATCC 33520 were harvested for 1 h, followed by 3 washing steps the presence of residual protease activity from the continuous culture vessel, in PBS Hereafter the monolayers were during the attachment assay, the washed twice by centnfugation (1800 χ incubated for 1 h m peroxidasc-conju- DMEM was supplemented with 1 mM g, 10 min) and resuspended in PBS gatcd swine immunoglobulins to rabbit phenylmethylsulfonyl fluoride immunoglobulins (Dakopatts, Copen supplemented with 25 mM MgCl2 and 1 mM CaCl (MBS, pH 7 4) After the hagen, Denmark) diluted I 100 in PBS 2 Alkylation different experimental treatments the supplemented with 1% bovine serum pellet was finally resuspended in Dul albumin After washing, the conjugated The spirochetes were suspended in becco modified Eagle medium immunoglobulin was visualized with phosphate-buffered 4% v/v formaldc- (DMEM) supplemented with Ham's diamino-benzidine (DAB) 0 5 mg/ml hylde or 1 7% v/v glutaraldehyde, pH F12 medium (DMFM/F12 3 1 v/v) PBS containing 0 03% (ν/ν) Η,Ο, 7 2 at room temperature for I h Con (blow Laboratories, Irvine, United As previously described (13), the trol bacterial suspensions without fixa Kingdom) The number of spirochetes attachment of Τ denticela ATCC 33520 tives were incubated under identical per millimeter was counted in a bac to a morphologically distinct popula conditions in MBS After incubation the terial counting chamber (Hawksley, tion of rounded RPE cells was scored spirochestcs were harvested bv centn Lancing, United Kingdom) by phase in randomly chosen microscopic fields fugation washed once in MBS sus contrast microscopy One millimeter of (magnification χ 1250) located approxi pended in DMEM and incubated with the suspension, containing approxi mately 0-2 mm from the edge of the the RPE cells
45 238 Keulers eí al.
20 C). After incubation, 100 μ\ sample served that, at the end of the incubation Heat treatment was diluted with 900 μ\ DMEM (final period, only some of the submitted Spirochetes, suspended in MBS. were concentration spirochetes 108/ml) and spirochetes had attached to the RPE incubated for 20 mm at 56 С in a shak incubated directly with the RPE cells cells. ing waterbath. Control samples were in for 3 h. Spirochetes treated with sialic The effect of proteinase-K and tryp cubated in MBS for 20 min at 37С acid and N-acetyl-D-glucosamine were sin treatment of T. denticola ATCC After incubation the spirochetes were incubated for 6 h. 33520 on the attachment to the rRPE harvested by centrifugation, suspended cells is shown in Table 1. The data for ín DMEM and incubated with the RPE proteinase-K and trypsin in Table 1 is Periodate oxidation of 7. denticola cells. the mean value of the 3 tested enzyme To determine the involvement of oxidiz- concentrations because regression stat able sugar moieties in the attachment of istics revealed no differences between Anti sera T. denticola ATCC 33520 to rounded the 3 tested enzyme concentrations Native polyclonal rabbit antisera RPE cells, the tréponèmes were incu (data not shown). Treatment with pro against sonicated extracts of T. denticola bated with 25 mM sodium metaperi- teinase-K resulted in a decrease of ATCC 33520 or T. vincenlii LA-1 spiro odate in 0.1 M acetate buffer pH 4.1 attachment to rRPE cells, as indicated chetes and native non-immune rabbit (NaI04, Boehringer, Mannheim, Ger by a significantly higher percentage of serum were examined for their ability many) for 2 h at 4 C, washed in MBS rRPE cells with 0 and 1-5 attached to affect attachment. Both immune sera and incubated with the RPE cells. Con spirochetes and a lower percentage of showed evident and distinguished reac trols were incubated in 0.1 M acetate rRPE cells with >5 attached spiro tivity to T. denticola ATCC 33520 in buffer (pH 4.1). chetes. Treatment of the tréponèmes immunoblots by numerous strongly re with trypsin had no significant effect acting bands. The spirochetes were in on the attachment. The presence of the Statistical analysis cubated in serum dilutions of 1:200, serine proteinase inhibitor phenyl- 1 :800 and 1 : 1600 for anti-Г denticola The counted scores were expressed in methylsulfonyl fluoride eliminated the ATCC 33520; 1: 50, 1 :800 and 1:1600 percentages. Statistical analysis was per proteinase-K effect (Table 1). for anti-T1 vincenlii LA-1 and 1:200, formed on the root square-transformed The effect of glutaraldehyde, form 1 :800 and 1 : 1600 for the non-immune percentages. The transformed data were aldehyde and heat treatment of T. den serum. The dilutions were made in analyzed either by the Student's Mests ticola ATCC 33520 on the attachment MBS. Control samples without serum for independent samples or a one-way to rRPE cells was tested in different were incubated under identical con analysis of variance (one-way ANOVA). assays (Table 1). Glutaraldehyde treat ditions. After incubation for 1 h at In all analyses a value of Ρ < 0.05 was ment resulted in a decrease of attach 37 C, the spirochetes were washed once accepted as significant. ment to rRPE cells, as indicated by a in MBS, resuspended in DMEM and significantly higher percentage of rRPE incubated for 3 h with the RPE cells. cells with 0 attached spirochetes and a Results significantly lower percentage of rRPE The attachment assay of T. denticola cells with >5 attached spirochetes. Carbohydrate and amino acid inhibition ATCC 33520 to RPE cells revealed a Formaldehyde treatment showed a A number of carbohydrates and amino microscopically distinct population of comparable tendency, although not sig acids were screened for their effect on rounded (rRPE) cells that was more re nificant. Heat treatment at 56 С for 20 the attachment of T. denticola ATCC ceptive to the attachment of spirochetes min resulted in decreased attachment, 33520 to the rounded RPE cells, to de than the flattened RPE cells (Fig. 1). In as indicated by a significantly higher termine the specificity of the putative all the attachment assays it was ob percentage of rRPE cells with 0 attached treponemal adhesin(s). The carbo hydrates D-mannose, D-galactose and N-acetyl-D-galactosamine (all from E. Merck) were added to spirochete cell suspensions (109 cells/ml DMEM) at the concentrations 5, 10, 25 and 50 mM. Sialic acid (N-acetylneuraminic acid; Janssen Chimica, Beerse, Belgium) was added at the concentrations 10 and 50 mM. N-acetyl-D-glucosamine (Janssen Chimica) was added at 50 mM. The amino acids L-alanine, L-argi- nine, L-lysine, L-proline, L-serine, L- phenylalanine and L-glutamine (all from E. Merck) were added to spiro chete cell suspensions (ΙΟ9cells/ml PBS) at a concentration of 150 mM. The Fig I Phase contrast micrograph of stained 7" denticola ATCC 33520 spirochetes attached spirochetes were incubated for 1 h at to a monolayer of rat palatal epithelial (RPF.) cells. Notice that the rounded RPE cells are 37 С except for sialic acid (2 h at 4 C) more receptive for the attachment of Τ denticola ATCC 33520 than the flattened cells. Bar: and N-acetyl-D-glucosamine (1.5 h at 25 μνη.
46 Attachment of Τ denticela to epithelial cells 239
Table 1 The effect of proteinase-K, trypsin, glutaraldehyde, formaldehyde and heat treatment serum or native non-immune scrum re of Τ denlicola ATCC 33520 on the attainment to rounded RPE cells sulted m a significant decrease in attach Mean percentage ± SEM1 of rRPE cells ment of Τ denticoia to rRPE cells (Table with score 2) The inhibition of attachment was Treatment 0 1-5 >5 related to the serum concentration Analysis of variance (one-way ANOVA) 11±1 22±4 67±5 Control revealed no differences between the Proteinase-K 29 ±2* 41±1' 30±1 Trypsin 12 + 2 29 ±4 59±4 three sera at comparable dilutions Proteinase-K + phenylmethylsulfonyl (luonde 17 + 6 27±9 56±3 The efTect of the carbohydrates on Trypsin + phenylmethylsulfonyl fluoride 10±2 29±3 61 ±3 the attachment of Τ denticela ATCC 33520 to rRPE cells is shown in Table Control 40±4 28±5 32±2 Glutaraldehyde (1 7% v/v, 1 h) 74 ±8* 19±7 8±1* 3 Of the 4 tested concentrations of D- mannose, only 50 mM inhibited the Control 5 + 5 17+17 79 + 22 attachment significantly, as indicated by 22±8 35+10 44±18 Formaldehyde (4% v/v, 1 h) a significantly higher percentage of Control 14±6 28±3 57±9 rRPE cells with 0 attached spirochetes Heat (56 C, 20 mm) 34±3* 39±6 27 ±3* and a significantly lower percentage of ' Standard error of the mean * Significantly different from control. Students Mest Ρ < 0 05 rRPE cells with >5 attached spiro chetes Both tested N-acetyl-D-galacto- samine concentrations, 25 mM and 50 Table 2 The eíTecl οΓ native polyclonal rabbit an lisera or non-immune rabbit serum pretreal- mM (data not shown), resulted in a sig ment of Τ denticoia ATCC 33520 on the attachment to rounded RPE cells nificantly higher percentage of rRPE Mean percentage ± SFM of rRPF cells cells with 0 attached spirochetes and a with score significantly lower percentage of rRPb cells with > 5 attached spirochetes D- Serum dilution 0 1-5 >5 galactose had no effect on the attach Control 19±5 31 ±4 50±9 ment (Table 3) In a separate assay, both Anti-Г denticoia 1 1600 42±9 32±3 26+10 tested sialic acid concentrations, 10 mM strain ATCC 33520 1 800 39±6 46 + 4 15 + 3* and 50 mM (data not shown), signifi 1 200 57 ±5* 34±3 9±5« cantly decreased the attachment, as in Antl Τ vincenti! 1 1600 27 + 3 39±1 34±4 dicated by a significantly higher percen strain LA 1 1 800 30+4 38 + 3 32±3 tage of rRPE cells with 0 attached spiro 1 50 44+8 39 + 3 17 ±6· chetes and a significantly lower percentage of rRPE cells with 1-5 Non-immune serum 1 1600 46±10 38±4 16±9· 1 800 39 ±6· 42±4 1914* and > 5 attached spirochetes N-acetyl- 1 200 57 + 3' 29±4 14±2* D-glucosamine had no efTect on the attachment (Table 3) * Significantly different from control, Students /-test P<0 05 Of all the amino acids tested, only L- phenylalanine influenced the attach ment of Τ denlicola ATCC 33520 to Table 3 The effect of carbohydrates, amino acids or periodate pretreatment of Τ denlicola rRPE cells Addition οΓ 150 mM L-phe- ATCC 33520 on the attachment to rounded RPE cells nylalamne resulted in a significantly Mean percentage + SEM of rRPfc cells higher percentage of rRPE cells with 0 with score attached spirochetes and a significantly Treatment 1-5 >5 lower percentage of rRPF cells with > Control 9±2 3l±l 61+3 5 spirochetes attached (Table 3) D-mannose 50 mM 34 ±7* 27±4 19 + 6* Periodate oxidation of Τ denlicola re N-acetyl-D-galactosamine 25 mM 39 ±5* 25±5 36±8* sulted in a inhibition of attachment
D-galactose 50 mM 15±1 20x6 65±5 There was a significantly higher percen Control 11±3 47 + 3 42±7 tage of rRPE cells with 0 attached spiro sialic acid 10 mM 46 + 3* 17+1* 17 + 3· chetes and a significantly lower percen N-acetyl-D-glucosamme 50 mM 22±4 32 + 4 46±5 tage of rRPE cells with 1-5 and > 5 attached spirochetes (Table 3) Control i + i 23±9 76±9 L-phenylalanine 25 + 7* 45±9 30 ±8* Control 47 + 6 42±5 H±l Discussion Periodate oxidation 75 ±2* 23 ±2* 2±1" 100 mM The growth of Τ denlicola ATCC 33520 " Significantly different from control, Students /-test /><0 05 ш continuous culture and the use of epithelial cells derived from a cell line enabled us to study the attachment of spirochetes and a significantly lower Exposure of Τ denticela ATCC 33520 spirochetes under controlled conditions percentage of rRPE cells with > 5 at to native anti-Г denticoia ATCC 33520 No evident motility was noted in the tached spirochetes serum, native anti-Г vmcenin LA-1 freshly harvested samples taken from
47 240 Keulers et al
the continuous culture However overt ruthenium red-sensitive surface layer, Acknowledgements attachment was observed indicating indicating the presence of glycosamino that treponemal motility did not seem glycans (17 23) was recently demon We gratefully acknowledge Sjoerd to be a prerequisite for [he in iitro strated on Τ denticela ATCC 33520 (4) Rijpkema (National Institute of Public attachment of Τ deniicola AICC 13520 The attachment of Τ denticola ATCC Health and Environmental Protection, to epithelial cells As noted in previous 33520 to epithelial cells seemed to be Billhoven, the Netherlands) for prepar studies (13 23). a distinct population of mediated by an adhesin(s) with affinity ing the polyclonal antisera M A van 't rounded cells in the monolayer of RPE for D-mannose, N-acetvl-D-galactos- Hof, Department of Medical Statistics, cells was more receptive to the attach amine, sialic acid and the amino acid L- University of Nijmegen, the Nether ment of a part of the Τ denticola popu phenylalanine Adhesins with affinities lands) for statistical analysis of the data lation for D-mannose and N-acetyl-D-galac- and Karin Eekelaar and Alphons Veld The attachment of Τ denticola to tosamine are also reported in studies kamp for skillful technical assistance fibroblasts and erythrocytes seems to be concerning the attachment of Τ dentico mediated by treponemal lectin-hke ad- la to human fibroblasts (29) Hemagglu hesin(s) with affinity for D-mannose, D- tination studies to human erythrocytes References galactose, D-glucosamine, N-acetyl-D- failed to reveal adhesins with affinities 1 Armitage GC, Dickinson WR, Jender- galactosamine and sialic acid (9,21, 29) for D-mannose and N-acetyl-D-galac- seck RS Levine SM. Chambers DW Re We attempted to characterize the tre tosamine, but revealed hemagglutinins lationship between the percentage of sub ponemal adhesin(s) involved in the with an affinity for sialic acid (21 ) Even gingival spirochetes and the seventy of attachment to epithelial cells by expos though Τ denticola ATCC 33520 ad periodontal disease J Penodontol 1982 53 550 556 ing Τ dentiiola ATCC 33520 to a variety hesins and hemagglutinins (21) share an of treatments Treatment of Τ denticola affinity for sialic acid it is unlikely that 2 Boehnnger HR Taichman NS Shenker BJ Suppression of fibroblast prolifer ATCC 33520 with protcinase-K. heat, the treponemal attachment and hemag ation by oral spirochetes Infect Immun alkylation by glutaraldchyde and, to a glutination are mediated by the same 1984 45 155-159 lesser extent formaldehyde resulted in a surface-located component, because of 3 Cimasoni G McBnde ВС Adherence of decrease in attachment, indicating that the trypsin sensitivity of the hemaggluti Treponema denticola to modified hy- treponemal attachment is mediated by nin and its lack of affinity for D-man droxyapatite J Dent Res 1987 66 protein-containing moieties The nose and N-acetyl-D-galactosamine (9, 1727 1729 attachment was not influenced by tryp 21) Indications for specific attachment 4 Cowan MM MikxFHM Keulers RAC, sin treatment The latter is in accord mechanisms of Τ denticola ATCC 35405 ßusscher HJ blectrophoretic mobility, ance with Weinberg & Holt (29), who have also been found by Haapasalo et ultraslructure and hemagglutination of Treponema denotóla ATCC 33520 Sub postulated that attachment involving al (II) mitted trypsin-sensitive proteins would not be The dose-response inhibition of the 5 Dawson JR Filen RP Tip-oriented ad ecologically sensible, because Τ dentico attachment of Τ denticola ATCC 33520 herence of Treponema denticola to fib la contains a trypsin-like enzyme (10, b> native rabbit antiserum or native ronectin Infect Immun 1990 58 18, 20) and trypsin-like activity is pres rabbit non-immune serum suggests that 1924-1928 ent in the periodontal pocket (16) another serum component than im 6 Dulbecco R Vogt M Plaque formation Periodate oxidation resulted also in a munoglobulin binds to the spirochetes and isolation of pure lines wilh poliomy decrease in attachment indicating the This scrum component inhibits attach elitis viruses J Fxp Med 1954 99 167 involvement of carbohydrate-contain ment and cannot be eliminated bv wash 7 Fit7gerald TJ Johnson RC Miller JN, Sykes JA Characterization oflhe attach ing moieties It is templing to speculate ing Comparable results were obtained ment of Treponema pallidum (Nichols that the involvement of protein- and in hemagglutination studies with 7 den- strain) to cultured mammalian cells and carbohydrate-moieties indicates the in tuola ATCC 33520 (21) and in attach the potential relationship to patho volvement of treponemal surface- ment studies with other Τ denticola genicity Infect Immun 1977 18 located glycoproteins in the attachment strains and human fibroblasts (29) 467 478 process Glycoproteins were recently Serum contains, among others, fib- 8 Gibbons RJ Bacterial adhesion to oral observed in the outer membrane of Τ ronectin and sialated proteoglycans tissues a model for infectious diseases J denticela (30) Although glycoproteins The degree of attachment has been re Dent Res 1989 68 750-760 have been implicated as adhesins (11). lated to the presence of fibronectin-spe- 9 Grenier D Characteristics of hemolytic and hemagglutmating activities of Tre their role in the attachment process of cific adhesins on Τ denticola (5 29), and ponema dentiiola Oral Microbiol Immu Τ denticola is unknown On the other an affinity of the Τ denticola adhcsin(s) nol 1991 6 246-249 hand the involvement of carbohydrate- for sialic acid was observed in this study 10 Grenier D, Ulllo V-J, McBnde ВС containing moieties m the attachment Whether fibronectin and /or sialated Cellular location of a Treponema dentico process could also indicate a role of gly- proteoglycans are involved in the inhi la chymotrypsinlike protease and im cosaminoglycans Glycosaminoglycans bition of attachment by the sera remains ponance ol the protease in migration have been demonstrated on the surface to be elucidated through the basement membrane Infect Immun 1990 58 347-351 of Treponema pallidum (7) and some Τ In conclusion, our results indicate denticola strains (4. 23), and their in that the attachment of Τ denticola 11 Haapasalo M Singh U McBnde ВС. volvement in the attachment process Unto V-J Sulfhydryl-dependent attach ATCC 33520 to epithelial cells is med ment of Treponema deniicola to laminin has been implied Although Τ denticola iated by trypsin-rcsistant adhesin(s) of and other proteins Infect Immun 1991 ATCC 33520 lacks a positive glycos- protein and carbohydrate nature with 59 423ÍM217 aminoglycan reaction with acid bovine affinity for D-mannose, N-acetyl-D-gal- 12 Keulers RAC Maltha JC, Mikx FHM, serum albumin (21), the presence of a actosamine and sialic acid Wolters Lutgerhorst JML Attachment
48 Attachment of Τ denticola to epithelial cells 241
of Treponema denticola strains to ma dentuola strains isolated from the hu 25 Riviere GR, Wcisz KS, Simonson LG. keratinocytemonolayers of different man periodontal pocket Сигт Microbiol Lukehart SA Pat hogen-reía led spiro origin Oral Microbiol Immunol 1993 1986 14 85 89 chetes identified within gingival tissue 8 73-77 19 Maltha JC, Mikx FHM. Kuijpers FJ from patients with acute necrotizing ul 13 Keulers RAC. Maltha JC. Milcx FHM. Necroii7ing ulcerative gingivitis in beagle cerative gingivitis Infect Immun 1991 Wolters-Lutgerhorsl JMI. Attachment dogs III Distribution of spirochetes in 59 2653-2657 of Treponema denticola strains ATCC interdental gingival tissue J Periodont 26 Sela MN, Weinberg A, Bonnsky R, Holt 13520. ATCC 3540S, Bll and Ny54l Res 1985 20 522-531 SC, Dishon Τ Inhibition of superoxide to a morphologically distinct popula 20 Mikx FHM Comparison of peptidase, production in human polymorpho tion of rat palatal epithelial cells Sub- glycosidasc and esterase activities of oral nuclear leukocytes by oral tre mi tied and non-oral Treponema species J Gen ponemal factors Infect Immun 1988 56 14 Lindhe J, Socransky SS Chemotaxis and Microbiol 1991 1.37 63 68 589 594 vascular permeability produced by hu 21 Mikx FHM Keulers RAC Hemaggluti 27 Shenker BJ. Listgarten MA. Taichman man periodontopathy bacteria J Peri nation activity of Treponema denticola NS Suppression of human lymphocv te odont Res 1979 14 138-146 grown in serum-lree medium in continu responses by oral spirochetes a mono- 15 Lislgarten MA Electron microscopic ous culture Infect Immun 1992 60 cyte-depended phenomenon J Immunol observations on the bacterial flora of 1761 1766 1984 132 2019-2045 acute necrotizing ulcerative gingivitis J 22 Mikx ГНМ. Maltha JC van Campen 28 Simonson LG, Goodman CH. Bui JJ. Penodontol 1965 36 68 79 GJ Spirochetes in early lesions of necro Morton HE Quantitative relationship of 16 Loesche WJ. Laughon BB Role of spiro tizing ulcerative gingivitis experimentally Treponema denticela to severity of peri chetes in periodontal disease In Genco induced in beagles Oral Microbiol Im odontal disease Infect Immun 1988 56 RJ. Mergenhagen SE, ed Host-parasite munol 1990 5 86 89 726 728 interactions in periodontal diseases 23 Olson I Attachment of Treponema den· 29 Weinberg A, Holt SC Interaction оГ7г*>- Washington, DC Am Soc Microbiol mola to cultured human epithelial cells ponema denticola TD-4, GM 1 and MS25 1982 62 75 Scand J Dent Res 1984 92 55 63 with human gingival fibroblasts Infect 17 1 uit JH Ruthenium red and violet II 24 Rennljens FMJ, Mikx FHM. Wollers- Immun 1990 58 1720-1729 bine structural localization in animal Lulgcrhorst JML, Maltha JC Adher 30 Yotis WW, Sharma VK, Gopalsami С et tissues Anal Ree 1971 171 169-416 ence of oral tréponèmes and their effect al Biochemical properties of the outer 18 Makmcn KK. Sycd SA, Makinen P-L, on morphological damage and detach membrane of Treponema dem и ola J Clin Loesche WJ Bcnzoylarginine peptidase ment of epithelial cells in atro Infect Microbiol 1991 29 1397 1406 and iminopcptidase profiles of Trepone Immun 1986 51 642 647
49
Chapter 5
Hemagglutination activity of Treponema denticola grown in serum-free medium in continuous culture
Mikx, F.H.M. and Keulers, R.A.C.
Infect Immun 1992: 60: 1761-1766. (Reprint) INACTION AND IMMUNITY, May 1992 ρ 1761 1766 V ol 60, No 5 0019 9567 92ΌΜ761 06$(J2 000 Copyright ¡t. 1992. American Society for Microbiology
Hemagglutination Activity of Treponema denticola Grown in Serum-Free Medium in Continuous Culture FRANS Η M ЧІКХ* AND ROB А С KEULERS Department ofPenodontoiogy and Pre\enti\e Dentistry, TRIKON Research Program Oral Microbiology, University of Nijmegen, Ρ Ο Βαχ 9101, NL 6500ИВ Nijmegen, The Netherlands Received 18 November 19У1 Accepted 21 January 1992
Hemagglutination by different Treponema denticola strains was observed for erythrocytes of human, horse, bovine, and rabbit origin. The growth of T. denticola ATCC 33520 in serum-free medium in continuous culture enabled us to study the hemagglutinating activity of freshly harvested spirochetes of a denned physiological status. The hemagglutinating activity was cell bound and not related to motility or appendages, such as fimbriae. The activity was destroyed by proteolytic enzymes, heat, and alkylation, indicating that the agglutinin is of a protemaceous nature. In addition, periodate oxidation of the spirochetes indicated the involvement of carbohydrate groups. Microscopic inspection of the hemagglutination mixtures dl the titration endpoints revealed that only a part of the spirochete population was involved in the hemagglutination process. The hemagglutinating activity was found to be growth phase related. The activity was blocked by serum, while of all tested amino acids and carbohydrates, only sialic acid blocked the activity at low concentrations. In conclusion, we found a hemagglutinating activity in Τ denticola which was cell bound and growth phase related. The agglutinin may be a glycoprotein, like lectin, that recognizes sialic acid as a receptor.
Oral spirochetes drc mainly found in the microbiota of the the adherence properties of spirochetes harvested from a gingival crevice and periodontal pockets in association with continuous culture in a scrum free medium periodontal disease (17, 18, 22, 30) Histological investiga tions have revealed spirochetes at the front of subgingival plaque in rapid progressive periodontitis, in the periodontal MATERIALS AND METHODS tissues of ulcerative gingivitis, and in the junctional cpilhe Baclena and cultures. Τ denticola ATCC 33520, ATCC hum of experimentally induced periodontal lesions (15, 21, 35405, and ATCC 15404 were obtained from the American 28) Oral Ireponema species possess a variety of enzymatic Type Culture Collection Strain LUD and Τ vtneentu LAI activities which may play a role in soft tissue and bone were obtained from N S Taichman, Dental School, Univer destruction (7, 14, 20, 24) The proteolytic activities of sity of Pennsylvania Τ denticola Fl, NY 545, MY 535, Ш1, Treponema denticola arc unique among those of the cultiva and B12 are our own isolates from human periodontitis (19) Ыс Ireponema species of oral and non oral origin (19) The The strains were grown in GM1 brolh supplemented with involvement of oral spirochetes in the progression ol pen Π 3*^ heat inactivated bovine scrum (1) or in Proteose- odontal lesions ind tissue invasion identifies these batteria Irypticase ycasi (Ρ7Ύ) medium (10 g of Proteose Peptone as putative pathogens in periodontal disease Successful no 2 (Dtfco, Detroit, Mich ], 5 g of Trypucase peptone treatment of periodontal pockets leads to a reduction of |BBI , Cockeysvillc, Md ], 2 5 g of KCl, and 0 5 g of spirochetes in the subgingival microbiota as well as to a ι cysteine HCl [Merck, Darmstadt, Germany] per liter) The reduction of specific proteolytic activities of Τ denticola in medium was titrated to pH 7 0 with 1 N KOH and heat the pockets (16, 18, 26) Little is known about the coloniza sterilized After the medium cooled, a filter sterilized mix tion of the subgingival area, although adherence is believed r ture of 5 ml of 10 r (wl/vol) NaHC О , 0 025 g of thiamine PP, to play a role in the colonization process (10) ч (Sigma, Amsterdam, The Netherlands), and 5 ml of volatile Recently, the adherence of Τ pallidum and 7" hyodysen fatty acid solution was added The volatile fatly acid solution tenae to different tell types and of Τ denticola to saliva contained 100 ml oí Π 1 N KOH and 0 5 ml of isobutync acid, coated hydroxyapalite, hbroblasts, and kcratinocylcs was 0 5 ml of οι 2 methylbulync acid, 0 5 ml of isovaleric acid, described (2. 3, 8, 25, 27, 29) Glycosaminoglycans and and 0 5 ml of valeric acid (all from Merck) probably fibronectin seem to play a role in the adherence of Batch-grown spirochetes were collected after 4 days of Τ pallidum^ while sialic acid (N acctylncuraminic acid) may culluring al 37°C in an anaerobic glove box (Coy, Ann he involved with the receptor binding ligands on Τ hvodys Γ Arbor, Mich ) in a nitrogen atmosphere with 5 ί H and 4% enteriae and Τ pallidum (2, 8, 29) Adhcsins recognizing 2 C0 Most tests were performed with Τ denticola ATCC galactosyl and mannosyl as well as fibronectin receptors 2 33520 grown in scrum free PTY medium in continuous have been indicated to play a role in the adherence of Τ culture in a chemostat at pH 7 0, at a redux potential of -532 denticola to fibroblasts and fibronectin coaled coverslips (5, mV, at 37°C, at a dilution rate of 0 04 h ', and in a nitrogen 32) r r atmosphere with 5 c H2 and 4 r CO,, resulting in a density of The hcmagglutinalmg activity of Τ denticola ATCC 33520 5 x 10* spirochetes per ml and an optical density at 550 nm is used m the present study as a model in the investigation of of 0 650 The spirochetes in the steady slate of this culture did not show motility in phase contrast microscopy Samples of spirochetes were obtained directly from the chemostat vessel or from the bdtch cultures The samples " Corresponding author were ccnlnfugcd lor 20 mm at 27,000 x g at 4°C 1 he pellets
52 MIKX AND KEULERS iNFtCT IMMUN were resuspended by being vortexed in phosphate buffered TABLE 1 Hemagglutination liters for Τ denticoia ATCC 13520 saline (PBS) (pH 7 2) with 25 mM MgCU (MBS) to reach a and erythrocytes of different origins 4 concentration of at least 4 x IO spirochetes per ml The Median tiler Mean brythrocyles actual spirochete concentration in each test was estimated in (range) rank a counting chamber (Hawksley, Lancing, United Kingdom) by phase contrast microscopy Human" Hemagglutination. Human« rabbit, horse, and bovine A type 64 (2-64) 9 erythrocytes were harvested from EDTA treated blood В type 12 (2-64) 75 О type 32 (2-64) 75 Packed erythrocytes were washed two times tn PBS (pH 7 2) and finally suspended in MBS to a concentration of 4 x 10 Animal* erythrocytes per ml, as microscopically estimated The Rabbit 64 (16-42) 65 erythrocyte suspensions were stored at 4°C and used within Horse 128(12 512) 75 7 days after preparation Serial twofold dilutions of spiro Bovine 16(8-64) 33 chete suspensions in 0 1 ml of MBS were made in multiwell '' Kruskal Wallis one wdy ANOVA chi square - 0 17 nol significant U bottom microtitcr plates (Hycull, Udcn, The Nether '' Kruskal Wallis one way ANOVA (.hi square = 2 R4 not significant lands) To each well was added 0 1 ml of erythrocyte suspension In most tests, human О type erythrocytes were used, unless otherwise indicated The trays were kept over by numerous strongly reacting bands They showed limited night at 4°C, and hemagglutination was read the next morn cross reactivity with Τ pallidum and Τ phagedena and no ing The endpoint was defined as the highest dilution show cross reactivity with Borrelia burgdorferi, I eptospira b\ ing complete hemagglutination The titer is reported as the flexa, and L interrogans In the inhibition tests, spirochetes reciprocal of the endpoint dilution, based on an initial were incubated in scrum dilutions of 1 50 and 1 800 in MBS spirochete concentration of 4 χ lO'Vml The tiler as a After incubation for 1 h at 20°C, the spirochetes were percentage of the control titer was calculated by dividing the harvested by centnfugation, washed once, and titrated titer of the control by the titer of the test and multiplying by Hemagglutination inhibition tests. Ammo acids and carbo 100 All tests were performed on at least two different hydrates were screened for inhibition of the hemagglu occasions in duplicate tinating activity D (+) Glucose, D-(-)-fructose, i>( + )ga Statistical analyses were performed with the Kruskal lactose, o(+)mannosc, L (-) fucose lactose, raffi nose, Wallis one way analysis of variance (ANOVA) ι alanine, L arginine, L lysine, L proline, L serine, L phen Heat treatment. Five milliliter portions of spirochete sus ylalaninc, L glutammc, glucuronic acid, galacturonic acid, pensions in MBS were incubated at 35, 40, 45, 50, and 55°C N dcctylglucosaminc N acetylgalactosamine, N acetyl Samples were taken from each portion at 5, 10, 20,40, and 80 neuraminic acid, gelatin, pectin, heparin, and chondroitin mm, cooled, and titrated on ice (all from Merck) were added to the spirochete suspensions at Alkylalion. Spirochetes were suspended in phosphate three concentrations, 50, 25, and 12 5 mM, unless otherwise buffered 2 and 49c (vol/vol) formaldehyde and 1 7"7f (vol/vol) indicated Human mixed saliva and sublingual saliva were glutaraldchydc (pH 7 2) At 15, 10, 60, 120, and 180 min, collected from two volunteers and centnfuged at 27,000 x g 0 2 ml samples were taken, added to 1 3 ml of MBS, for 15 min The saliva supernatant was used as an incubation centnfuged, resuspended in MBS, and titrated fluid After 1 h of incubation of the suspensions at 20°C, the Periodate oxidation. Spirochetes were suspended m 0 025 spirochetes were harvested by centnfugation, suspended in M NaI04 in 0 1 M acetate buffer (pH 4 6) and in the same MBS to the original concentration, and titrated buffer as a control After incubation for 2 h at 4"C, the spirochetes were harvested by centrifugal ion, washed once, suspended in MBS to the original concentration, and ti RESULTS Iratcd Hemagglutination Human A , В , and О type erythro Treatment with proteolytic and hydrolytic enzymes. For cytes were examined in live tests In addition, rabbit, horse, attempted digestion of the agglutinin, the following enzymes and bovine erythrocytes were examined in four tests All were used type I trypsin (10 00 BAEE units per mg), type tests were performed in duplicate with Г denticoia ATCC VII chymotrypsin (40 to 60 U/mg), type VI protease (8 to 10 33520 harvested from a continuous culture in the steady U mg), type XI protease (2 to 10 U/mg) β N acetylglu state No ¡significant difference in nonagglutinating activity cosaminidase (74 U/mg), and chondroitinasc ABC (0 2 to 1 among human A , В , and О type or animal erythrocytes was U/mg) (all from Sigma), pronasc E (70 PL/mg) and hyal observed (Table 1) Washing of the spirochetes in PBS uroniddsc (250 LSP E/mg) (both from Merck) and papain instead of in MBS resulted in a 50Ύ reduction m hemagglu (10 U/mg) and neuraminidase (25 U/mg) (both from Bochr tinating activity Hemagglutinatmg activity was only ob inger |Mannheim, Germany]) Spirochetes (4 χ 10v cells per served in the spirochetal fraction and not in the spent culture ml of MBS) were incubated in a final enzyme concentration supernatant or washings Microscopic inspection of the of 1 mg ml for 2 h at 374 as described by Bowdcn el al (2) contents of the hemagglutination wells at the titration end The controls were incubated in M BS only After incubation, points revealed many nonadhenng spirochetes (Fig 1) the spirochetes were harvested by centnfugation, washed Incubation with lcK bovine scrum albumin in 2 M acetic once, suspended in MBS to the original concentration, and acid (pH 4) resulted in no microscopic indications of gly titrated cosaminoglycans in the form of precipitates on the spiro An tisera. Native nonimmune and polyclonal ¿misera were chetai surface, as found for Τ pallidum by Fitzgerald el a! kindly provided by S Rijpkema (The Nation il Institute of (8) Public Health and Environmental Protection Bilthoven The The hemagglutinatmg activity of nine Τ denticoia strains Netherlands), who immunized rabbits with sonicates of was examined in seven different tests The strains were whole Τ dent ¡cola ATCC 33520 or Τ vmtentu I Al cells harvested from 5 day old batch cultures in GM1 broth The immune sera showed evident reactivity in immunoblots Strains with a relatively high median tiler above the mean of
53 VOL 60 1992 HEMAGGLUTINATION OF Τ DENTICOLA 1763
HA tter (ring·)
О 20 40 ВО вО WO 120 140 «О hour· FIG 2 Growth curve of Τ denticola ATCC 31S20 and median hemagglutination (HA) titers Symbols • optical density at 550 nm Δ lest times
FIG 1 Hemagglutination of О type erythrocytes with Τ denti formaldehyde resulted in a significant loss of activity which cola ATCC 33520 at the titration endpoint Interference microscopy was proportional to the formaldehyde concentration (Fig 4) Incubation with 1 7% glutaraldehyde resulted in an immedi ate inhibition of the hemagglutmating activity Enzyme and periodate treatments. The influence of proteo ranks of 27 5 were Fl, ATCC 35405, ATCC 33520, and lytic and hydrolytic enzymes on the hemagglutmating activ LUD A median tiler below the mean of ranks of 27 5 was ity of Τ denticola ATCC 13520 was examined in duplicate in found for strains NY 545, NY 545, B12, ATCC 35404, and two tests Most of the proteolytic enzymes reduced the Bll (Table 2) hemagglutmating activity lo 12% of the control level Pro Growth phase. Culture vessels containing 100 ml of prere nase Ь and papain resulted m a reduction to 25% of the duccd PTY broth were inoculated at 24 h intervals with 5 ml control level Phenylmethylsulfonyl fluoride neutralized the samples of the continuous culture of Τ denticola ATCC effect of trypsin Incubation of the spirochetes with a num 33520 in three different tests Inoculation and incubation ber of hydrolytic enzymes resulted in no reduction of the were donc m the anaerobic glove box At the end of the hemagglutmating activity (Tabic 3) incubation period, the optical density of the culture and the Treatment with sodium periodate in 0 1 M acetate buffer hemagglutination titers were estimated Figure 2 shows the reduced the hemagglutmating activity to 12% of the control growth curve and the median hemagglutination titers for level acetate buffer alone did not influence the activity three separate tests at various times The median titer of the (Table 3) spirochetes harvested during the early growth phase was Hemagglutination inhibition tests. Different carbohydrates significantly lower than the median titers of the spirochetes and amino acids were investigated in a number of tests for harvested at the end of the exponential growth phase and the blocking of the hemagglutmating activity of Τ denticola stationary phase (Wilcoxon rank test, Ρ = 0 02) Motility ATCC 33520 Of all the tested substances only N acetyl was not observed in any sample neuraminic acid resulted in a significant decrease m the Heat treatment and alky lat ion. The effect of heat on the hemagglutmating activity (Table 4) Human mixed salivi and hem agglutinating activity was tested by incubation of Τ sublingual saliva resulted in a consistent 50% reduction in denticola ATCC 33520 in MBS al 35, 40 45 50 and 55°C the hemagglutmating activity (Fig 3) The hemagglutmating activity was found to be Native polyclonal antiscra against Τ denticola ATCC sensitive to temperatures above 40"C Incubation of Τ denticola ATCC 33520 in 2 and 4% HA fn %
TABLE 2 Hemagglutination titers for different Τ denticola strains
Strain Median Mean titer (range) tank" Fl 32 (16-64) 47 ATCC 35405 32(8-64) 43 ATCC 33520 32 (2-64) 42 HID 16(4-64) 36 NY 535 4(2-16) 24 NY 545 4(4-16) 23 B12 8(1-16) 22 О 20 40 0 ATCC 35404 4(1 32) 22 minutes Bll 1(1-64) 15 FIG 3 Effect of heat on Τ denticola ATCC 33520 hemaggtu " Kruskal Wallis one way ANOVA chi square = 3 93 F - Ό 02 Mean οΓ tinating activity (HA) Symbols • 35°C + 40°C * 45°C U 50"C ranks 27 5 χ э5°С
54 1764 MIKX AND KEULERS INFECT. IMMUN.
TABLE 4 Hemagglutinating activity of 7. dcnticola ATCC 33520 treated with carbohydrates, amino acids, and other organic substrates Hemagglulination „ , Substrate Dose (% of control level") D (+) Glucose, D-(-)-inictose, 50 mM 100 4 D-(+)-galactose, D-(+)-man- nose, L-(-)-fucose, lactose, and raffinose Glucuronic acid and galactur- 50 mM 100 2 onic acid N-Acetylneuraminic acid 10 mM 6Л 3 N-Acetylglucosamine and 50 mM 100 2 iV-acerylga lac tosami ne l-Alanine, l-arginine, ι-lysine, 50 mM 100 4 FIG 4. Effect of formaldehyde and glutaraldehyde on T. denti- ι -proline, ι -senne, L-phcnyl- cola ATCC 33520 hemagglulinaling activity (НЛ). Symbols* Л, 2% a Ian me, and L-glulaminc formaldehyde, O, 4-V formaldehyde; ·, 1.7*V glutaraldehyde. Gela I in, pectin, heparin, and 0 2% 100 2 chondroitin Mixed saliva and sublingual 100% 50A 2 33520 and T. vincenttt LAI were tested in three different saliva tesis with, as the test organism, T. dcnticola ATCC 33520 " Control treatment was MBS harvested from the continuous culture. The hemagglu h Results for the different tests were identical tinating activity of scrum-treated spirochetes was signifi cantly reduced. The reduction was related to the serum concentration and was of the same magnitude for spiro the tested Τ dcnticola strains, a result which could indicate chetes treated with the two different polyclonal sera or a difference tn the expression of the putative hemagglutinin. nonimmune serum (Table 5). The hcmagglutinating activity was found to be growth phase related. A relationship between hcmagglutinating ac DISCUSSION tivity and growth was also observed for cultures oiPorphy- romonas gmgtvalis (12), while in Escherichia colt the pro The agglutination of erythrocytes and attachment to other duction of adhesms was found to be a function of growth rate cells have been described for different oral and non-oral (31). The low titer of spirochetes harvested during the early bacteria and have been related to specific adhesin-receptor growth phase indicates that the amount of agglutinin or the interactions (6,9,23). The attachment of Treponema species to fraction of agglutinating spirochetes is at least 15 times lower various cell types has been investigated (2, 8, 25, 27, 29). in these spirochetes than in spirochetes harvested at the However, hemagglutination by Treponema species was, at stationary phase. Fluctuations m the adherent population least at the time of review of this paper, not described, have been observed in many lectin-producing bacteria (23) possibly because of growth conditions, the difficulty of and can be of importance in the persistence of a species in a growing these organisms in large enough amounts, and the desquamating habitat, such as the gingival sulcus, from tested Treponema species and strains. For example, we which adhercnl bacteria are readily removed with the shed found no hemagglutination by T. phagedena and T. hyodys- epithelial cells and in which at least some of the progeny entenae strains (data not shown). The present findings have to colonize new available surfaces (9). revealed a difference in hcmagglutinating activity between Microscopic inspection of the hemagglutination mixtures al Ihe titration endpoints revealed many unattached spiro chetes, indicating that only a part of the spirochete popula TABLE 1 Effect on hcmagglutinating activity of enzyme and tion is involved in the hemagglutination process. This phe periodate treatments of Τ dentuda ATCC 33520 nomenon was also noted in attachment studies with Hemagglutination keratmocytcs (13). Treatment" (
55 VOL 60, 1992 HEMAGGLUTINATION OF Τ DENT1COLA 1765
a defined physiological status The hemagglutinating activity ACKNOWLEDGMENTS was cell bound and not related to appendages, such as We lhank Kelli Cowan of the State University of Groningen and fimbriae (Electron microscopic examination failed to reveal Jan Lukassen of the Katholieke Universiteit Nijmegen for their the presence of such appendages [4| ) The activity seemed useful remarks and practical assistance and Sjoerd Rijpkema of the not to depend on motility, because no motility was observed National Institute of Public Health and Environmental Protection, in samples taken in the different stages of the batch grown Bilthuven, The Netherlands for the preparation οΓ sera cultures as well as in the steady state of the continuous REFERENCES culture The hemagglutinating activity was destroyed by 1 Blakemore, R. P., and E. Canale-Parola. 1976 Arginine cata proteolytic enzymes, heat, and alkylation, indicating that the bolism by Treponema denticola J Baclenol 128:616-622 agglutinin is of a protcinaccous nature In addition, the 2 Bernden, С. Α., L. A. J осп s, and L. M. Kelley. 1989 Character periodale sensitivity indicated the involvement of carbohy nation of the attachment of Treponema hyodysentenae to drate groups Glycosammoglycan has been found on the líenle intestinal epithelial cells in vitro Am J Vet Res surface of Τ pallidum strains and some Τ denticola strains 50:1481-1485 and probably originates from the host or scrum in the culture 3 Cimasonf, G., and В. C. McBridc. 1987 Adherence of medium (8, 25) In the present study, Τ denticola ATCC Treponema denticola lo modified hydroxy apatite J Dent Res 33520 was grown in scrum free medium This strain did not 66:1727 1729 4 Cowan, M. M. (Slate University of Groningen). 1991 Personal show a glycosammoglycan positive reaction, and its hemag communication glutinating activity was not influenced by hyaluronidase or 5 Dawson, J. R., and R. P. Ellen. 1990 Tip oriented adherence of chondroitinase treatment, indicating that surface-bound gly Treponema denticola to hbronectin Infect Immun 58:3924- cosaminoglycan was probably not involved in hemagglutina 4928 tion by Τ denticola ATCC 1VÏ20 6 Dehazya, P., and R. S. Coles. 198Ü Agglutination of human Incubation of Τ denticola ATCC 33520 with native rabbit erythrocytes by Fusobactenum nut lea tum factors influencing nonimmune serum or native rabbit antiserum resulted in a hemagglutination and some characteristics of the agglutinin J dose-response inhibition of the hemagglutinating activity Bactenol 143.205 211 7 bchn, N. E. 1986 Enzyme prohlcs from eight small sized oral This result was obtained after washing of the scrum treated spirochetes Scand J Dent Res 94.132-140 tréponèmes and suggests that a serum factor(s) binds to the 8 Fitzgerald, T. J., R. С Johnson, J. N. Miller, and J. A. Sykes. spirochetes, causing an inhibition of hemagglutination A 1977 Characterization of the attachment of Treponema palli dose-response inhibition of the adherence of Τ denticola to dum (Nichols strain) to cultured mammalian cells and the human gingival fibroblasts was found by Weinberg and Holt potential relationship of attachment to pathogenicity Infecí (32) with hcat-inactivatcd fetal bovine scrum In addition, Immun 18:467-478 Weinberg and Holt (32) postulated that there were Icclin-Iikc 9 Gibbons, R. J. 1984 Microbial ecology Adherent interactions adhcsins on the Τ denticola surface with affinities for which may affect microbial ecology in the mouth J Dent Res galaclose and mannose A different lectin seems to play a 63:378-385 role in Τ denticola ATCC ЧЭ520, since galactose and man- 10 Gibbons, R. J. 1989 Bacterial adhesion to oral tissues a model for infectious diseases J Dent Res 68.750-76(1 nose had no effect on the hemagglutinating activity and of all 11 Grenier, D. 1991 Characteristics of hemolytic and hemagglu the tested carbohydrates, only N acetylneuraminic acid tinating activities of Ireponcma denticola Oral Microbiol (sialic acid) blocked the activity at low concentrations Immunol 6:246-249 Comparable results were found in studies of the adherence 12 looshila, L·., A Amano, Т. Напіокв, H. Tamagawa, S. Shizuku- of Τ denticola to keratinocytcs (13) and in a hemagglulina Ishi, and A. Tsunemitsu. 1986 Isolation and some properties of tion study (11) published during the review process for this exohtmaggluiinin from ihe culture medium of Bacterotdes gin manuscript gnaiis 381 Infect Immun 52:421 -427 Sialoglycoprotems and sialoglycolipids are present on 13 Keulers, R. А. С. (University of Nijmegen I. 1991 Personal communication different cell surfaces, including the erythrocyte membrane 14 Laughton, В. E., S. A. Syed,andW. J Loesche 1981 API ZYM Also, serum and salivary proteoglycans carry sialic acid system for identification of Вас temide ν spp , Capnocvtophaga molecules, possibly explaining the inhibition by nonimmune spp , and spirochetes of oral origin J Clin Microbiol 15:97- serum and the low but consistent inhibitory effect of saliva in 102 the hemagglutination tests Sialic acid has been implicated in 15 Ustgarten, M. A. 1965 Flectron microscopic observations on the attachment process for Τ pallidum, Τ hyodysentenae, the bacterial Dora of acute necrotizing ulcerative gingivitis J and other microorganisms (2, 23, 29) It is postulated that, in Penodontol 136:328 339 the development of supragmgival plaque, oral streptococci 16 Ustgarten, M. A. 1986 Direct micioscopy of periodontal pat ho adhere to sialoglycoconjugatcs of the pellicle on the tooth gens Oral Microbiol Immunol 1-31-36 surface and that Actinomyces species then adhere to galac 17 Unarten, Μ Α., and L. Hellden. 1978 Relative distribution of bacteria at clinically heahhy and pcnodontally diseased sites in tose residues made available by their neuraminidase activity humans J Clin Penodontol 5 115 132 (10) These oral bacteria and the sialated salivary proteogly 18 Loesche, W. J., J. Giordano, and P. P. Hujoel. 1990 The utility cans seem to make the supragmgival environment unattrac of the ΒΑΝΑ test for monitoring anaerobic infections due to tive for the attachment of Τ denticola However, sialic acid spirochetes (Treponema denticola) in periodontal disease J receptors may be available on the desquamating junctional Dent Res 69-1696-1702 epithelium in the subgingival environment, the habitat of the 19 Mikx, Ι. Η. M. 1991 Comparison of peptidase, glycosidase and oral spirochetes Whether the adhesins of Τ denticola play a esterase activities of oral and non oral Treponema species J role in the ecology of the subgingival microbiota remains to Gtn Microbiol 137:63-68 be proven 20 Mikx, Í.H. M., and M H. de Jong. 1987 Keralinolylic activity of cutaneous and oral bacteria Infect Immun 55:621-625 In conclusion, we found a hemagglutinating activity in Τ 21 Mikx. F. H. M., J. С Maltha, and G. J. van Campen. 1990 denticola which was cell bound and growth phase related Spirochetes in early lesions of necrotizing ulcerative gingivitis The hemagglutinating activity seems to be of protein and experimentally induced in beagle dogs Oral Microbiol Immu carbohydrate nature and may be a glycoprotein, like lectin, nol 5:86 89 that recognizes sialic acid as a receptor 22 Moore, W. E. C. 1987 Microbiology of periodontal disease J
56 1766 MIKX AND KEULERS lN>EtT IMMUN
Periodontal Res 22:33S-141 epithelial cells in vitro Infect Immun 51:642-647 23 Ofek, I., and N. Sharon. 1990 Adhesins as lectins specificity 28 Saghe, R., M. G. Newman, F. A. Carranza, and G. L· Pattlson. and role in infection CUIT Top Microbiol Immunol 151:91- 1982 Bacterial invasion of gingiva in advanced periodontitis in 1Π humans J Penodontol 53:217-222 24 Ohla, K., K. K. Makinen, and W. J. Loesche. 1986 Purification 29 Stelner, В. M., S. Sell, and R. F. Schell. 1987 Treponema and charactenzdiion of an enzyme produced by Treponema pallidum attachment to surface and matrix proteins of cultured denticola capable of hydrolyzing synthetic trypsin substrates rabbit epithelial cells J Infect Dis 155:742 748 Infect Immun 53:213 22Ü 30 Thellade. E., W. H. Wright, S. Boglum Jensen, and H. Loc. 1966 25 Olson, I. 1984 Attachment of Treponema denticola to cultured Experimental gingivitis in man II A longitudinal clinical and human epithelial cells Stand J Dent Res 92*SS-63 bacteriological investigation J Periodontal Res 1:1 13 26 Oosterwaal, P. J. M.. M. I. Malee, F. H. M. Mikx, M. A. Van 'I 31 van Verseveld, H. W., P. Bakker, T. van der Wonde, С. TerfeÜi, Hof, and H. H. Renggli. 1966 The effect of subgingival débride and F. К. de Graaf. 1985 Production of fìmbria! adhesins K99 ment with hand and ultrasonic instruments on the subgingival and F41 by enterotoxogenic bschenchia coli as a function of microflora J Clin Penodontol 14:528-533 growth rate domain Infect Immun 49:149-163 27 Reijnljens, F. M. J., F. H. M. Mlkx, J. M. L. Wolters Lutger- 32 Weinberg, Α., and S. C. Holt. 1990 Interaction of Treponema horst, and J. С Maltha. 1986 Adherence of oral treponemas denticola TD 4, GM I, and M 325 with human gingival fibro and their effect on morphological damage and detachment of blasts Infect Immun 58:1720-1729
57
Chapter 6
Development of an in vitro model to study the invasion of oral spirochetes: A pilot study. Introduction.
Oral spirochetes are a major component of the subgingival microflora associated with several forms of periodontal disease (8,24,25). Besides in plaque, spirochetes have been observed within gingival tissues from diseased sites (3,4,7,10). Invaded spirochetes in oral tissues are mostly identified as being of the intermediate type (6,13) and occasionally of the small (26) or large type (6). Treponema denticola (5,11,15) and Treponema vincentii (9) possess degradative enzymes that can contribute to deterioration of epithelial barriers and, in theory, to invasion of tissue. However, the capability of these and other oral spirochetes to invade multilayered tissue remains to be elucidated. Different models have been developed to study the invasive capability of oral and non-oral spirochetes in vitro. Grenier et al. (5) evaluated the invasive capability of T. denticola by studying the degradation of a reconstructed basement membrane (Matrigel) by this microorganism in combination with the release of spirochetes from the gel. Thomas et al. (27) tested whether T. pallidum and T. phagedenis biotype Reiter could invade host cells by determine the fate of radiolabeled spirochetes added to aortic endothelial cells grown on membrane filters. However, these models lack multilayered tissues in which, in vivo, invasion takes place. Multilayered tissue is used in the invasion model developed by Riviere et al. (21) who studied the invasion of T. pallidum and Τ phagedenis biotype Reiter in double-sided culture chambers, created by mounting abdominal walls excised from mice between two halves of small dialysis cells.
The aim of the present study is the development of an in vitro model in which oral spirochetes interact with multilayered epithelial tissue under controlled conditions. This was achieved by reconstructing an epidermis of human foreskin keratinocytes and studying the (invasive) interaction with a group of oral and non- oral spirochetes.
60 Materials and methods.
- Cell culture. Submerged tissue cultures from human foreskin keratinocytes were prepared using the Rheinwald-Green feeder layer technique (18). Briefly, human foreskin keratinocytes were isolated from juvenile foreskin and cultured together with irradiated (3000 Rad) mouse 3T3 fibroblast feeder cells in Dulbecco's modification of Eagles medium (DMEM) and Ham's nutrient mixture F12 (DMEM/F12 3:1) supplemented with 10% (v/v) fetal calf serum (FCS), 1% L-glutamine (200 mM), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.4 ц§/тЬ hydrocortisone, 10 μΜ isoproterenol, and 10 ng/mL epidermal growth factor (EGF). Incubation was performed at 37°C in air containing 9% CO,. After trypsinisation at confluence, 0.5 χ 106 normal human keratinocytes were cultured air-exposed on de-epidermized human dermis (DED) as described by Régnier et al. (16). Briefly, pieces of human skin (approximately 3x5cm) were taken from the thigh of deceased patients and placed in sterile phosphate-buffered saline (PBS) supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin, and 2.5 μg/mL fungizone (PBSsup). After washing for 1 hour, by repeatingly refreshing the PBSsup, the skin fragments were incubated in fresh PBSsup for 6 days at 37 °C. During this incubation period the PBSsup solution was replaced once after 3 days. At the end of the 6-days incubation period the epidermis was mechanically scraped off with the use of sterile forceps, and the DED was placed in 85% (v/v) glycerol and stored for a minimum of 3 weeks at 4°C before use. For air-exposed tissue cultures, the DED was thoroughly and carefully washed in PBSsup to remove glycerol, irradiated (3000 Rad), and washed in culture medium (DMEM/Ham F12 3:1). Thereafter, the DED was placed, with its basal membrane side up, on a stainless steel grid, and 200 \iL culture medium, containing 0.5 χ IO6 normal human keratinocytes (second or third passage), were inoculated inside a stainless steel ring (diameter 1 cm) placed on top of the DED. After 24-48 hours the ring was removed, and the level of culture medium was adjusted to just reach the hight of the grid. This method ensured that the cells were
61 exposed to air throughout the remaining culture period. Incubation was performed in the same medium and under identical conditions as described for the submerged tissue cultures. 24 hours before the addition of spirochetes, the air-exposed tissue cultures were carefully washed once with culture medium without antibiotics, whereafter the tissue cultures remained, air-exposed, in this medium.
- Spirochetes. For this pilot study, the following Treponema strains were used. Nine T. denticola strains: Bll, В12, S3, Sil, Fl (all isolated in our laboratory from human periodontitis patients (12)), LUD (obtained from N.S. Taichman, Dental School, University of Pennsylvania), and ЛТСС 35404, ATCC 35405, ATCC 33520 (obtained from the American Type Culture Collection); two human intestinal spirochetes HRM-4 and HRM-14, (isolates obtained from G. Dettori, Istituto di Microbiologia, Facoltà di Medicina e Chirurgia "A. Gemelli", Università Cattolica del Sacro Cuore, Roma, Italia); one swine intestinal tréponème, Serpulina hyodysenteriae strain ATCC 31287 (obtained from the American Type Culture Collection). All strains, except HRM-4 and ATCC 31287, were batch-cultured in GM-1 broth supplemented with 0.3% heat-inactivated bovine serum (1) or in Proteose- Trypticase-Yeast (PTY) medium (see Chapter 5). Isolates HRM-4 and ATCC 31287 were cultured on a blood-agar plate containing 25g Brain Heart infusion, 10g Bacto-
Peptone, 20g Bacto-Agar (all from Difco), lg KN03, lg Na-succinate, lg Na- formiate, lg Cystein-HC1.H20 (all from Merck), 5 mL haemin solution (0.1%), 1 mL Menadion-solution (0.05%), and 100 mL Sheep blood per litre medium. The haemin solution consisted of 100 mL KOH (0.1 M), 50 mL Ethanol (95%), 50 mL demin, and 200 mg haemin. The menadione solution consisted of 25 mg menadione in 50 mL ethanol (95%). All strains were cultured at 37°C in an anaerobic glove box (Coy, Ann Arbor,
Mich.) in a nitrogen atmosphere with 5% H2 and 4% C02 Samples of spirochetes
62 were obtained directly from the batch cultures or scraped from the blood-agar plate, suspended in MBS, pelleted by centrifugation for 5 min. at 4500 rpm. and resuspended in DMEM/F12 without antibiotics. Different strains were pooled into four mixtures. Mixture 1 consisted of the T. denticola laboratory strains В11, В12, S3, SI 1, Fl, and LI ID; mixture 2 consisted of the T. denticola ATCC strains 35404, 35405, and 33520; mixture 3 consisted of the two human intestinal spirochetes HRM-4 and HRM-14, and mixture 4 consisted of the swine intestinal tréponème, S. hyodysenteriae ATCC 31287.
- Invasion assay. Aliquots of 100 μι mixture (approximately 109spirochetes/mL) were inoculated into a sterile metal ring (diameter 0.6 cm), that was placed on the air-exposed tissue
cultures, and incubated 15 hours at 37°C in air containing 9% C02.
- Fixation and staining. After incubation, the tissue cultures were cautiously washed three times with PBS, immersed in 4% formaldehyde, dehydrated in a graded series of ethanol solutions, impregnated with xylol and embedded in paraffin. Serial sections of 7 μτη were prepared on a base sledge microtome. Sections selected ad random were deparaffined and immuno-stained as previously described (see Chapter 3), with the modification that in this assay a ten-fold concentration of the polyclonal antisera was used. The epithelial cultures were counter-stained with haematoxylin and eosine.
The stained sections were studied with the light microscope at phase contrast (magnification 1250x) for the presence of spirochetes attached to or invaded in the air-exposed cultures.
63 Results and discussion.
Light microscopy showed that seeding of the human foreskin keratinocytes on a dermal substrate and subsequently lifting the culture to the air-liquid interface, resulted in a reconstructed epidermis covered with a cornified layer after 20 days of culture (Figure 6.1). The epithelial cells seemed to have their characteristic appearances, ranging from columnar (basal cells) to completely flattened (corneocytes), resembling results obtained by others (2,16).
Figure 6.1: Vertical paraffin section stained with 'Л^ haematoxylin and eosin of air-exposed cultures of human foreskin keratino cytes grown for 20 days on de-epidermized dermis. (Magnification 200x). C: cornified layer; E: epi DED dermis; DED: de-epider mized dermis.
Tf
The investigated sections revealed that co-incubation of the reconstructed epidermis with the different mixtures of spirochetes resulted in a low number of superficially attached spirochetes in case of the T. denticola laboratory strains {mixture 1; Figure 6.2), and, in case of the human intestine spirochetes HRM-4 and HRM-14 (mixture 3; Figure 6.3). Only very incidental S. hyodysenteriae ATCC 31287 spirochetes were encountered on the reconstructed epidermis. No attachment was observed after co-incubation of the reconstructed epidermis with the T. denticola ATCC strains (mixture 2). Invasion of spirochetes was not observed in the investigated sections. Invasion of oral spirochetes was anticipated because in a previous study (14) we
64 isolated T. denticola and T. vincentii out of gingival biopsies of animal and human origin. In addition, Uitto et al. (28) reported the penetration of some T. denticola spirochetes in cultured porcine periodontal ligament epithelial cells. However, in the present model invasion of the applied spirochetes was not observed. This might be due to the abundant keratinization that developed in this model. The use of different culture techniques, like reduction of the calcium concentration in the culture medium in combination with the use of epithelial cells derived from non-keratinizing tissue, might reduce the abundant keratinization and create more favourable conditions for invasion of attached spirochetes. Moreover, variations in the incubation conditions, for example lower oxygen tensions, could induce spirochetal invasion. However, these alterations of the developed invasion model could not be pursued due to ending of the project.
Figure 6.2: A phase contrast light Figure 6.3: A phase contrast light microscopic view of T. denticola microscopic view of human intestine spirochetes (arrows; mixture 1) HRM-4 and HRM-14 spirochetes superficially attached to fragments of (arrows; mixture 3) superficially cultured epidermis. attached to fragments of de- (Magnification: 1320x). epidermized dermis. (Magnification: 1320x).
65 It is also possible that the applied spirochetes are incapable to invade. Riviere et al. (21) did not observe invasion of different cultivable Treponema species, including T. denticola and Treponema phagedenis, in their in vitro model. They did find, however (20,21,22), that dental plaque harvested from sites of periodontitis contains so called non-cultivable pathogen-related oral spirochetes (PROS) which are capable of penetrating and migrating through a mouse abdominal wall placed in a dialysis cell. The role of these PROS in periodontal disease remains to be elucidated.
To elucidate the mechanism of invasion of oral and non-oral spirochetes further research is necessary. In this context we initiated the development of an in vitro invasion model. The present study was primarily focused on the culture of a reconstructed epidermis and on recapture of treponemal species in combination with this multilayered tissue at the light microscopic level. Now these primary goals have been achieved, further exploration of culture and incubation conditions can be pursued.
Acknowledgements. We thank Kees Jansen (Department of Pathology, Medical Sciences, University of Nijmegen, Nijmegen, The Netherlands) for providing the human foreskins, and Jos Groenen, René van Rheden and Pia Helmich for their technical assistance.
66 References.
1. Blakemorc, R.P., and Canale-Parola. 1976. Arginine catabolism by Treponema denticola. J. Bacteriol. 128: 616-622. 2. Boddé, H.E., Holman, В., Spies, F., Wecrheim, Α., Kempenaar, J., Mommaas, M, and Ponec, M. 1990. Freeze-fracture electron microscopy of in vitro reconstructed human epidermis. J. Invest. Dermatol. 95: 108-116. 3. Carranza Jr., F.A., Saglie, R., Newman, M.G., and Valentin, P.L. 1983. Scanning and transmission electron microscopic study of tissue-invading microorganisms in localized juvenile periodontitis. J. Periodontol. 54: 598-617. 4. Courtois, G.J., Cobb, СМ., and Killoy, W.J. 1983. Acute necrotizing ulcerative gingivitis. A transmission electron microscope study. J. Periodontol. 54: 671- 679. 5. Grenier, D., Uitto, V-J., and McBride, B.C. 1990. Cellular location of a Treponema denticola chymotrypsinlike protease and importance of the protease in migration through the basement membrane. Infect. Immun. 58: 347-351. 6. Listgarten, Μ.Α. 1965. Electron microscopic observations on the bacterial flora of acute necrotizing ulcerative gingivitis. J. Periodontol. 36: 328-339. 7. Listgarten, M.A., and Lewis, D.W. 1967. The distribution of spirochetes in the lesion of acute necrotizing ulcerative gingivitis: an electron microscopic and statistical survey. J. Periodontol. 38: 379-386. 8. Loesche, W.J. 1988. The role of spirochetes in periodontal disease. Adv. Dent. Res. 2: 275- 283. 9. Makinen, K.K., Syed, S.A., Loesche, W.J., and Makincn, P.-L. 1988. Proteolytic profile of Treponema vincentii ATCC 35580 with special reference to collagenolytic and arginine aminopeptidase activity. Oral Microbiol. Immunol. 3: 121-128. 10. Manor, Α., Lebendiger, M., Shiffer, Α., and Tovel, Η. 1984. Bacterial invasion of periodontal tissues in advanced periodontitis in humans. J. Periodontol. 55: 567-573. 11. Mikx, F.H.M. 1991. Comparison of peptidase, glycosidase and esterase activities of oral and non-oral Treponema species. J. Gen. Microbiol. 137: 63- 68. 12. Mikx, F.H.M., and De Jong, M.H. 1987. Keratinolytic activity of cutaneous and oral bacteria. Infect. Immun. 55: 621-625. 13. Mikx, F.H.M., Maltha, J.C., van Campen G.J. 1990. Spirochetes in early lesions of necrotizing ulcerative gingivitis experimentally induced in beagles. Oral
67 Microbiol. Immunol. 5: 86-89. 14. Mikx, F.H.M., Maltha, J.C., and Keulers, R.A.C. 1989. Are there differences between oral treponema in dental plaque and in gingival tissue? J. Dent. Res. CED, 68: pp. 620, Abstr. no. 87. 15. Ohta, K., Makinen, K.K., and Loesche, W.J. 1986. Purification and characterization of an enzyme from Treponema denticola capable of hydrolyzing synthetic trypsin substrates. Infect. Immun. 53: 213-220. 16. Régnier, M., Pruniéras, M., and Woodley, D. 1981. Growth and differentiation of adult human epidermal cells on dermal substrates. Front. Matrix Biol. 9: 4- 35. 17. Reijntjens, F.M.J., Mikx, F.H.M., Wolters-Lutgerhorst, J.M.L., and Maltha, J.C. 1986. Adherence of oral tréponèmes and their effect on morphological damage and detachment of epithelial cells in vitro. Infect. Immun. 51: 642-647. 18. Rheinwald, J.G., and Green, II. 1975. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331-344. 19. Riviere, G.R., Thomas, D.D., and Cobb, CM. 1989. In vitro model of Treponema pallidum invasiveness. Infect. Immun. 57: 2267-2271. 20. Riviere, G.R., Wagoner, M.A., Sharon, Α., Baker-Zander, M.S., Weisz, K.S., Adams, D.F., Simonson, L.G., and Lukehart, S.A. 1991. Identification of spirochetes related to Treponema pallidum in necrotizing ulcerative gingivitis and chronic periodontitis. N. Engl. J. Med. 325: 539-543. 21. Riviere, G.R., Weisz, K.S., Adams, D.F., and Thomas, D.D. 1991. Pathogen- related oral spirochetes from dental plaque are invasive. Infect. Immun. 59: 3377-3380. 22. Riviere, G.R., Weisz, K.S., Simonson, L.G., and Lukehart, S.A. 1991. Pathogen- related spirochetes identified within gingival tissue from patients with acute necrotizing ulcerative gingivitis. Infect. Immun. 59: 2653-2657. 23. Siboo, R., Al-Joburi, W., Gornitsky, M., and Chan, E.C.S. 1989. Synthesis and secretion of phospholipase С by oral spirochetes. J. Clin. Microbiol. 27: 568- 570. 24. Simonson, L. G., Goodman, C.H., Bial, J. J., and Morton H.E. 1988. Quantitative relationship of Treponema denticola to severity of periodontal disease. Infect. Immun. 56: 726-728. 25. Simonson, L.G., Goodman, C.H., and Morton, H.E. 1990. Quantitative immunoassay of Treponema denticola serovar С in adult periodontitis. J. Clin. Microbiol. 28: 1493-1496.
68 26. Taylor Heylings, R. 1967. Electron microscopy of acute ulcerative gingivitis (Vincent's type). Br. Dent. J. 122: 51-56. 27. Thomas, D.D., Navab, M, Haake, D.A., Fogelman, A.M., Miller, J.N., and Lovett, M.A. 1988. Treponema pallidum invades intercellular junctions of endothelial cell monolayers. Proc. Natl. Acad. Sci. USA 85: 3608-3612. 28. Uitto, V.-J., Pan, Y.-M., and Firth, J. 1992. Development of an in vitro model for periodontal epithelium. J. Dent. Res. 71: pp. 605, IADR Abstract no. 714.
69
Chapter 7
General discussion Table of contents chapter 7 Page
7.1. Introduction 73
7.2. Inter-strain differences 74
7.3. Intra-strain differences 75
7.4. Non-uniform distribution 76
7.5. Attachment assay 78
7.6. Attachment mechanisms 78
7.7. Motility 80
7.8. Tip-associated attachment 81
7.9. Possible attachment models for T. denticola 82
7.10. Concluding remarks 83 7.1. Introduction.
Oral spirochetes are members of the periodontal microflora which is associated with periodontal disease (2,87,94,115). The main reason to consider oral spirochetes as putative pathogens is based on their abundant increase in number in the subgingival microflora at sites affected by periodontal destruction (146,147). Also the observed decrease in the number of spirochetes after successful treatment of periodontally affected sites (84) and the presence of antibodies against oral spirochetes in periodontal patients (17,62,71,104,162,163,168) indicate an association between these microorganisms and periodontal disease. Moreover, oral spirochetes have been identified within affected oral epithelial tissue, ahead of other types of invading oral bacteria (82,167). However, from the literature it is not possible to draw decisive conclusions on the role of oral spirochetes in the aetiology of periodontal disease. It should be realized that their increase and invasion at affected sites might be the result rather than the cause of the disease. In vivo, oral spirochetes arc mainly present in the outermost zone of the subgingival microflora, where they are in intimate contact with the sulcular or pocket epithelium (64,82,83,95,137). Attachment to these epithelial tissues might be one way by which oral spirochetes maintain in this area. Studies regarding the mechanism of attachment of oral spirochetes to epithelial tissue might, in the long run, result in methods to inhibit colonization and thereby prevent or limit periodontal disease. The main objective of the research described in this thesis was to obtain information about the attachment mechanism of oral spirochetes to cells of epithelial origin.
As the object of study Treponema denticola was chosen. This is the only cultured oral spirochete species with a positive correlation between its presence in subgingival dental plaque and periodontitis in humans (146,147). T. denticola is likely to be an
73 important pathogen because it has the potential to mediate tissue destraction by direct action against host cells and matrix proteins. T. denticola is cytotoxic for epithelial cells (3,4,132,178), suppresses fibroblastic (10,164) and endothelial (165) cell proliferation, induces actin rearrangements in fibroblasts (5), lyses human red blood cells (47), enhances bone resorption (44), and modulates the host responses of neutrophils (9,60,80,81,143,164) and lymphocytes (144,164). In addition, this oral spirochete possesses proteolytic (124) as well as fibrinolytic (121) and collagenolytic (101,176,179) enzymes and produces endotoxins (51,80,107,190) and a variety of potentially toxic metabolites (14,29,30,80,95).
7.2. Inter-strain differences.
The first goal of the present study was to establish attachment of T. denticola under in vitro conditions because at the time our studies were initiated, a controversy existed as to whether T. denticola was able or not to attach to epithelial cells in vitro. Olson (123) and Reijntjens et al. (132) described the in vitro attachment of T. denticola to epithelial cells, while Fitzgerald et al. (33,35) failed to find any attachment of this microorganism to a wide variety of cultured cell types. The second goal was to develop an assay for treponemal attachment to cells of epithelial origin. The development of such an assay was necessary because investigations on interactions between oral spirochetes and cultured epithelial cells were primarily focused on the detrimental effects of the spirochetes on these cells (4,123,132,178) and assays focused on the attachment of T. denticola to epithelial cells were lacking. Whether or not T. denticola was able to attach to epithelial cells was tested using ten different T. denticola strains and monolayers of keratinocytes derived from rat palatal epithelium, guinea pig ear, human buccal epithelium or human corneal epithelium {Chapter 2). These in vitro experiments clearly indicated the ability of T. denticola to attach to cells of epithelial origin. However, differences between the T. denticola strains were observed. These inter-strain differences were not only found for the attachment of T. denticola to cultured epithelial cells, but also in
74 hemagglutination experiments {Chapter 5). It was observed that some T. denticola strains attached good while others did to a lesser extend or not at all. These results indicated a heterogeneity in surface characteristics between the different strains. Several studies (20,49,106,180,184,190) have shown that different strains of T. denticola express different serological active proteins on their surface, but only Haapasalo et al. (49) and Weinberg and Holt (185) payed attention to the biological activity of these proteins and found that some are involved in the attachment process. Our studies revealed differences in attachment between three serologically different T. denticola strains ATCC 35404, ATCC 35405 and ATCC 33520. This might indicate that the three different serotypes have different, strain specific adhesins with immunological activity.
7.3. Intra-strain differences.
In addition to inter-strain differences, T. denticola displayed intra-strain differences in attachment. Microscopic observations at the end of incubation revealed many unattached spirochetes. This phenomenon was observed in all assays, using T. denticola with either epithelial cells or erythrocytes. The presence of many unattached spirochetes at titration endpoint in hemagglutination tests indicates the existence of a certain subpopulation of spirochetes able to attach while the remainder is not and indicates surface heterogeneity among the spirochetes within one T. denticola strain. Intra-strain treponemal surface heterogeneity was also observed at the ultrastructural level {Appendix). Both transmission and scanning electron microscopy revealed a Ruthenium-red positive layer on some, but not all, T. denticola ATCC 33520 cells in one assay. Involvement of Ruthenium-red positive material in the attachment of T. denticola (123) and T. pallidum (31,34) has been suggested. Although it is tempting to speculate that the intra-strain heterogeneity in respect to attachment might be related to the presence or absence of the Ruthenium-red positive layer, its origin as well as its role in attachment remains to be elucidated.
75 The existence of subpopulations with different surface characteristics might be a prerequisite for oral spirochetes to persist in an environment like the gingival crevice. In this environment bacteria are readily removed by the shedding of epithelial cells. Treponemal surface heterogeneity in combination with changes in cell surface characteristics in time might enable, at least part of the progeny, to colonize new available epithelial surfaces.
In addition to the inter-strain and intra-strain differences among the T. denticola strains, attachment experiments also revealed differences in the attachment of each strain to epithelial cells derived from different sources {Chapter 2). For instance, 7! denticola strain ATCC 35404 attached in relative high numbers to human buccal epithelial cells, to a lesser extend to rat palatal epithelial cells, and not at all to cells derived from guinea pig ear or human corneal epithelium. These differences in attachment indicate differences in the amount or structure of receptors expressed on the different epithelial cell surfaces. This issue was, however, not further pursued in this study.
7.4. Non-uniform distribution.
T. denticola did not attach to every epithelial cell in a certain monolayer and as a consequence attached spirochetes were non-uniformly distributed over the monolayers {Chapter 2). It is very unlikely that this non-uniform distribution of the spirochetes was caused by local differences in the density of the inoculum since microscopical examination prior to incubation showed no clumping or uneven distribution of the spirochetes in the inoculum. Therefore, it was concluded that the non-uniform distribution of the attached spirochetes was caused by a keratinocyte related factor. Uneven distributions of attached spirochetes were also observed by Fitzgerald et al. (33) and Wong (188,189) in their studies with T. pallidum. Though Fitzgerald et al. stated that differences in growth rates between cells were not involved in
76 enhanced attachment, Wong revealed more attachment of T. pallidum to actively growing cells than to quiescent ones. Also Olson (123) described a preference of T. denticola to attach to epithelial cells with a rounded appearance. It appears therefore that monolayers of epithelial cells are heterogeneous and that they contain subpopulations of keratinocytes which are more receptive for T. denticola attachment. In the first experiments, however, such distinct subpopulations of cells could not be distinguished in the monolayers at the light microscopic level. Substitution of the modified Warthin silver staining by an indirect immunohistochemical staining showed that T. denticola attached preferably to a subpopulation of "rounded-up" epithelial cells {Chapter J). Additional scanning electron microscopy revealed that the spirochetes attached in particular to microvilli of these rounded RPE cells (this thesis). As outlined by Erickson et al. (26) and Porter et al. (131), rounded RPE cells are probably in their mitotic phase because cells that grow in monolayers are flattened during interphase and rounded during mitosis. The rounding-up is accompanied by an increase in the number of microvilli, which might influence the attachment of T. denticola. Another important factor might be redistribution of cell membrane components during the cell cycle. Flattened inter-phase cells have an uneven distribution of cell membrane receptors because membrane components involved in attachment will be located mainly at the side of the cell which is in contact with the culture vessel. These membrane components are therefore not available as receptors for the attachment of tréponèmes. Transposition of cell membrane components during cell division might increase the amount of available receptors. This, as well as de novo synthesis of cell membrane components during late inter-phase (45), might increase the amount of cell membrane receptors available for T. denticola attachment. In context of the latter it is of interest that mitotic cells have been shown to express relatively more sialic acid than do cells in inter-phase (43) and that we presume sialic acid to play a role in the attachment of T. denticola to epithelial cells and erythrocytes {Chapters 4 and 5). It is, however, difficult to translate these in vitro results to the in vivo situation.
77 In vivo oral spirochetes are present in the vicinity of the junctional epithelium, a non- keratinizing multilayered tissue in which only the cells in the basal layer divide. Therefore the dividing cells themselves are not readily available for attachment of T. denticola. The junctional epithelium, however, contains only few cell layers and it is possible that the suprabasal cells of the junctional epithelium still express the receptors favouring treponemal attachment.
7.5. Attachment assay.
The preference of T. denticola ATCC 33520 to attach to rounded rat palatal epithelial (RPE) cells and the accidental possibility to distinguish these cells by an immunohistochemical staining, enabled the development of an assay in which treponemal factors involved in the attachment could be studied (Chapter 3). To enhance the reproducibility between experiments performed at different times, T. denticola ATCC 33520 was cultured in a continuous culture. First optimal conditions for the attachment of T. denticola ATCC 33520 to the rounded RPE cells were determined by studying the influence of incubation time, incubation temperature, and pH on attachment. This revealed that the attachment of T. denticola ATCC 33520 to the rounded RPE cells was optimal after an incubation period of 6 hours at 37°C and pH 7. To test the sensitivity of the assay, batch-cultured T. denticola strains ATCC 35405, Bl 1 and Ny541 were tested, using ATCC 33520 as a reference. The observed differences in attachment between different strains was in agreement with previously obtained results and indicated that the assay was sensitive enough to discriminate between T. denticola strains.
7.6. Attachment mechanisms.
Attachment of T. denticola ATCC 33520 depended on incubation time, incubation temperature and pH, indicating that attachment was not achieved by non-
78 selective mechanical trapping of the spirochetes. To characterise the treponemal factors involved in the attachment of T. denticola ATCC 33520 to rounded RPE cells, the spirochetes were pre-exposed to proteolytic enzymes, alkylation, heat, periodate or different sugars {Chapter 4). Analysis of these treatments on the attachment revealed that T. denticola ATCC 33520 attached to rounded RPE cells by means of a trypsin-resistant treponemal adhesin, probably of a protein and carbohydrate nature, which has an affinity for D- mannose, N-acetyl-D-galactosamine and sialic acid. Extension of the epithelial attachment studies to hemagglutination studies revealed a hemagglutinin at the treponemal surface of a protein and carbohydrate nature, which recognized sialic acid as a receptor (Chapter 5). Because the hemagglutinin appeared to be trypsin-sensitive and lacked affinity for D-mannose and N-acetyl-D-galactosamine, it is not likely that attachment to epithelial cells and hemagglutination are mediated by the same surface-located components, This again suggests heterogeneity among surface located components of T. denticola ATCC 33520 and the existence of specific attachment mechanisms. Specific mechanisms in the attachment of T. denticola have also been indicated by other authors. Weinberg and Holt (184,185) assumed the presence of lectin-like adhesins on the T. denticola surface with affinity for galactose and mannose. In strain MS25 it appeared to be treponemal major outer sheath proteins of 58 and 64 kDa which are probably involved in the attachment to human gingival fibroblasts. However, not all strains have the same proteins. For example, T. denticola strain ATCC 35404 (TD-4) and SR-4 contained only the outer sheath protein of 58 kDa. Specific attachment mechanisms by T. denticola have also been postulated by Haapasalo et al. (49,50) who studied the attachment of T. denticola ATCC 33405 to several proteins. Their data indicate that the attachment of T. denticola to different proteins is mediated by a sulfhydryl-containing, glycoprotcin-like adhesin with no affinity to galactose and mannose.
In addition to the attachment mediated by glycoprotein-like adhesins, surface-located
79 glycosaminoglycans might be involved. Glutaraldehyde fixation of T. denticola ATCC 33520 in the presence of Ruthenium-red indicated acid glycosaminoglycans on the surface of some, but not all, spirochetes (Appendix). Glycosaminoglycans are extremely water soluble and may be involved in the attachment of T. denticola ATCC 33520 since we observed a 50% reduction in hemagglutinating activity after washing of the spirochetes in PBS (Chapter 5). The periodate sensitivity of the attachment of T. denticola ATCC 33520 to rat palatal epithelial cells or erythrocytes might, in part, be explained by the involvement of glycosaminoglycans, since overnight exposure of spirochetes to sodium metaperiodate resulted in a collapse of the Ruthenium-red positive layer. Treponemal surface located glycosaminoglycans have been implicated in the attachment process of T. pallidum (31,34) and T. denticola (123). In the case of T. pallidum, these surface located glycosaminoglycans have been identified as being composed of hyaluronic acid, chondroitin sulfate, or glycosaminoglycans which are closely related to these two substances (31). Exposure of T. denticola to hyaluronidase indicated the presence of hyaluronic acid (123). We, however, did not observe an influence on the hemagglutinating activity after hyaluronidase or chondroitinase treatment of T. denticola ATCC 33520 (Chapter 5). Whether this difference is due to different incubation times [18 hours incubation (123) versus a 2 hours incubation in our experiment] has to be investigated.
7.7. Motility.
In none of our experiments any of the T. denticola strains, although alive, showed motility prior to incubation. Fitzgerald et al. (35) considered the motility of T. pallidum a prerequisite for attachment, as non-motile heat-treated spirochetes failed to attach to cultured cells. On the other hand, Cimasoni and McBride (18) observed abundant attachment of chloroform-treated non-motile T. denticola strains 51B2, CD-I, RitzA and LA-1 to apatite beads. Though in none of our experiments any of the T. denticola strains showed
80 motility prior to incubation, they did attach and the attachment was heat-sensitive. The fact that heat-killed T. pallidum failed to attach to cells (35) might therefore be explained by denaturation of adhesins rather than to non-motility.
7.8. Tip-associated attachment.
Data concerning tip-associated attachment of T. denticola are inconsistent. Our scanning electron microscopic studies revealed that T. denticola ATCC 33520 attached primarily at random points on the treponemal surface to the microvilli of the RPE cells {Chapter 2). However, Olson (123) observed that the T. denticola strains B2 and Tl attach preferably by their tips to epithelial cells, which is in accordance with the predominantly tip-associated attachment of the T. denticola strains ATCC 33520, ATCC 35404, ATCC 35405, b, d and e to fibronectin-coated coverslips as observed by Dawson and Ellen (23). On the other hand, Cimasoni and McBride (18) observed that besides tip-associated attachment, T. denticola CD-I, RitzA and LA-1 attached also along their whole length to apatite beads. Weinberg and Holt (184,185) also observed that attachment of T. denticola GM-1, MS25 and ATCC 35404 to human gingival fibroblasts was not exclusively tip-associated, but appeared to be at at random sites on the treponemal surface. Discrepancies in the treponemal attachment sites may partly be explained by experimental designs and T. denticola inter-strain differences. However, other factors may play a role. Cimasoni and McBride (18) observed tip-associated attachment more frequently in motile spirochetes than in non-motile spirochetes. Dawson and Ellen (23) observed a reduction of tip-associated attachment in less motile spirochetes after incubation at 4°C in comparison to incubation at 37°C. This is in accordance with the results obtained in our studies in which all spirochetes used, although freshly harvested from a continuous culture, were non-motile and the attachment was never obviously tip-associated.
81 7.9. Possible attachment models for T. denticola.
Based on studies performed so far, the following mechanisms regarding the in vitro attachment of the oral spirochete T. denticola may be proposed:
T. denticola attachment may be mediated by non-specific hydrophobic interactions. Weinberg and Holt (185) described the involvement of a hydrophobic major outer sheath protein of 64 kDa in the attachment of some T. denticola strains to human gingival fibroblasts. However, it is uncertain where the hydrophobic domains reside in the intact polypeptide and if they are part of the active sites for attachment. Attachment of T. denticola may be mediated by attractive electrostatic interactions. Cowan et al. (22) described the presence of two distinct pH- dependent electrophoretic mobilities within single cultures of T. denticola ATCC 33520. Adsorption of the spirochetes with an excess of erythrocytes removed most of the strongly negative subpopulation of spirochetes. They concluded that the strongly negative spirochetes are capable of attachment to erythrocytes and hypothesized that the interaction is mediated either between oppositely charged domains on the interacting surfaces or through the bridging of divalent cations between both the negatively charged spirochetes and erythrocytes. Attachment of T. denticola may be mediated by intermediary ligands like fibronectin. Studies performed by Haapasalo et al. (50), Dawson and Ellen (23), and Weinberg and Holt (184) revealed that T. denticola attaches to fibronectin. Because fibronectin also contains a cell-binding domain it might form a bridge between T. denticola and its substrate. Attachment of T. denticola may be mediated by surface-located (acid) glycosaminoglycans. Fixation of T. denticola ATCC 33520 in the presence of Ruthenium-red indicated acid glycosaminoglycans on the surface of some, but not all, spirochetes (Appendix). Since glycosaminoglycans are extremely water
82 soluble, their involvement might explain the 50% reduction in hemagglutinating activity after washing of T. denticola ATCC 33520 in PBS. Moreover, the observed periodate sensitivity in the attachment of T. denticola to rat palatal epithelial cells or erythrocytes might, in part, be explained by involvement of glycosaminoglycans, since overnight exposure of spirochetes to sodium metaperiodate appeared to cause the Ruthenium-red positive layer on the cellsurface to collapse. Attachment of T. denticola may be mediated through specific adhesin-receptor interactions. Different studies (47,49,50,184,185, Chapters 4 and 5) describe the involvement of a lectin-like, treponemal located adhesin of protein and carbohydrate nature, with different carbohydrate receptor specificity.
7.10. Concluding remarks.
Although different strains of T. denticola have many characteristics in common, differences are observed in the ability to attach and in motility which is a prerequisite for attachment. Moreover, there are differences in the way spirochetes attach. Therefore, experimental results obtained with specific strains of T. denticola should be evaluated with care. It is likely that the attachment of T. denticola is mediated through a combination of different mechanisms as outlined above. The existence of multiple attachment mechanisms offers a great advantage to oral spirochetes in vivo where they encounter different surfaces to colonize. On the other hand, the multitude of attachment mechanisms may frustrate the development of methods to prevent attachment of this microorganism to epithelial cells in the oral cavity.
For references see chapter 8.3.
83
Chapter 8
Summary Samenvatting References 8.1. Summary.
Periodontal disease finds its origin in the bacteria that form the dental, and in particular, the subgingival microflora. However, in the subgingival microflora of affected patients a wide range of organisms can be observed by dark-field or phase- contrast microscopy. This makes it difficult to "pin-point" periodontal disease to specific microorganisms or groups of microorganisms. Oral spirochetes are members of the periodontal microflora which is associated with periodontal disease. As outlined in detail in Chapter 1, the main reason to implicate oral spirochetes as putative pathogens is based on the increase of these microorganisms in the subgingival microflora at sites affected with periodontal disease. Also, the observed decrease in the number of spirochetes after successful treatment of periodontal affected sites, and the raised antibody levels against oral spirochetes in affected patients, speak for an association between these microorganisms and periodontal disease. Moreover, oral spirochetes have been identified within affected oral epithelial tissue. However, it is not yet known whether spirochetes contribute to the disease process or are simply opportunists exploiting the new anaerobic, nutrient-rich, ecological niche, created in the developing periodontal pocket. Therefore, it should be realized that their increase and invasion might be the result of the disease rather than the cause. Whether or not oral spirochetes are pathogenic, they are able to persist in the oral cavity. In vivo, oral spirochetes are mainly present in the outermost zone of the subgingival microflora. This positions them in intimate contact with the sulcular or pocket epithelium. Attachment to this oral tissue might be the way by which oral spirochetes retain themselves in this area. Therefore, studies into the mechanism of attachment of oral spirochetes to epithelial tissue might, in the long run, result in methods to inhibit colonization by these microorganisms and thereby prevent or limit periodontal disease. The main objective of the research described in this thesis was to obtain knowledge about the attachment of oral spirochetes to cells of epithelial
86 origin.
All oral spirochetes identified so far have been assigned to the genus Treponema. The best-known species associated with periodontal disease is Treponema denticola (T. denticola). This is a small spirochete which has been cultivated on a regular basis from pockets affected by periodontal disease. It is likely to be an important pathogen because it has the potential to mediate tissue destruction by direct action against host cells and matrix proteins. T. denticola is cytotoxic for epithelial cells, suppresses fibroblastic and endothelial cell proliferation, induces actin rearrangements in fibroblasts, lyses human erythrocytes, enhances bone resorption, and modulates the host responses of neutrophils and lymphocytes. In addition, this oral spirochete possesses proteolytic, fibrinolytic and collagenolytic enzymes and it produces a variety of potentially toxic metabolites. Moreover, T. denticola is until now the only cultured oral spirochete species of which a positive correlation has been found between numbers of a particular serotype of this microorganism in the subgingival microflora and severe periodontal disease.
To achieve information concerning the attachment of T. denticola to epithelial cells two primary goals were set: establishment of attachment of T. denticola under in vitro conditions, and development of an assay in which treponemal factors involved in attachment to epithelial cells can be studied. The first goal was necessary because at the onset of the studies described in this thesis there was discrepancy in the literature as to whether T. denticola was able to attach to epithelial cells in vitro. The second goal was set because investigations regarding the interaction between oral spirochetes and cultured epithelial cells were primarily focused on the detrimental effects of spirochetes on these cells, and assays focused on the attachment mechanisms of T. denticola to epithelial cells were lacking.
The first goal was approached by screening the degree of attachment of ten different T. denticola strains to monolayers of four types of epithelial cells derived
87 from rat palatal epithelium, guinea pig ear, human buccal epithelium or human corneal epithelium (Chapter 2). In this assay the attached T. denticola strains were stained with a modified Warthin silver staining. Screening of attached spirochetes was performed by phase-contrast microscopy. These in vitro experiments clearly indicate the ability of most T. denticola strains to attach to cells of epithelial origin. Because the monolayers were washed after incubation with the spirochetes, haphazard trapping of the spirochetes was eliminated. Additional scanning-electron microscopic studies with rat palatal epithelial cells revealed that the attachment of T. denticola was not primarily tip- associated. Most T. denticola spirochetes gave the impression of being attached to the microvilli of the epithelial cells at random points along the treponemal surface. Of interest are the differences that were observed between the studied T. denticola strains. Some T. denticola strains attached good where other strains attached to a lesser extend or not at all. These differences indicate surface- heterogeneity between different T. denticola strains. Besides inter-strain differences, T. denticola displayed intra-strain differences in attachment. Microscopical observations at the end of the incubation period revealed many unattached spirochetes. The presence of many unattached spirochetes indicated surface-heterogeneity among the spirochetes of one T. denticola strain. Another striking observation was the non-uniform distribution of attached spirochetes over the monolayers. This was not caused by clumping or uneven distribution of the spirochetes in the inoculum, as was shown by microscopical examination prior to incubation. It was concluded that the non-uniform distribution of the attached spirochetes over the monolayers indicated that monolayers of cultured epithelial cells are heterogenic in nature and contain a subpopulation of epithelial cells more receptive for the attachment of T. denticola. Quantification of the degree of attachment by actual counting confirmed differences in attachment between different strains. However, due to the non-uniform distribution of attached spirochetes, the number of attached spirochetes per cell per
88 microscopie field was very low and showed relative wide ranges.
The non-uniform distribution of attached spirochetes over the epithelial cell monolayers, the impossibility to distinguish the more receptive epithelial cells at the light microscopic level in the first assays, and the low number of attached spirochetes calculated per cell per microscopic field, urged the need for modification of the attachment assay {Chapter 3). Firstly, T. denticola ATCC 33520 was cultured in a continues culture and the attachment assay was pursued with a cell line originating from rat palatal epithelium, to enhance the reproducibility between experiments performed at different times. Secondly, the modified Warthin silver staining was replaced by an indirect immunohistochemical staining in which we used native rabbit polyclonal antisera directed against T. denticola ATCC 33520 and T. vincentii LA-1, to reduce background staining. By accident, it appeared that the applied immunohistochemical staining method enabled the distinction between two rat palatal epithelial cell populations in the monolayer at the light microscopic level, the one consisting of flattened and the other of rounded cells. The modified attachment assay clearly visualized that T. denticola attaches preferably to the rounded cells. The preference of attachment of T. denticola ATCC 33520 to these cells and the possibility to distinguish these cells by immunohistochemical staining, enabled study of the treponemal factors involved in the attachment of T. denticola. The optimal conditions for the attachment of T. denticola ATCC 33520 to the rounded rat palatal epithelial cells were determined by study the influence of the incubation time, incubation temperature and pH on the attachment process {Chapter 3). This revealed that the attachment of T. denticola ATCC 33520 to these cells is optimal after an incubation period of 6 h at 37 °C at pH 7. To test if the modified assay was sensitive enough to discriminate between the attachment levels of different strains, the assay was applied to three batch-cultured T. denticola strains ATCC 35405, Bl 1 and Ny541 with ATCC 33520 as a reference.
89 The assay revealed strain differences in attachment. Especially the poor attachment of strain Ny541 to rounded cells was in agreement with previous results (Chapter 2).
The dependence of the attachment of T. denticola ATCC 33520 to incubation time, incubation temperature, and pH, indicated that it is not established by means of aselect mechanical trapping of the spirochetes. Our next goal was to characterise the treponemal factors involved in the attachment of T. denticola ATCC 33520 to the rounded rat palatal epithelial cells. This was achieved by pretreatment of the spirochetes with several compounds, exerting different types of activities, and subsequent investigation of their effects on attachment (Chapter 4). These studies revealed the involvement of a heat-sensitive, trypsin resistant treponemal adhesin of a probably protein and carbohydrate nature with an affinity for D-mannose, N-acetyl-D-galactosamine, and sialic acid. Attachment studies of T. denticola ATCC 33520 to rounded rat palatal epithelial cells were extended by hemagglutinating studies (Chapter 5). These studies revealed a hemagglutinating activity for T. denticola which was cell-bound and growth-phase related. The heat-sensitive, treponemal haemagglutinin is probably of protein and carbohydrate nature and recognizes sialic acid as receptor. As the hemagglutinin appeared to be trypsin sensitive and as it lacked affinity for D- mannose and N-acetyl-D-galactosamine, it is not likely that attachment to epithelial cells and hemagglutination are mediated by the same surface located components. This again implicates heterogeneity in surface located components of T. denticola ATCC 33520 and the existence of specific attachment mechanisms.
Treponemal intra-strain surface heterogeneity was also observed at the ultrastructural level (Appendix). Both transmission and scanning electron microscopic studies revealed a Ruthenium red positive layer on some, but not all, T. denticola ATTC 33520 cells. Fixation of T. denticola with acid bovine albumin failed to reveal the presence of such a layer (Chapter 5). However, its origin as well
90 as its role in attachment processes remains to be elucidated.
Besides in dental plaque, spirochetes have been observed within gingival tissues at affected sites. Therefore we initiated the development of an in vitro model to study the putative invasion activity of spirochetes (Chapter 6). Co-incubation of cultured reconstructed epidermis with different oral and non-oral spirochetes resulted in a low number of superficially attached spirochetes. Invasion of spirochetes was not observed. Lack of invasion of the applied spirochetes might either be due to the abundant cornified layer that covered the reconstructed epidermis, invalid experimental conditions or incapability of the cultured spirochetes to invade. Termination of the project prohibited further exploration into this issue.
As may have become clear from the results described above, the use of different T. denticola strains (in combination with cultured tissue cells of different origin) results in variable outcomes regarding the in vitro attachment of these microorganisms. Though many characteristics may be general present among different strains of T. denticola, inter-strain differences are observed in the ability to attach. Due to these strain specific attachment of T. denticola, extrapolation of experimental results obtained with specific strains, to T. denticola in general, should be performed with caution.
91 8.2. Samenvatting
Orale spirocheten worden zeer vaak aangetroffen in de subgingivale microflora die geassocieerd is met tandvleesontstekingen. Zoals beschreven in Hoofdstuk 1, is het belangrijkste argument om orale spirocheten te beschouwen als mogelijk pathogeen gelegen in het feit dat hun aantal zeer sterk kan toenemen in de subgingivale microflora gerelateerd aan ontstoken plekken. Tevens blijkt dat succesvolle behandeling van tandvleesontstekingen gepaard gaat met een sterke afname in het aantal spirocheten in de subgingivale microflora. Bovendien blijken orale spirocheten in staat het tandvlees te invaderen. Tenslotte vertonen patiënten met tandvleesontstekingen een positieve immuunrespons tegen orale spirocheten wat duidt op een associatie tussen dit microorganisme en parodontale ontstekingen. Bovenstaande argumenten bewijzen echter de betrokkenheid van orale spirocheten bij de initiatie van tandvleesontstekingen niet. Het is ook mogelijk dat de toename van orale spirocheten in de subgingivale microflora en de invasie van deze microorganismen op aangetaste plaatsen het gevolg zijn van plaatselijke veranderingen in het milieu ten gevolge van de ziekte. Of ze nu wel of niet de oorzaak zijn van parodontale ontstekingen, orale spirocheten blijken in staat zich te handhaven nabij de parodontale weefsels en kunnen dus een rol spelen in de etiologie van deze ziekten. In vivo bevinden orale spirocheten zich voornamelijk in de periferie van de subgingivale microflora. Hierdoor staan ze in nauw contact met het sulcus- of pocketepitheel. Het is mogelijk dat hechting aan het epitheel de spirocheten in staat stellen zich in dit gebied te handhaven. Daarom zouden studies gericht op de hechting van orale spirocheten aan epitheelweefsels op langere termijn kunnen bijdragen tot het vinden van methoden om kolonisatie van orale weefsels door deze bacteriën te verhinderen en zodoende het ontstaan van ziekten van het parodontium te verminderen dan wel te voorkomen. Het onderzoek zoals beschreven in dit proefschrift is primair gericht op het verkrijgen van informatie omtrent de hechting van orale spirocheten aan cellen van epitheliale oorsprong.
92 Alle tot nu toe geïdentificeerde orale spirocheten behoren tot het genus Treponema. Treponema denticola is tot op heden de best onderzochte orale spirocheten-soort die geassocieerd wordt met parodontale ziekten. Dit microorganisme wordt zeer regelmatig geïsoleerd uit de subgingivale microflora afkomstig van aangetaste plaatsen. Bovendien is T. denticola tot nu toe de enige gekweekte soort waarvan een positieve correlatie is aangetoond tussen het aantal aanwezige spirocheten van een specifiek serotype in de subgingivale plaque en de mate van tandvleesontsteking.
T. denticola komt in aanmerking als mogelijk pathogeen omdat in vitro onderzoek heeft uitgewezen dat dit microorganisme in staat is weefsels aan te tasten door aan te grijpen op cellen of op eiwitten van de extracellulaire matrix. Zo blijkt T. denticola cytotoxisch voor epitheel cellen, onderdrukt dit microorganisme de proliferatie van fibroblasten en endotheelcellen, induceert het veranderingen in het actine-cytoskelet van fibroblasten, lyseert het humane erythrocyten, bevordert het botafbraak en moduleert dit microorganisme de respons van lymfocyten en neutrofiele granulocyten. Daarnaast is gebleken dat dit microorganisme verscheidene (potentieel) toxische metabolieten produceert en enzymen bezit met protease-, fibrinogenase- of collagenase-achtige activiteiten.
Om meer informatie te verkrijgen over de wijze waarop T. denticola aan epitheelcellen kan hechten, heeft het onderzoek zich in de eerste plaats gericht op de mogelijkheid om T. denticola onder in vitro omstandigheden te laten hechten aan epitheelcellen. Het volgende doel behelsde de ontwikkeling van een assay waarmee factoren die betrokken zijn bij de hechting van T. denticola aan epitheelcellen kon worden bestudeerd. Het eerste doel werd gesteld omdat op het moment dat ons onderzoek werd gestart het nog niet duidelijk was of Τ denticola in staat was te hechting aan epitheelcellen. De ontwikkeling van een hcchtingsmodcl was noodzakelijk omdat het onderzoek naar de interactie tussen orale spirocheten en epitheelcellen zich tot dan toe voornamelijk richtte op de schadelijke effecten van deze bacteriën op de cellen en niet op de hechtingsmechanismen die hieraan ten
93 grondslag liggen.
Het eerste doel is benaderd door de hechting te bestuderen van tien verschillende T. denticola stammen aan monolayers van epitheelcellen afkomstig van rat palatum-epitheel, cavia oor-epitheel, humaan wang-epitheel of humaan cornea- epitheel {Hoofdstuk 2). In deze tests werden de spirocheten na hechting gekleurd met een gemodificeerde zilver nitraat kleuring (volgens Warthin). De gehechte spirocheten werd gescoord d.m.v. fase-contrast microscopie. Uit deze in vitro experimenten bleek duidelijk dat T. denticola tot hechting aan epitheelcellen in staat is. Door de monolayers na incubatie met de spirocheten te wassen, werd het aselect "plakken" aan de cellen beperkt. Tevens gaf additioneel scanning electronen-microscopisch onderzoek indicaties dat de orale spirocheten hechten aan de microvilli van epitheelcellen. Deze interacties tussen spirocheet en microvilli vonden verspreid over de spirocheet plaats en waren niet beperkt tot de tip van de spirocheet. Interessant was de waarneming dat er verschillen waren tussen de verschillende T. denticola stammen met betrekking tot hun hechting aan de epitheelcellen. Sommige stammen hechtten goed terwijl andere stammen slechter of helemaal niet hechtten. Deze verschillen duidden op heterogeniteit in het oppervlak van de verschillende T. denticola stammen. Behalve een heterogeniteit tussen de stammen werd eveneens heterogeniteit binnen de stammen waargenomen. Bovendien waren de gehechte spirocheten niet homogeen over de monolayers verdeeld. Omdat microscopische inspectie van de inocula, voorafgaande aan de hechtingsassay, geen samenklontering of niet-homogene verdeling van de spirocheten liet zien, kan de niet-homogene verdeling niet worden geweten aan de verdeling van de spirocheten in de inocula. Waarschijnlijk spelen aan epitheelcel-gerelateerde factoren een rol. Echter, in de eerste hechtings-assays kon de niet-homogene verdeling van de spirocheten over de monolayers niet gerelateerd worden aan een op lichtmicroscopisch niveau onderscheidbare populatie epitheelcellen.
94 Ten gevolge van de niet-homogene verdeling bleek het aantal gehechte spirocheten berekend per epitheelcel erg laag met een relatief grote spreiding. De assay bleek daarom niet geschikt om aan spirocheet gerelateerde factoren betrokken bij de hechting te onderzoeken. Dit maakte modificatie van de tot dan toe gebruikte assays noodzakelijk {Hoofdstuk 3). Als eerste werd besloten de hechtingsassay te vervolgen met maar één stam nl. T. denticola ATCC 33520. Om de fysiologische condities van T. denticola zo constant mogelijk te houden werd een zgn. continue-culture opgezet. Bovendien is gekozen om te werken met een cellijn, nl. rat palatum epitheel. De tweede modificatie bestond uit het gebruik van een immunohistochemische kleuring in plaats van de gemodificeerde zilvemitraat kleuring volgens Warthin. Het bleek dat het gebruik van de immunohistochemische kleuring het ook mogelijk maakte om twee populaties in de monolayers te onderscheiden nl. platte en bolvormige cellen. De gemodificeerde hechtingsassay toonde duidelijk aan dat T. denticola ATCC 33520 bij voorkeur hechtte aan de bolvormige cellen. Hierdoor werd onderzoek naar de aan treponema gerelateerde factoren betrokken bij het hechtingsproces mogelijk. Allereerst werden de optimale condities bepaald waaronder T. denticola ATCC 33520 hecht aan de bolvormige rat palatum epitheelcellen. Het bleek dat optimale hechting plaats heeft na een incubatietijd van 6 uur bij 37 °C en pH 7. Daarnaast werd getest of de gemodificeerde hechtingsassay gevoelig genoeg was om verschillen in hechting tussen verschillende T. denticola stammen te registreren. Hiertoe werden drie batch-gekweekte T. denticola stammen, nl. ATCC 35405, Bil en Ny541, geïncubeerd met monolayers van rat palatum epitheelcellen. De mate van hechting van deze stammen bleek te verschillen. Vooral de matige hechting van stam Ny541 aan de bolvormige rat palatum epitheelcellen was overeenkomstig eerder gevonden resultaten (vergi. Hoofdstuk 2).
Het wassen van de monolayers na incubatie met de spirocheten tezamen met de invloed van de incubatietijd, de incubatietemperatuur en de pH op de hechting,
95 impliceerde dat de hechting niet het gevolg was van het aselect achterblijven van spirocheten op de cellen. De volgende stap bestond uit het karakteriseren van de aan treponema- gerelateerde factoren betrokken bij het hechtingsproces. Identificatie van deze factoren gebeurde door de spirocheten voor incubatie met de epitheelcellen verschillende behandelingen te laten ondergaan en het effect hiervan op de hechting te registreren {Hoofdstuk 4). Uit deze experimenten bleek dat T. denticola ATCC 33520 hecht aan de bolvormige rat palatum epitheelcellen door middel van een hitte- gevoelig, trypsine-resislcnt adhesine, waarschijnlijk van proteïne- en koolhydraatachtige signatuur, met een affiniteit voor D-mannose, N-acetyl-D- galactosamine en siaalzuur. Tenslotte werden naast de hechtingsexperimenten aan de bolvormige rat palatum epitheelcellen, hemagglutinatie experimenten uitgevoerd door bestudering van de hechting van T. denticola ATCC 33520 aan erythrocyten {Hoofdstuk 5). Hieruit bleek dat T. denticola tot agglutinatie van erythrocyten in staat is. Nader onderzoek leerde dat hemagglutinatie slechts plaats vond in de aanwezigheid van spirocheten en dat die afhankelijk was van de groeifase van de spirocheten. Het aan de spirocheet-gerelateerde hemagglutinine bleek hitte gevoelig en waarschijnlijk van eiwit- en koolhydraatachtige signatuur. Het vertoonde een affiniteit voor siaalzuur. Omdat het hemagglutinine gevoelig was voor het enzym trypsine en het geen affiniteit vertoonde voor D-mannose en N-acetyl-D-galactosamine, spelen waarschijnlijk specifieke adhesines een rol bij de hechting aan rat palatum epitheelcellen en erythrocyten. Dit impliceert dat er aan het oppervlak van T.denticola ATCC 33520 verschillende adhesines geëxposeerd worden met specifieke bindingsaffiniteiten.
Behalve de aanwezigheid van verschillende adhesines, beschikt een fractie van de T. denticola ATCC 33520 spirocheten over een "Ruthenium-rood positieve" buitenlaag {Appendix). Zowel transmissie- als scanning-electronenmicroscopisch onderzoek toonde deze Ruthenium-rood gevoelige buitenlaag aan op sommige
96 spirocheten. Na fixatie van T. denticola in de aanwezigheid van runder serum albumine (BSA) was deze laag niet waarneembaar (vergi. Hoofdstuk 5).
Orale spirocheten komen niet alleen voor in de subgingivale microflora nabij aangetaste plaatsen, maar zij zijn ook geïnvadeerd waargenomen. Om dit fenomeen nader te bestuderen werd de ontwikkeling van een in vitro model geïnitieerd {Hoofdstuk 6). Van enkele orale- en niet orale treponema stammen resulteerde de in vitro co-incubatie met gereconstrueerde epidermis in oppervlakkige hechting van een zeer laag aantal spirocheten. Invasie werd niet waargenomen. Het uitblijven van invasie door spirocheten in ons model zou gerelateerd kunnen zijn aan de uitbundige keratinisatie van de gereconstrueerde epidermis, aan de niet juiste experimentele omstandigheden gedurende de incubatie, of aan het onvermogen tot invasie van de door ons onderzochte spirocheten.
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112 Appendix
Ultrastructure of T. denticola ATCC 33520
Cowan M.M.', Keulers R.A.C.2, Mikx F.H.M.2, and Busscher H.J1.
' Laboratory for Materia Technics, University of Groningen, Groningen, The Netherlands " Division of Dentistry, Tnkon Research Program in Oral Microbiology, University of Nijmegen. Nijmegen, The Netherlands Introduction.
Attachment of spirochetes to substrata can be envisaged as an interaction between their outermost surface and the substrate. It is therefore of interest to identify the structures on their cell surface. Spirochetes possess an outer sheath, endoflagella and a protoplasmic cylinder (3,4,5). The outer sheath is the most external layer and envelops the endoflagella and protoplasmic cylinder. The outer sheath of many spirochete strains consists of an unit-membrane which carries a regular protein array resembling an S-layer within the outer leaflet of the membrane as opposed to superimposed on top of it (6). Thus, the outer leaflet of the outer membrane constitutes the outermost surface of the treponemal cell (5,8). No fimbrial appendages extending beyond this sheath have been described. Conversely, the presence of a slime layer external to the sheath structure has been suggested (3) and indirectly demonstrated (7). In this study transmission as well as scanning electron microscopy was used to investigate the surface of T. denticola ATCC 33520.
Materials and methods.
- Culture conditions of rat palatal epithelial cells and Treponema denticola ATCC 33520.
The rat palatal epithelialcells (RPE) were a gift of Dr. A. Arenholt (University of Arhus, Arhus, Denmark). The RPE cells were cultured aerobically (95% air and
5%C02) in Eagle Minimal Essential Medium (MEM) (Gibco, Breda, The Netherlands) with Earle's salts and 25mM Hepes buffer, supplemented with 6% fetal calf serum. The cells were grown on 13-mm circular glass coverslips placed in Petri dishes (diameter 6 cm), (Nunc, Roskilde, Denmark). Before the attachment assay, the growth medium was drained and the confluent monolayers were washed twice in
114 TC-Dulbecco's phosphate buffered saline (140 mM NaCl, 2.7 mM KCl, 8 mM
Na2HP04, 1.8 mM KH2P04, 1 mM CaCl2 and 1 mM MgCl2) (PBS, pH 7.2) and placed in multidish 24-well plates (Nunc, Roskilde, Denmark)
Treponema denticola ATCC 33520 was grown in serum-free proteose trypticase yeast broth (PTY) of the following composition: 10 g/L proteose peptone No.2 (Difco, Detroit, MI, USA), 5 g/L trypticase peptone (BBL, Cockeysville, MD, USA), 2,5 g/L KCl and 0,5 g/L L-cysteine HCl (Merck, Darmstadt, Germany). Medium was titrated to pH 7.0 with 1 N KOH and heat sterilized. After cooling a filter sterilized component of 5 mL 10% w/v NaHC03 , 0.025 g thiamine pyrophosphate (Sigma, Amsterdam, The Netherlands) and 5 mL volatile fatty acid (VFA) solution was added. The VFA solution contained 100 mL 0,1 N KOH and 0,5 mL iso-butyric acid, 0,5 mL DL-2-methyl butyric acid, 0,5 mL iso-valeric acid and 0,5 mL valeric acid (all from Merck). All cultures were first grown in continuous culture in a chemostat at pH 7.0,
1 Rh -532 mV, 37°C, a dilution rate of 0.04 h" and a nitrogen atmosphere with 5% H2 and 4% C02, resulting in an optical density at 550 nm of 0.650. Samples of spirochetes were transferred from the chemostat vessel to batch culture. Batch cultures were grown at 37°C in an anaerobic glove box (Coy, Ann Arbor, MI, USA) in a nitrogen atmosphere with 5% II2 and 4% C02. Early exponential phase cultures were harvested at 16 h, and stationary phase cultures at 80 h. Cultures were harvested by centrifugation at 10,000 χ g at 4°C for ten minutes and washed once and resuspended either in phosphate buffered saline
(pH 7.4) with 1 mM CaCl2 and 25 mM MgCl2 (MBS) or in 10 mM potassium phosphate buffer (pH 7.0). In some cases 80 h cultures of T. denticola were suspended in sodium acetate buffer (pH 4.5) and treated with 0.1 M sodium meta- periodate overnight at 4 °C. Controls were treated with buffer alone.
- Transmission electron microscopy.
Treponemal suspensions in water were placed on a carbon-coated grid and
115 stained with 2% (w/v) methylamine tungstate as described by Handley (2) to detect surface appendages. Ruthenium-red (RR) staining was performed as follows: bacterial cells were fixed in a mixture of 0.5 mL 3.6% (w/vol) glutaraldehyde, 0.5 mL 0.2 M cacodylate buffer (pH 6.5) and 0.5 mL RR (1.5 mg/mL in water) for 1 h on ice. After three washings in 0.07 M cacodylate buffer, cells were fixed in a 0.5 mL RR solution for 3 h at 27 °C. Cells were washed once in 0.07 M cacodylate buffer and suspended in 2% agarose in the same buffer. Samples were then dehydrated in a graded series of ethanol and embedded in Epon after which sections of approximately 70 nm were made. All preparations were viewed with a Philips 201 transmission electron microscope operating at 80 kV.
- Scanning electron microscopy.
Tréponème cultures were washed twice by centrifugation (1800g, 10 min) and resuspended in MBS. The pellet was then resuspended in Dulbecco's Essential Medium supplemented with Ham's F12 medium (DMEM/F12 3:1 v/v) (Flow Laboratories, Irvine, UK). One mL of the suspension, containing approximately 108 spirochetes per mL, was added to microtiter wells containing washed monolayers of rat palatal epithelialcells. Incubation took place for 5 hours in air, containing 7.5%
C02 and a relative humidity of 95%. Incubation was followed by washing of the monolayers three times in PBS, and fixation in 3% glutaraldehyde for 1 h at 4°C. After washing in PBS, the specimens were postfixed in 1% osmium tetroxide in 14 mM Pallade buffer (pH 7.4) for 16 h at 4°C. Some monolayers were fixed in the above fixatives supplemented with 5 mg/mL RR. All monolayers were dehydrated in a series of ethanol, followed by a graded series of ethanol/acetone and a critical point drying procedure with carbon dioxide. After drying, the monolayers were attached to copper stubs, sputtercoated with gold palladium and examined in a Phillips 500 scanning electron microscope at 15-25 kV.
116 Results.
Negatively stained whole cells of T. denticola ATCC 33520 display no fibrillar appendages (Figure 1A). The flagellar filaments and outer sheath characteristics of this species are clearly visible, as is the regularly patterned S-layer type material present within the outer sheath of the organism (Figure IB). Thin sections of RR-treated cells revealed a thin irregular layer of stained material outside the sheath of some cells which may be comprised partly of carbohydrate (Figure 1С). This layer was only visible on a small proportion of cells (see arrows). The remaining cells were bare. No ultrastructural differences were detectable among bacteria cultures of varying ages.
Figure 1: Transmission electron microscopy ofT. denticola ATCC 33520. A and B: Methylamine tungstate (2.0%) stained whole cells; C: Ruthenium-red stained thin sections. Arrows indicate RR-stained material. Bars represent 100 nm.
117 Scanning electron micrographs of RR-stained preparations of T. denticola ATCC 33520 attaching to rat palatal epithelial cells demonstrate the presence of an acidic polysaccharide distributed on the surfaces of some spirochetes (Figure 2A). This layer was not detected in case preparations were not treated with RR during fixation (Figure 2B).
Figure 2: Scanning electron microscopy ofT. denticola ATCC 33520 attached to rat palatal epithelial cells fixed in the presence of Ruthenium-red (A) or fixed without Ruthenium-red (B). Arrows indicate RR-stained material. Bar represents 10 micron.
Negative staining detected no difference between periodate-treated, buffer treated, and control cells of an 80 h culture (Figure ЗА). However, RR-stained thin sections of periodate treated cells revealed dark areas of collected stain on some cells indicating disruption in the RR-staining layer (Figure 3B).
118 Figure 3: Transmission electron microscopy of sodium meta-periodate treated T. denticola ATCC 33520. A. methylamine tungstate (2.0%) stained whole cells; B: Ruthenium-red stained thin sections. Arrow indicates RR-stained material. Bars represent 100 nm.
In conclusion.
This study used transmission as well as scanning electron microscopy to investigate the surface of T. denticola ATCC 33520. Cultures of pure T. denticola ATCC 33520 present two ultrastructural distinct kinds of surfaces to their environment. Both transmission and scanning electron micrographs revealed a RR- positive layer present on some, but not all, T. denticola cells. Sodium meta-periodate treatment appeared to collapse the RR-staining layer on the cell surface.
119 References.
1. Cockayne, Α., Sanger, R., Ivic, A, Strugnell, R.A., MacDougallJ.H. , Russell, R.B.B., and Penn, C.W. 1989. Antigenic and structural analysis of Treponema denticela. J. Gen. Microbiol. 135: 3209-3218. 2. Handley, P.S. 1991. Negative staining. In: Mozes, N., Handley, P.S., Busscher, HJ., and Rouset, P.G. (eds.), Microbial cell surface analysis - structural and physicochemical methods. VCH Publishers Inc., New York. pp. 63-87 3. Holt, S.C. 1978. Anatomy and chemistry of spirochetes. Microbiol. Rev. 42: 114- 160. 4. Johnson, R.C. 1977. The spirochetes. Ann. Rev. Microbiol. 31: 89-106. 5. Listgarten, M.A., and Socransky, S.S. 1964. Electron microscopy of axial fibrils, outer envelope, and cell division of certain oral spirochetes. J. Bacteriol. 88: 1087-1103. 6. Masuda, K., and Kawata, T. 1982. Isolation, properties and reassembly of outer sheath carrying a polygonal array from an oral tréponème. J. Bacteriol. 150: 1405-1413. 7. Olson, I. 1984. Attachment of Treponema denticela to cultured human epithelial cells. Scand. J. Dent. Res. 92: 55-63. 8. Yotis, W.W., Sharma, V.K., Gopalsami, C, Chegini, S., McNulty, J., Hoerman, K, Keen Jr., J., and Simonson, L.G. 1991. Biochemical properties of the outer membrane oí Treponema denticola. J. Clin. Microbiol. 29: 1397-1406.
120 Dankwoord.
In de jaren dat ik aan dit proefschrift heb gewerkt (en dat waren er nogal wat), heb ik het genoegen gehad om met verschillende mensen samen te werken. Al deze personen hebben, ieder op hun eigen wijze, aan de totstandkoming van dit proef schrift een bijdrage geleverd. Aangezien het onmogelijk is om iedereen persoonlijk in dit dankwoord te vermelden, wil ik hierbij diegene wiens naam hier niet vermeld wordt als eerste hartelijk bedanken. Ik ga ervan uit dat wie de schoen past deze zelf wel kan aantrekken. Enkele van hen wil ik echter in het bijzonder hier noemen en bedanken. Mijn promotor Prof. Dr. Klaus König en co-promotores Dr. Frans Mikx en Dr. Jaap Maltha. Frans en Jaap, de paar regels in dit dankwoord zijn niet representatief voor jullie bijdrage aan dit proefschrift, maar toch. Ik dank jullie voor jullie geduld bij het bespreken van de manuscripten, de stimulerende opmerkingen en de vrijheid die jullie mij geboden hebben gedurende het onderzoek. Dr. An Wolters-Lutgerhorst. An, ik dank je voor de belangstelling voor het werk, je bijdragen aan de discussies over de experimenten en het kritisch lezen van de manuscripten. Servaas, Pia, René, Lony en Thea. Jullie bedankt voor jullie collegialiteit, hulp, belangstelling en de 'crypto-koffie/thee pauzes'. "Cue-boy" René, naast het werk denk ik met plezier terug aan de met bier doordrenkte snookeravonden en onze gesprekken over muziek en het lab. De stagiaires Birgitte, William, Alphons en Karin. Vooral de hoeveelheid bollen die jij, Karin, gescoord hebt, hebben een belangrijke bijdrage geleverd aan het onderzoek. Ik ben blij dat je behalve werkervaring ook nog iets anders van de afdeling hebt opgepikt. Nu we het toch over Kees hebben, ook jou en de andere medewerkers van preventieve tandheelkunde t.w. Peter, Thijs, Pieter, Hans en Jan ben ik zeer erkente lijk.
121 De medewerkers van Dermatologie. Jullie waren voortreffelijke buren, humorvol en altijd tot helpen bereid. Zonder de anderen tekort te doen wil ik met name Gijs bedanken voor het delen van zijn ongelooflijke kennis omtrent het kweken van cellen. Hans Smits bedank ik hartelijk voor zijn hulp tijdens het gebruik van de electronenmicroscoop. Jos Groenen voor het opzetten van het gereconstrueerd epidermis-model. Kees Jansen voor zijn bemiddelende rol bij de levering van de voorhuidjes. Sjoerd Rijpkema voor de antisera. Hans, Louis en Roos voor hun hulp in de bibliotheek. Dr. Martin van 't Hof voor de statistische verwerking van de gegevens. Dr. Kelly Cowan en Dr. Henk Busscher voor hetgeen beschreven in de appendix. Mijn ex-huisgenoten Sjaak, Silke, Genie en Irma. Een aparte vermelding voor jullie, Sjaak en Silke, is hier zeker op zijn plaats. Ik denk dat jullie door de jaren heen nog wel het meest betrokken zijn geweest bij het wel en wee van ondergetekende. Ik denk dan ook met veel genoegen terug aan onze tijd op de Parkweg en dank jullie voor jullie steun, interesse en nog veel meer (wat hier verder geen vermelding behoeft). Boudewijn, voor jouw geldt in hoge mate hetzelfde en ik vind het dan ook zeer leuk dat Sjaak en jij als mijn paranimfen bij het geheel betrokken zijn. Arko "parko de pitbull van de parkweg" die regelmatig kwam zeuren dat hij weer uit wilde en er zodoende mede verantwoordelijk voor was dat ik tussen het schrijven en lezen door een frisse neus haalde. "Last but not least" mijn ouders, familie en vrienden. Ik beschouw mijzelf een bevoorrecht mens dat ik omringt ben door zulke fijne mensen, ¡tol Leiden, 25 maart 1995.
122 Curriculum vitae.
4 aprii 1957: Geboren te Geertruidenberg
1969-1976: VWO aan het Boschveldcollege te Venray
1976-1985: Studie biologie aan de Katholieke Universiteit van Nijmegen met als doctoraal programma: Ontwikkelingsbiologie der Dieren (hoofdvak), Biochemie en Fysiologische Chemie (bijvakken) Kandidaats-examen: 31 maart 1981 Doctoraal-examen: 25 juni 1985
1987-1991: Als assistent in opleiding de hechting onderzocht van orale spi- rocheten, in het bijzonder T. denticola ATCC 33520, aan cellen van epitheliale oorsprong en erythrocyten. Dit onderzoek vond plaats in de laboratoria van de vakgroep Periodontologie en Preventieve Tandheelkunde, en de vakgroep Orthodontie en Orale Histologie, Tandheelkunde, Faculteit der Geneeskunde en Tandheelkunde, Katholieke Universiteit te Nijmegen
1993-heden: Als wetenschappelijk medewerker verbonden aan het Laboratorium voor Stralengenetica en Chemische Mutagenese, Rijksuniversiteit Leiden. Hier wordt, onder de auspiciën van Dr. A.D. Tates, samen met de analiste Angélique de Roon, het in vitro clastogene effect van ultraviolette-B straling op huidcellen bestudeerd (EEG-contract no: EV-5-VCT-910034).
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