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Strongyloides venezuelensis:Adhesion of Adult Worms to Culture Vessels by Orally Secreted Mucosubstances
EXPERIMENTAL PARASITOLOGY ARTICLE NO. PR964100
85, 10–15 (1997)
Strongyloides venezuelensis: Adhesion of Adult Worms to Culture Vessels by Orally Secreted Mucosubstances HARUHIKO MARUYAMA
AND
YUKUFUMI NAWA
Department of Parasitology, Miyazaki Medical College, Miyazaki 889-16, Japan MARUYAMA, H., AND NAWA, Y. 1996. Strongyloides venezuelensis: Adhesion of adult worms to culture vessels by orally secreted mucosubstances. Experimental Parasitology 85, 10–15. Adults worms of Strongyloides venezuelensis were cultured in vitro. After overnight incubation, about 60% of the worms adhered firmly to the bottom of culture vessels by secreting adhesive substances from the mouth. A single worm produced 24.5 ± 10.1 of the adhesion spots overnight. When they were transferred to new culture vessels, they still produced new spots comparable to those produced for first 24 hr. The adhesion spots were positively stained with Coomassie brilliant blue and also with mucicarmine, periodic acid–Schiff, and alcian blue, pH 2.5, but not with alcian blue, pH 0.3, indicating their glycoprotein nature. The substances were amorphous and did not contain cells or nuclei. Histologic staining with a panel of lectins showed that the adhesive substances were rich in mannose, N-acetyl galactosamine, and N-acetyl glucosamine, but devoid of sialic acid. These characteristics were distinct from those of jejunal goblet cell mucins of rats. Adhesive substances contained antigenic components recognized by sera from infected rats. Thus, the adhesive substances secreted from the mouth of S. venezuelensis were clearly of parasite origin. We consider the production/secretion of the adhesive substances by S. venezuelensis adult worms a key step for the parasites to invade and establish the host epithelial layer. © 1997 Academic Press INDEX DESCRIPTORS: Strongyloides venezuelensis; parasitic; adhesion; mucosubstances; lectin.
INTRODUCTION
the host mucosa are poorly understood. Strongyloides venezuelensis adult worms parasitize in between intestinal villi, close to the surface of the mucosa (Wertheim 1970). They invade the intercellular space of the epithelial cells and move actively between adjoining cells. They leave tunnels as they migrate through the epithelium (Dawkins et al. 1983). Compared to such morphological study on the behavior of parasites, functional approaches on the establishment of parasites have not been fully explored. The aim of the present study is to investigate the adhesion of S. venezuelensis in vitro. When S. venezuelensis adult worms were cultured in vitro, they adhered firmly to the bottom of the culture vessels by secreting mucinous adhesive substances from their mouths. We presume that these adhesive substances could be key materials for the parasites to attach to the host intestinal mucosa upon the invasion of the epithelial layer.
Establishment of an infectious agent in a host depends on attributes of both pathogen and host organisms. To understand the host–parasite interface, both parasite-derived and host-derived components should be analyzed. In the case of intestinal helminth infections, immune-mediated mucosal responses of the host have been fairly well segregated into regulatory and effector systems at a cellular level (Wakelin 1989). Our recent series of work revealed that at least two distinct effector mechanisms are operating selectively against Nippostrongylus brasiliensis and Strongyloides spp., goblet cells to the former and mucosal mast cells to the latter (Nawa et al. 1994). In Strongyloides infection in mice and rats, intestinal mastocytosis was observed and a substantial portion of the mast cells migrate into the epithelial layer around the time of immune-mediated expulsion, so that mastcell-derived substances are considered the effector molecules. In contrast to the detailed studies on host mucosal responses, strategies of the parasites as to how they reach and remain in
MATERIALS
10 0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
AND
METHODS
Animals and parasites. Male Wistar rats 8 weeks old were purchased from Kyudo Co. (Kumamoto, Japan). The
ADHESIVE SUBSTANCES PRODUCED BY S. venezuelensis used in this study was kindly supplied by Professor Y. Sato, Department of Parasitology, School of Medicine, University of the Ryukyus, and maintained in our laboratory by serial passage in Wistar rats. Third-stage infective larvae (L3) were obtained from fecal culture by the filter paper method as previously described (Sato and Toma 1990). Animals were infected by subcutaneous inoculation with 20,000 L3 for maintenance and recovery of adult worms. Adult worms were obtained from the intestine of Wistar rats at 8 days postinfection. In brief, the upper 2⁄3 of the small intestine was removed from infected rats, cut open longitudinally, and incubated at 37°C in phosphate-buffered saline (PBS) for 1 hr. The worms migrated out from the intestine, were extensively washed with sterile PBS, and adjusted to the required concentration. In vitro culture of adult worms. As a preliminary experiment, we examined the capability of S. venezuelensis adult worms to adhere to various plastic and glass culture wares after incubation at 37°C overnight in PBS. The culture wares examined were 25-cm2 culture flasks (Nunc, Roskilde, Denmark; Corning, Inc., Corning, NY), a 96-well cell culture plate (Nunc, Sumitomo, Tokyo, Japan), glass chamber slides (Lab-Tek chamber, Nunc), and a polypropylene tube (Nunc). Subsequently, we used 96-well cell culture plates (Nunc) for the following experiments and adult worms were plated at a density of 70 ± 10 worms/well, except as otherwise stated. At the end of the incubation, wells were vigorously washed with PBS and then ethanol was added to the wells for histochemical examinations or colorimetric assays. Histochemical examinations. To characterize histochemically the nature of adhesive substances produced by S. venezuelensis adult worms, they (70 ± 10 worms/well) were cultured in eight-well chamber slides in PBS at 37°C overnight. After incubation, the chambers were removed and the slides were washed with PBS and stained with Coomassie brilliant blue for protein, with mucicarmine, which stains mucin in red, and periodic acid–Schiff (PAS) followed by alcian blue, pH 2.5, which stains acid mucopolysaccharide in blue, neutral mucin in red to red purple, and sialomucin in red purple to blue. Adhesive substances were also stained with alcian blue, pH. 0.3, alone, which stains only sulfated mucosubstances. Binding to lectins. The biotinylated lectins used in the present study were as follows: Agaricus bisporus agglutinin (ABA), Jack bean agglutinin (Con A), Grifonia simplicifolia agglutinin-II (GSA-II), Helix pomatia agglutinin (HPA), Maackia amurensis agglutinin (MAA), Sambucus sieboldiana agglutinin (SSA), and Ulex europaenus agglutinin-I (UEA-I). HPA and GSA-II were purchased from Sigma (St. Louis, MO) and E-Y Laboratories, Inc. (San Mateo, CA), respectively. All other lectins were from Hohnen Co. (Tokyo, Japan). The known saccharides specifically recognized by them were given in Table I in the results. S. venezuelensis adult worms (about 70 worms/well) were cultured in PBS in 96-well microculture plates at 37°C overnight. Wells were extensively washed with PBS to remove
S. venezuelensis ADULT WORMS
11
TABLE I Binding of Lectins to Rat Goblet Cell Mucins and Adhesion Spots
Lectin
Sugar specificity
Goblet cell mucins
Adhesion spots
ABA Con A GSA-II HPA MAA SSA UEA-I
Galb1 → 3Gal NAc a Man GlcNAc aGalNAc Siaa2 → 3Gal Siaa2 → 6Gal aFuc
±∼+ − + − − ++ −
+ ++ + ++ − − −
Note. Abbreviations: Gal, galactose; Man, mannose; GlcNAc, N-acetyl glucosamine; GalNAc, N-acetyl galactosamine; Sia, sialic acid; Fuc, fucose.
adult worms, fixed with ethanol, and then were added with methanol containing 0.3% H2O2 to block endogenous peroxidase activities. After washing with PBS, wells were incubated with PBS containing 1% bovine serum albumin for 2 hr at room temperature, washed, and then incubated with biotinylated lectins for 1 hr. Wells were then incubated with horseradish peroxidase (HRP)-conjugated streptavidin (GIBCO BRL, Gaithersburg, MD) for 1 hr. Substrate ABTS (2,29-azino-di[3-ethyl-benzthiazoline sulfonate] ) was added after washing and optical densities were read in a Labsystems Multiskan Bichromatic (Labosystems Oy, Helsinki, Finland). To demonstrate binding of lectins to adhesive substances histochemically, chamber slides prepared as above were stained with lectins in the same manner for the colorimetric assay except that metal-enhanced DAB (Pierce, Rockford, IL) was employed instead of ABTS. As the reference of lectin binding, tissue sections of rat small intestines fixed in Carnoy’s fluid were stained in parallel with the slides of the parasite-derived adhesive substances. Enzyme-linked immunosorbent assay (ELISA). Binding of rat sera to the the adhesive substances produced by S. venezuelensis adult worms was tested in ELISA. After incubation with adult worms at 37°C overnight, the cell culture plates were fixed in ethanol and blocked for endogenous peroxidase activity. Then various concentrations of sera obtained from S. venezuelensis-infected and uninfected rats were added to the wells. Wells were then washed and HRP-conjugated anti-rat immunoglobulins (PO450, DAKO, Glostrup, Denmark) were added. After incubation for 1 hr and washing three items, ABTS was added and optical densities were read.
RESULTS Adhesion of S. venezuelensis adult worms to culture vessels. When adult worms of S. venezuelensis were cultured in vitro, they adhered
12
MARUYAMA AND NAWA
firmly to the bottom of glass as well as plastic culture flasks and dishes of almost all types except for the polypropylene tube. After overnight incubation in chamber slides, the chambers were gently washed with PBS. About 60% of S. venezuelensis adult worms adhered to the glass surface by secreting adhesive substances from the mouth, more than 95% of them actively moving (Figs. 1A and 1B). In addition to the actual binding sites, numerous spots of adhesive substances without worms were observed, indicating that the worms attached and detached repeatedly during culture. In fact, when a single worm was placed in a well of 96-well culture plate and incubated overnight, the average number of adhesive spots produced was 24.5 ± 10.1 in 14 hr. They were able to make new spots after 24 hr of culture as well as in the first 24 hr. It was likely that they could mark spots as long as they were alive. There was no noticeable difference among adhesion spots made by worms collected on different days of infection. Characterization of adhesive substances. Af-
FIG. 1. Adhesion of S. venezuelensis adult worms by mucosubstances secreted from the mouth.
ter adult worms were washed off by vigorous pipetting with PBS, the secreted substances remained on the bottom were positively stained with Coomassie brilliant blue with mucicarmine, PAS, and alcian blue, pH 2.5, but not with alcian blue, pH 0.3, indicating their glycoprotein nature. The substances were amorphous and neither cells nor nuclei were observed within them even after staining. Thus, adhesive substances were of extracellular materials which were considerably glycosylated with some acidic sugar residues. Since general staining properties of the adhesive substances were similar to those of rat goblet cell mucins, there was a possibility that these mucinous substances were ingested and excreted goblet cell mucins. To test this point, the nature of adhesive substances was further characterized by binding to a panel of biotinylated lectins. The results were summarized in Table I. From these binding profiles, the adhesive substances were rich in mannose, N-acetyl galactosamine, and Nacetyl glucosamine, but devoid of sialic acids. They were distinct from rat intestinal goblet cell mucins, suggesting that they were of parasite origin. Typical shapes of adhesive substances stained by HPA are shown in Fig. 2. We next quantified the amount of adhesion spots in a lectin binding assay using biotinylated HPA. When different numbers of adult worms were cultured and the binding of HPA to the adhesive substances was measured, a good correlation [y 4 0.444 log(x) + 0.154; r 4
FIG. 2. Adhesion spots produced by S. venezuelensis adult worms. Adhesion spots were stained with biotinylated HPA, followed by HRP-conjugated streptavidin.
ADHESIVE SUBSTANCES PRODUCED BY
0.935] was obtained between the number of worms and the optical densities (Fig. 3). Adult S. venezuelensis worms seemed to produce adhesive substances continuously, because when they were kept in PBS in a nonadhesive polypropylene tube for 2 hr and then transferred into a 96-well culture plate, substantial amounts of adhesive substances were detected (Fig. 4). Production/excretion was thought to be metabolically active processes because adhesive substances were not produced when worms were incubated at 4°C (Fig. 4). Antigenicity of adhesive substances. To confirm further that adhesive substances were of parasite origin, we tested the binding of rat sera to the substance in ELISA. Adult worms were cultured in PBS overnight at 37°C in wells of microtiter plates. Wells were then washed to remove the worms and then S. venezuelensisinfected and uninfected rat sera were added after blocking of endogenous peroxidase activities and nonspecific binding. As shown in Fig. 5, sera from infected, but not uninfected, animals significantly bound to adhesive substances. Thus, adhesive substances contain components immunogenic in rats, indicating again that they were of parasite origin. While the worm expulsion occurred between 3 and 4 weeks postinfection (data not shown), the anti-
FIG. 3. Correlation between the number of worms added per well and the amount of adhesion substances produced by the worms.
S. venezuelensis ADULT WORMS
13
FIG. 4. Comparison of production of adhesive substances under different conditions of culture. Adult worms (∼70/ well) were cultured in wells of microtiter plates and the amount of adhesive substances was measured in a lectin binding assay with HPA. Each bar represents the mean ± SD of triplicate assays. P < 0.01.
body titer against adhesive substances began to rise in 2 weeks and reached a peak in 6 weeks postinfection. DISCUSSION The gastrointestinal tract is undoubtedly the most favored niche for adult metazoan parasites represented by the digenetic trematodes, cestodes, nematodes, and acanthocephalans (Mettrick and Podesta 1974). While the great
FIG. 5. Binding of rat sera to adhesion spots. Rats were infected with 20,000 infective larvae of S. venezuelensis. Sera were collected before and after infection and binding of the sera (diluted at 1 to 25,000) to adhesion spots was measured in ELISA.
14
MARUYAMA AND NAWA
majority of platyhelminths and larger nematodes such as Ascaris reside in the intestinal lumen, smaller nematodes tend to invade mucosa at various degrees. Capillaria philippinensis, for example, burrow in the mucosa of the small intestine repeatedly, causing severe tissue damage and violent diarrhea (Cross et al. 1978; Nawa et al. 1988). Trichuris also invade mucosa. Strongyloides adult worms locate between intestinal villi, invade the epithelial layer, and move actively between adjoining epithelial cells. They leave tunnels as they migrate through the epithelium (Dawkins et al. 1983). They do not penetrate the lamina propria across the basement membrane. It is suggested that they move in and out of the epithelial layer repeatedly over the course of infection (Dawkins 1989). In the present study, we showed that S. venezuelensis adult worms adhered to the surface of culture vessels by orally secreted adherent substances (Fig. 1). Amorphous material projecting from the buccal capsule was previously noticed in adult Strongyloides ratti, though it was interpreted as immunoglobulins from the host (Speare 1989). Our results of histochemical study on adhesive spots including the lectin binding profile (Table I) and immunogenicity in rats (Fig. 5) indicate that the substances were clearly of parasite origin and have a glycoprotein nature. The substances were rich in carbohydrates, especially mannose and N-acetyl galactosamine, although they were devoid of sialic acid (Table I). The lack of sialic acid might be a common characteristic of nematodes (Bacic et al. 1990; Borgonie et al. 1994). The substances seemed to be produced through active metabolic processes (Fig. 4). Although the secretion of adhesive substances in vivo has yet to be demonstrated, it is likely that ‘‘a fibrillar– granular electron-dense deposit’’ between the cuticle of S. ratti adult worms and the surrounding intestinal cells observed in the electron micrograph (Dawkins et al. 1983) could possibly be the adhesive substances we described here. Attachment to the surface of the host is one of the logically required steps for the parasite in the invasion process (Dubremetz and McKer-
row 1995). In Schistosoma mansoni infection, cercariae emit sticky mucus secretions from the posterior acetabular glands, which allow them to attach to and penetrate skin (Stirewalt 1974). Cercarial glands contain sialomucin (Stirewalt and Walter 1973), which binds to lectins such as wheat germ agglutinin (Linder 1986). Interestingly, cercariae leave tracks of ‘‘footprints’’ or ‘‘kissing marks’’ on the lipid-coated glass surface (Linder 1986), as we observed with S. venezuelensis adults (Fig. 2). Since S. venezuelensis adult worms invade the epithelial layer repeatedly during infection (Dawkins 1989), any mucosal change that affects attachment of S. venezuelensis adult worms to epithelial cells and/or subsequent entry should result in a decrease in infectivity or worm expulsion. In this regard, at around the time of Strongyloides expulsion from mice and rats, mucosal mast cells proliferate and migrate into the epithelium, where they are supposed to release effector molecules for worm expulsion (Nawa et al. 1994). Ishikawa et al. (1995) reported that reserpine-treated rats gained resistance against implantation of S. venezuelensis adult worms and concluded that reserpineinduced sulfated goblet cell mucins were responsible for their resistance. Related to this point, hamsters having highly sulfated goblet cell mucins could expel S. venezuelensis more quickly than other species of hamsters having less sulfated goblet cell mucins (Shi et al. 1994). Therefore, it is conceivable that highly sulfated mast-cell-derived proteoglycans and/or goblet cell mucins on the surfaces of epithelial cells might prevent attachment of S. venezuelensis and subsequent invasion. Examination of the effects of mast-cell-derived mediators and sulfated mucins on the adhesion of S. venezuelensis should cast a new light on the mechanism of worm expulsion. Whether the worm-derived mucosubstances could induce protective immunity or could protect worms from being seen by the host should be explored in the future. We presume that the production/secretion of the adhesive substances by S. venezuelensis adult worms is a key step for the parasites to invade and establish in the host epithelial layer.
ADHESIVE SUBSTANCES PRODUCED BY
REFERENCES BACIC, A., KAHANE, I., AND ZUCKERMAN, B. M. 1990. Panagrellus redivius and Caenorhabditis elegans: Evidence for the absence of sialic acids. Experimental Parasitology 71, 483–488. BORGONIE, G., VAN DRIESSCHE, E., LINK, C. D., DE WAELE, D., AND COOMANS, A. 1994. Tissue treatment for whole mount internal lectin staining in the nematodes Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus. Histochemistry 101, 379–384. CROSS, H. J., BANZON, T., AND SINGSON, C. 1978. Further studies on Capillaria philippinensis. Development of the parasite in the Mongolian gerbil. Journal of Parasitology 64, 208–213. DAWKINS, H. J. S., ROBERTSON, T. A., PAPADIMITRIOU, J. M., AND GROVE, D. I. 1983. Light and electron microscopical studies of the location of Strongyloides ratti in the mouse intestine. Zeitschrift fu¨r Parasitenkunde 69, 357–370. DAWKINS, H. J. S. 1989. Strongyloides ratti infections in rodents: Value and limitations as a model of human strongyloidiasis. In ‘‘Strongyloidiasis: A Major Roundworm Infection of Man’’ (D. I. Grove, Ed.), pp. 287–332. Taylor & Francis, Philadelphia. DUBREMETZ, J. F., AND MCKERROW, J. H. 1995. Invasion mechanisms. In ‘‘Biochemistry and Molecular Parasitology’’ (J. J. Marr and M. Mu¨ller, Eds.), pp. 307–322. Academic Press, San Diego. ISHIKAWA, N., SHI, B.-B., KHAN, A. I., AND NAWA, Y. 1995. Reserpine-induced sulphomucin production by goblet cells in the jejunum of rats and its significance in the establishment of intestinal helminths. Parasite Immunology 17, 581–586. LINDER, E. 1986. Fluorochrome-labelled lectins reveal secreted glycoconjugates of Schistosoma larvae. Parasitology Today 2, 219–221. METTRICK, D. F., AND PODESTA, R. B. 1974. Ecological and physiological aspects of helminth–host interactions in the
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mammalian gastrointestinal canal. Advances in Parasitology 12, 183–1279. NAWA, Y., IMAI, J., ABE, T., KISANUKI, H., AND TSUDA, K. 1988. A case report of intestinal capillariasis—The second case found in Japan. Japanese Journal of Parasitology 37, 47–52. NAWA, Y., ISHIKAWA, N., TSUCHIYA, K., HORII, Y., ABE, T., KHAN, A. I., SHI, B.-B., ITOH, H., IDE, H., AND UCHIYAMA, F. 1994. Selective effector mechanisms for the expulsion of intestinal helminths. Parasite Immunology 16, 333– 338. SATO, Y., AND TOMA, H. 1990. Effects of spleen cells and serum on transfer of immunity to Strongyloides venezuelensis infection in hypothymic (nude) mice. International Journal for Parasitology 20, 63–67. SHI, B.-B., ISHIKAWA, N., ITOH, H., IDE, H., TSUCHIYA, K., HORII, Y., UCHIYAMA, F., AND NAWA, Y. 1994. Goblet cell mucins of four genera of the subfamily Cricetinae with reference to the protective activity against Strongyloides venezuelensis. Parasite Immunology 16, 553–559. SPEARE, R. 1989. Identification of species of Strongyloides. In ‘‘Strongyloidiasis: A Major Roundworm Infection of Man’’ (D. I. Grove, Ed.), pp. 11–83. Taylor & Francis, Philadelphia. STIREWALT, M. A., AND WALTERS, M. 1973. Schistosoma mansoni: Histochemical analysis of the postacetabular gland secretion of cercariae. Experimental Parasitology 33, 56–72. STIREWALT, M. A. 1974. Schistosoma mansoni: Cercaria to schistosomule. Advances in Parasitology 12, 115–180. WAKELIN, D. 1980. Immunity to helminths. Current Opinion in Immunology 1, 448–453. WERTHEIM, G. 1970. Experimental concurrent infections with Strongyloides ratti and S. venezuelensis in laboratory rats. Parasitology 61, 389–395. Received 25 April 1996; accepted with revision 14 August 1996
85, 10–15 (1997)
Strongyloides venezuelensis: Adhesion of Adult Worms to Culture Vessels by Orally Secreted Mucosubstances HARUHIKO MARUYAMA
AND
YUKUFUMI NAWA
Department of Parasitology, Miyazaki Medical College, Miyazaki 889-16, Japan MARUYAMA, H., AND NAWA, Y. 1996. Strongyloides venezuelensis: Adhesion of adult worms to culture vessels by orally secreted mucosubstances. Experimental Parasitology 85, 10–15. Adults worms of Strongyloides venezuelensis were cultured in vitro. After overnight incubation, about 60% of the worms adhered firmly to the bottom of culture vessels by secreting adhesive substances from the mouth. A single worm produced 24.5 ± 10.1 of the adhesion spots overnight. When they were transferred to new culture vessels, they still produced new spots comparable to those produced for first 24 hr. The adhesion spots were positively stained with Coomassie brilliant blue and also with mucicarmine, periodic acid–Schiff, and alcian blue, pH 2.5, but not with alcian blue, pH 0.3, indicating their glycoprotein nature. The substances were amorphous and did not contain cells or nuclei. Histologic staining with a panel of lectins showed that the adhesive substances were rich in mannose, N-acetyl galactosamine, and N-acetyl glucosamine, but devoid of sialic acid. These characteristics were distinct from those of jejunal goblet cell mucins of rats. Adhesive substances contained antigenic components recognized by sera from infected rats. Thus, the adhesive substances secreted from the mouth of S. venezuelensis were clearly of parasite origin. We consider the production/secretion of the adhesive substances by S. venezuelensis adult worms a key step for the parasites to invade and establish the host epithelial layer. © 1997 Academic Press INDEX DESCRIPTORS: Strongyloides venezuelensis; parasitic; adhesion; mucosubstances; lectin.
INTRODUCTION
the host mucosa are poorly understood. Strongyloides venezuelensis adult worms parasitize in between intestinal villi, close to the surface of the mucosa (Wertheim 1970). They invade the intercellular space of the epithelial cells and move actively between adjoining cells. They leave tunnels as they migrate through the epithelium (Dawkins et al. 1983). Compared to such morphological study on the behavior of parasites, functional approaches on the establishment of parasites have not been fully explored. The aim of the present study is to investigate the adhesion of S. venezuelensis in vitro. When S. venezuelensis adult worms were cultured in vitro, they adhered firmly to the bottom of the culture vessels by secreting mucinous adhesive substances from their mouths. We presume that these adhesive substances could be key materials for the parasites to attach to the host intestinal mucosa upon the invasion of the epithelial layer.
Establishment of an infectious agent in a host depends on attributes of both pathogen and host organisms. To understand the host–parasite interface, both parasite-derived and host-derived components should be analyzed. In the case of intestinal helminth infections, immune-mediated mucosal responses of the host have been fairly well segregated into regulatory and effector systems at a cellular level (Wakelin 1989). Our recent series of work revealed that at least two distinct effector mechanisms are operating selectively against Nippostrongylus brasiliensis and Strongyloides spp., goblet cells to the former and mucosal mast cells to the latter (Nawa et al. 1994). In Strongyloides infection in mice and rats, intestinal mastocytosis was observed and a substantial portion of the mast cells migrate into the epithelial layer around the time of immune-mediated expulsion, so that mastcell-derived substances are considered the effector molecules. In contrast to the detailed studies on host mucosal responses, strategies of the parasites as to how they reach and remain in
MATERIALS
10 0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
AND
METHODS
Animals and parasites. Male Wistar rats 8 weeks old were purchased from Kyudo Co. (Kumamoto, Japan). The
ADHESIVE SUBSTANCES PRODUCED BY S. venezuelensis used in this study was kindly supplied by Professor Y. Sato, Department of Parasitology, School of Medicine, University of the Ryukyus, and maintained in our laboratory by serial passage in Wistar rats. Third-stage infective larvae (L3) were obtained from fecal culture by the filter paper method as previously described (Sato and Toma 1990). Animals were infected by subcutaneous inoculation with 20,000 L3 for maintenance and recovery of adult worms. Adult worms were obtained from the intestine of Wistar rats at 8 days postinfection. In brief, the upper 2⁄3 of the small intestine was removed from infected rats, cut open longitudinally, and incubated at 37°C in phosphate-buffered saline (PBS) for 1 hr. The worms migrated out from the intestine, were extensively washed with sterile PBS, and adjusted to the required concentration. In vitro culture of adult worms. As a preliminary experiment, we examined the capability of S. venezuelensis adult worms to adhere to various plastic and glass culture wares after incubation at 37°C overnight in PBS. The culture wares examined were 25-cm2 culture flasks (Nunc, Roskilde, Denmark; Corning, Inc., Corning, NY), a 96-well cell culture plate (Nunc, Sumitomo, Tokyo, Japan), glass chamber slides (Lab-Tek chamber, Nunc), and a polypropylene tube (Nunc). Subsequently, we used 96-well cell culture plates (Nunc) for the following experiments and adult worms were plated at a density of 70 ± 10 worms/well, except as otherwise stated. At the end of the incubation, wells were vigorously washed with PBS and then ethanol was added to the wells for histochemical examinations or colorimetric assays. Histochemical examinations. To characterize histochemically the nature of adhesive substances produced by S. venezuelensis adult worms, they (70 ± 10 worms/well) were cultured in eight-well chamber slides in PBS at 37°C overnight. After incubation, the chambers were removed and the slides were washed with PBS and stained with Coomassie brilliant blue for protein, with mucicarmine, which stains mucin in red, and periodic acid–Schiff (PAS) followed by alcian blue, pH 2.5, which stains acid mucopolysaccharide in blue, neutral mucin in red to red purple, and sialomucin in red purple to blue. Adhesive substances were also stained with alcian blue, pH. 0.3, alone, which stains only sulfated mucosubstances. Binding to lectins. The biotinylated lectins used in the present study were as follows: Agaricus bisporus agglutinin (ABA), Jack bean agglutinin (Con A), Grifonia simplicifolia agglutinin-II (GSA-II), Helix pomatia agglutinin (HPA), Maackia amurensis agglutinin (MAA), Sambucus sieboldiana agglutinin (SSA), and Ulex europaenus agglutinin-I (UEA-I). HPA and GSA-II were purchased from Sigma (St. Louis, MO) and E-Y Laboratories, Inc. (San Mateo, CA), respectively. All other lectins were from Hohnen Co. (Tokyo, Japan). The known saccharides specifically recognized by them were given in Table I in the results. S. venezuelensis adult worms (about 70 worms/well) were cultured in PBS in 96-well microculture plates at 37°C overnight. Wells were extensively washed with PBS to remove
S. venezuelensis ADULT WORMS
11
TABLE I Binding of Lectins to Rat Goblet Cell Mucins and Adhesion Spots
Lectin
Sugar specificity
Goblet cell mucins
Adhesion spots
ABA Con A GSA-II HPA MAA SSA UEA-I
Galb1 → 3Gal NAc a Man GlcNAc aGalNAc Siaa2 → 3Gal Siaa2 → 6Gal aFuc
±∼+ − + − − ++ −
+ ++ + ++ − − −
Note. Abbreviations: Gal, galactose; Man, mannose; GlcNAc, N-acetyl glucosamine; GalNAc, N-acetyl galactosamine; Sia, sialic acid; Fuc, fucose.
adult worms, fixed with ethanol, and then were added with methanol containing 0.3% H2O2 to block endogenous peroxidase activities. After washing with PBS, wells were incubated with PBS containing 1% bovine serum albumin for 2 hr at room temperature, washed, and then incubated with biotinylated lectins for 1 hr. Wells were then incubated with horseradish peroxidase (HRP)-conjugated streptavidin (GIBCO BRL, Gaithersburg, MD) for 1 hr. Substrate ABTS (2,29-azino-di[3-ethyl-benzthiazoline sulfonate] ) was added after washing and optical densities were read in a Labsystems Multiskan Bichromatic (Labosystems Oy, Helsinki, Finland). To demonstrate binding of lectins to adhesive substances histochemically, chamber slides prepared as above were stained with lectins in the same manner for the colorimetric assay except that metal-enhanced DAB (Pierce, Rockford, IL) was employed instead of ABTS. As the reference of lectin binding, tissue sections of rat small intestines fixed in Carnoy’s fluid were stained in parallel with the slides of the parasite-derived adhesive substances. Enzyme-linked immunosorbent assay (ELISA). Binding of rat sera to the the adhesive substances produced by S. venezuelensis adult worms was tested in ELISA. After incubation with adult worms at 37°C overnight, the cell culture plates were fixed in ethanol and blocked for endogenous peroxidase activity. Then various concentrations of sera obtained from S. venezuelensis-infected and uninfected rats were added to the wells. Wells were then washed and HRP-conjugated anti-rat immunoglobulins (PO450, DAKO, Glostrup, Denmark) were added. After incubation for 1 hr and washing three items, ABTS was added and optical densities were read.
RESULTS Adhesion of S. venezuelensis adult worms to culture vessels. When adult worms of S. venezuelensis were cultured in vitro, they adhered
12
MARUYAMA AND NAWA
firmly to the bottom of glass as well as plastic culture flasks and dishes of almost all types except for the polypropylene tube. After overnight incubation in chamber slides, the chambers were gently washed with PBS. About 60% of S. venezuelensis adult worms adhered to the glass surface by secreting adhesive substances from the mouth, more than 95% of them actively moving (Figs. 1A and 1B). In addition to the actual binding sites, numerous spots of adhesive substances without worms were observed, indicating that the worms attached and detached repeatedly during culture. In fact, when a single worm was placed in a well of 96-well culture plate and incubated overnight, the average number of adhesive spots produced was 24.5 ± 10.1 in 14 hr. They were able to make new spots after 24 hr of culture as well as in the first 24 hr. It was likely that they could mark spots as long as they were alive. There was no noticeable difference among adhesion spots made by worms collected on different days of infection. Characterization of adhesive substances. Af-
FIG. 1. Adhesion of S. venezuelensis adult worms by mucosubstances secreted from the mouth.
ter adult worms were washed off by vigorous pipetting with PBS, the secreted substances remained on the bottom were positively stained with Coomassie brilliant blue with mucicarmine, PAS, and alcian blue, pH 2.5, but not with alcian blue, pH 0.3, indicating their glycoprotein nature. The substances were amorphous and neither cells nor nuclei were observed within them even after staining. Thus, adhesive substances were of extracellular materials which were considerably glycosylated with some acidic sugar residues. Since general staining properties of the adhesive substances were similar to those of rat goblet cell mucins, there was a possibility that these mucinous substances were ingested and excreted goblet cell mucins. To test this point, the nature of adhesive substances was further characterized by binding to a panel of biotinylated lectins. The results were summarized in Table I. From these binding profiles, the adhesive substances were rich in mannose, N-acetyl galactosamine, and Nacetyl glucosamine, but devoid of sialic acids. They were distinct from rat intestinal goblet cell mucins, suggesting that they were of parasite origin. Typical shapes of adhesive substances stained by HPA are shown in Fig. 2. We next quantified the amount of adhesion spots in a lectin binding assay using biotinylated HPA. When different numbers of adult worms were cultured and the binding of HPA to the adhesive substances was measured, a good correlation [y 4 0.444 log(x) + 0.154; r 4
FIG. 2. Adhesion spots produced by S. venezuelensis adult worms. Adhesion spots were stained with biotinylated HPA, followed by HRP-conjugated streptavidin.
ADHESIVE SUBSTANCES PRODUCED BY
0.935] was obtained between the number of worms and the optical densities (Fig. 3). Adult S. venezuelensis worms seemed to produce adhesive substances continuously, because when they were kept in PBS in a nonadhesive polypropylene tube for 2 hr and then transferred into a 96-well culture plate, substantial amounts of adhesive substances were detected (Fig. 4). Production/excretion was thought to be metabolically active processes because adhesive substances were not produced when worms were incubated at 4°C (Fig. 4). Antigenicity of adhesive substances. To confirm further that adhesive substances were of parasite origin, we tested the binding of rat sera to the substance in ELISA. Adult worms were cultured in PBS overnight at 37°C in wells of microtiter plates. Wells were then washed to remove the worms and then S. venezuelensisinfected and uninfected rat sera were added after blocking of endogenous peroxidase activities and nonspecific binding. As shown in Fig. 5, sera from infected, but not uninfected, animals significantly bound to adhesive substances. Thus, adhesive substances contain components immunogenic in rats, indicating again that they were of parasite origin. While the worm expulsion occurred between 3 and 4 weeks postinfection (data not shown), the anti-
FIG. 3. Correlation between the number of worms added per well and the amount of adhesion substances produced by the worms.
S. venezuelensis ADULT WORMS
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FIG. 4. Comparison of production of adhesive substances under different conditions of culture. Adult worms (∼70/ well) were cultured in wells of microtiter plates and the amount of adhesive substances was measured in a lectin binding assay with HPA. Each bar represents the mean ± SD of triplicate assays. P < 0.01.
body titer against adhesive substances began to rise in 2 weeks and reached a peak in 6 weeks postinfection. DISCUSSION The gastrointestinal tract is undoubtedly the most favored niche for adult metazoan parasites represented by the digenetic trematodes, cestodes, nematodes, and acanthocephalans (Mettrick and Podesta 1974). While the great
FIG. 5. Binding of rat sera to adhesion spots. Rats were infected with 20,000 infective larvae of S. venezuelensis. Sera were collected before and after infection and binding of the sera (diluted at 1 to 25,000) to adhesion spots was measured in ELISA.
14
MARUYAMA AND NAWA
majority of platyhelminths and larger nematodes such as Ascaris reside in the intestinal lumen, smaller nematodes tend to invade mucosa at various degrees. Capillaria philippinensis, for example, burrow in the mucosa of the small intestine repeatedly, causing severe tissue damage and violent diarrhea (Cross et al. 1978; Nawa et al. 1988). Trichuris also invade mucosa. Strongyloides adult worms locate between intestinal villi, invade the epithelial layer, and move actively between adjoining epithelial cells. They leave tunnels as they migrate through the epithelium (Dawkins et al. 1983). They do not penetrate the lamina propria across the basement membrane. It is suggested that they move in and out of the epithelial layer repeatedly over the course of infection (Dawkins 1989). In the present study, we showed that S. venezuelensis adult worms adhered to the surface of culture vessels by orally secreted adherent substances (Fig. 1). Amorphous material projecting from the buccal capsule was previously noticed in adult Strongyloides ratti, though it was interpreted as immunoglobulins from the host (Speare 1989). Our results of histochemical study on adhesive spots including the lectin binding profile (Table I) and immunogenicity in rats (Fig. 5) indicate that the substances were clearly of parasite origin and have a glycoprotein nature. The substances were rich in carbohydrates, especially mannose and N-acetyl galactosamine, although they were devoid of sialic acid (Table I). The lack of sialic acid might be a common characteristic of nematodes (Bacic et al. 1990; Borgonie et al. 1994). The substances seemed to be produced through active metabolic processes (Fig. 4). Although the secretion of adhesive substances in vivo has yet to be demonstrated, it is likely that ‘‘a fibrillar– granular electron-dense deposit’’ between the cuticle of S. ratti adult worms and the surrounding intestinal cells observed in the electron micrograph (Dawkins et al. 1983) could possibly be the adhesive substances we described here. Attachment to the surface of the host is one of the logically required steps for the parasite in the invasion process (Dubremetz and McKer-
row 1995). In Schistosoma mansoni infection, cercariae emit sticky mucus secretions from the posterior acetabular glands, which allow them to attach to and penetrate skin (Stirewalt 1974). Cercarial glands contain sialomucin (Stirewalt and Walter 1973), which binds to lectins such as wheat germ agglutinin (Linder 1986). Interestingly, cercariae leave tracks of ‘‘footprints’’ or ‘‘kissing marks’’ on the lipid-coated glass surface (Linder 1986), as we observed with S. venezuelensis adults (Fig. 2). Since S. venezuelensis adult worms invade the epithelial layer repeatedly during infection (Dawkins 1989), any mucosal change that affects attachment of S. venezuelensis adult worms to epithelial cells and/or subsequent entry should result in a decrease in infectivity or worm expulsion. In this regard, at around the time of Strongyloides expulsion from mice and rats, mucosal mast cells proliferate and migrate into the epithelium, where they are supposed to release effector molecules for worm expulsion (Nawa et al. 1994). Ishikawa et al. (1995) reported that reserpine-treated rats gained resistance against implantation of S. venezuelensis adult worms and concluded that reserpineinduced sulfated goblet cell mucins were responsible for their resistance. Related to this point, hamsters having highly sulfated goblet cell mucins could expel S. venezuelensis more quickly than other species of hamsters having less sulfated goblet cell mucins (Shi et al. 1994). Therefore, it is conceivable that highly sulfated mast-cell-derived proteoglycans and/or goblet cell mucins on the surfaces of epithelial cells might prevent attachment of S. venezuelensis and subsequent invasion. Examination of the effects of mast-cell-derived mediators and sulfated mucins on the adhesion of S. venezuelensis should cast a new light on the mechanism of worm expulsion. Whether the worm-derived mucosubstances could induce protective immunity or could protect worms from being seen by the host should be explored in the future. We presume that the production/secretion of the adhesive substances by S. venezuelensis adult worms is a key step for the parasites to invade and establish in the host epithelial layer.
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REFERENCES BACIC, A., KAHANE, I., AND ZUCKERMAN, B. M. 1990. Panagrellus redivius and Caenorhabditis elegans: Evidence for the absence of sialic acids. Experimental Parasitology 71, 483–488. BORGONIE, G., VAN DRIESSCHE, E., LINK, C. D., DE WAELE, D., AND COOMANS, A. 1994. Tissue treatment for whole mount internal lectin staining in the nematodes Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus. Histochemistry 101, 379–384. CROSS, H. J., BANZON, T., AND SINGSON, C. 1978. Further studies on Capillaria philippinensis. Development of the parasite in the Mongolian gerbil. Journal of Parasitology 64, 208–213. DAWKINS, H. J. S., ROBERTSON, T. A., PAPADIMITRIOU, J. M., AND GROVE, D. I. 1983. Light and electron microscopical studies of the location of Strongyloides ratti in the mouse intestine. Zeitschrift fu¨r Parasitenkunde 69, 357–370. DAWKINS, H. J. S. 1989. Strongyloides ratti infections in rodents: Value and limitations as a model of human strongyloidiasis. In ‘‘Strongyloidiasis: A Major Roundworm Infection of Man’’ (D. I. Grove, Ed.), pp. 287–332. Taylor & Francis, Philadelphia. DUBREMETZ, J. F., AND MCKERROW, J. H. 1995. Invasion mechanisms. In ‘‘Biochemistry and Molecular Parasitology’’ (J. J. Marr and M. Mu¨ller, Eds.), pp. 307–322. Academic Press, San Diego. ISHIKAWA, N., SHI, B.-B., KHAN, A. I., AND NAWA, Y. 1995. Reserpine-induced sulphomucin production by goblet cells in the jejunum of rats and its significance in the establishment of intestinal helminths. Parasite Immunology 17, 581–586. LINDER, E. 1986. Fluorochrome-labelled lectins reveal secreted glycoconjugates of Schistosoma larvae. Parasitology Today 2, 219–221. METTRICK, D. F., AND PODESTA, R. B. 1974. Ecological and physiological aspects of helminth–host interactions in the
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mammalian gastrointestinal canal. Advances in Parasitology 12, 183–1279. NAWA, Y., IMAI, J., ABE, T., KISANUKI, H., AND TSUDA, K. 1988. A case report of intestinal capillariasis—The second case found in Japan. Japanese Journal of Parasitology 37, 47–52. NAWA, Y., ISHIKAWA, N., TSUCHIYA, K., HORII, Y., ABE, T., KHAN, A. I., SHI, B.-B., ITOH, H., IDE, H., AND UCHIYAMA, F. 1994. Selective effector mechanisms for the expulsion of intestinal helminths. Parasite Immunology 16, 333– 338. SATO, Y., AND TOMA, H. 1990. Effects of spleen cells and serum on transfer of immunity to Strongyloides venezuelensis infection in hypothymic (nude) mice. International Journal for Parasitology 20, 63–67. SHI, B.-B., ISHIKAWA, N., ITOH, H., IDE, H., TSUCHIYA, K., HORII, Y., UCHIYAMA, F., AND NAWA, Y. 1994. Goblet cell mucins of four genera of the subfamily Cricetinae with reference to the protective activity against Strongyloides venezuelensis. Parasite Immunology 16, 553–559. SPEARE, R. 1989. Identification of species of Strongyloides. In ‘‘Strongyloidiasis: A Major Roundworm Infection of Man’’ (D. I. Grove, Ed.), pp. 11–83. Taylor & Francis, Philadelphia. STIREWALT, M. A., AND WALTERS, M. 1973. Schistosoma mansoni: Histochemical analysis of the postacetabular gland secretion of cercariae. Experimental Parasitology 33, 56–72. STIREWALT, M. A. 1974. Schistosoma mansoni: Cercaria to schistosomule. Advances in Parasitology 12, 115–180. WAKELIN, D. 1980. Immunity to helminths. Current Opinion in Immunology 1, 448–453. WERTHEIM, G. 1970. Experimental concurrent infections with Strongyloides ratti and S. venezuelensis in laboratory rats. Parasitology 61, 389–395. Received 25 April 1996; accepted with revision 14 August 1996