Vol. 58, No. 9
INFECTION AND IMMUNITY, Sept. 1990, p. 3143-3146
0019-9567/90/093143-04$02.00/0 Copyright © 1990, American Society for Microbiology
Endogenous Carbohydrate-Binding Proteins in Pneumocystis cariniit MATHIAS VIERBUCHEN,l* MONIKA ORTMANN,'
AND
GERHARD UHLENBRUCK2
Institutes of Pathology1 and Immunobiology,2 University of Cologne, D-5000 Cologne 41, Federal Republic of Germany Received 22 February 1990/Accepted 13 June 1990
By using biotinylated neoglycoproteins, the in situ occurrence of endogenous carbohydrate-binding proteins (lectins) in Pneumocystis carinii has been glycohistochemically demonstrated in lung tissue specimens from acquired immunodeficiency syndrome patients with P. carinii pneumonia. While the parasite possessed only a weak to moderate density of receptors for L-fucose and N-acetylated amino sugars, a strong specific binding of (P-D-galactoside and D-mannoside neoglycoproteins was observed on the cyst surface and within intracystic bodies. It is suggested that these endogenous lectins may be involved in the adhesion of P. carinii to the target.
iv; (vi) localization of peroxidase activity with 3-amino-9ethylcarbazole (8). Control experiments to ascertain the specificity of neoglycoprotein binding have been described elsewhere (6, 25). To test the effect of enzymatic digestion of tissue on neoglycoprotein binding, prior to the staining sequence (step iii), tissue sections were incubated with various proteinases and glycosidases as described elsewhere (26). Incubation step iii of the staining sequence and enzymatic digestion of tissue sections were performed at 37°C for 60 min. All other incubations were performed at room temperature for 30 min. Intense staining of the parasites was observed with P-Dgalactoside and D-mannoside neoglycoproteins (Fig. 1 and 2), whereas the parasite possessed only a moderate to weak binding capacity for the L-fucose- and the amino sugarcontaining neoglycoproteins. Binding of the neoglycoproteins was concentrated to the cell surface and in many instances to the intracystic bodies (Fig. 2). Even in the same tissue sections, the individual parasites showed a variation in the density of endogenous carbohydrate-binding sites ranging from moderate to very strong staining in samples stained by D-galactoside and Dmannoside neoglycoproteins and from negative staining to moderate staining with neoglycoproteins containing amino sugars. However, this intrasample variation did not influence the overall grading of the endogenous carbohydrate-binding capacities of P. carinii for the different neoglycoproteins. Pretreatment of tissue sections with trypsin abolished the binding of neoglycoproteins, while pretreatment of tissue sections with glycosidases exerted no effect on the staining. This indicates that proteins rather than carbohydrates are involved in the binding of the neoglycoproteins. The presence of an excess of the appropriate nonbiotinylated neoglycoprotein completely inhibited specific staining (Fig. 3). In comparison to simple sugars, which also impaired neoglycoprotein binding, the multivalent neoglycoproteins possessed stronger inhibitory activity. These results are in agreement with the observation that lectin binding extends beyond the single monosaccharide, and multivalency of the carbohydrate ligand induces a significant increase in lectin-binding reactivity in comparison to the simple saccharide (bonus effect of multivalence). Although certain studies have been performed on the binding of P. carinii to the alveolar surface, it is unclear
The nonspecific binding of microorganisms to the target is due to mechanisms including electrostatic forces and hydrophobic-hydrophilic interactions. In contrast, specific attachment is mainly mediated by microbial carbohydrate-binding proteins, which may also be called adhesins but are more generally termed lectins (15, 23). According to this concept, the tissue tropism of the infectious agent is mediated by the carbohydrate specificity of the microbial lectin and the presence of the appropriate carbohydrate in the target. The occurrence of endogenous lectins can be detected biologically, biochemically, and cytochemically (10, 11, 16-20). Recently, neoglycoproteins labeled with biotin have been extensively used for the histochemical (in situ) detection of endogenous lectins in paraffin-embedded tissue specimens (5-7, 25, 27). In the present study, this glycohistochemical technique was used for the detection of endogenous carbohydrate-binding proteins of Pneumocystis carinii in lung tissue sections from 20 male acquired immunodeficiency syndrome patients (21 to 53 years old) presenting with an initial P. carinii infection. The lung biopsy specimens were fixed in neutral buffered formaldehyde (6%) for 24 h. Sections (5 p.m thick) cut from the paraffin-embedded samples were used in these glycohistochemical studies. Bovine serum albumin (BSA) covalently linked to several carbohydrates (D-galactose, D-mannose, Lfucose, D-N-acetylglucosamine, and N-acetylgalactosamine) was obtained from Medac (Hamburg, Federal Republic of Germany [F.R.G.]) and Janssen Biochimica (Russelsheim, F.R.G.). Biotinylation of the neoglycoproteins was performed as described elsewhere (2). Before staining, the labeled neoglycoproteins were dissolved in phosphate-buffered saline (PBS) containing 0.2% BSA. Endogenous carbohydrate-binding proteins were detected in histological sections after deparaffination and rehydration as follows: (i) incubation in 1% H202-methanol in order to block endogenous peroxidase activity; (ii) floating with 1% BSA in PBS in order to reduce unspecific background staining; (iii) incubation with the labeled neoglycoprotein (10 ,ug/ml of PBS); (iv) washing in three changes of fresh PBS and incubation with peroxidase-labeled streptavidin (Dakopatts; 5 ,ug/ml of PBS); (v) repeating steps iii and * Corresponding author. t Dedicated to R. Fischer in honor of his 60th birthday.
3143
INFECT. IMMUN.
NOTES
3144
:.4 .S.K *::
A.
.rS *; i..i
N-E
_.0,
Am,
FIG. 1. Lung from a human immunodeficiency virus-positive patient with P. carinii pneumonia. Intraalveolar clusters of numerous cysts of P. carinii labeled with biotinylated and mannosylated BSA along the surface of the cysts. Arrows indicate weak to moderate mannose-binding activity of alveolar macrophages. Magnification, x 1,200.
whether nonspecific factors or specific forces like carbohydrate-lectin interaction are involved in the binding of the parasite (1, 4, 24, 28-30). This glycohistochemical study revealed that P. carinii contained a distinct number of lectin specificities. The pneumocytes have been shown to possess a distinct pattern of complex carbohydrates (9, 12-14). It has been shown that pneumocyte I especially contains galactoseand mannose-type glycoconjugates (3), which may serve as receptors for the microbial lectins occurring at highest density in P. carinii, as shown in this study. On the other hand, multiple soluble lectins have been detected in human lung tissue (21) and P. carinii has been shown to possess a broad spectrum of complex carbohydrates which could act as ligands for these lung lectins (22, 31). As proposed for the adhesion of Escherichia coli (27), a dual recognition mechanism employing a second set of complementary molecules from the parasite (carbohydrate) and the lung (lectin) may be involved in P. carinii binding. FIG. 2. Section of a lung tissue specimen showing a cluster of P. carinii attached to the alveolar surface. Intense binding of galactoseBSA-biotin along the surface of the cysts as well as of intracystic bodies in some cysts. Magnification, x2,400.
VOL. 58, 1990
NOTES
3145
FIG. 3. Lung tissue section from the same sample as shown in Fig. 1 stained in the presence of an excess of unlabeled neoglycoprotein (mannose-BSA) preventing the staining of P. carinii cysts. Magnification, x 1,200. We thank E. Vierkotten for her expert technical assistance. Supported by Deutsche Forschungsgemeinschaft grant Vi 106/1-1. LITERATURE CITED 1. Barton, E. G., and W. G. Campbell. 1969. Pneumocystis carinji in the lungs of rats treated with cortisone acetate. Ultrastructural observations related to the life cycle. Am. J. Pathol. 54:209-236. 2. Bayer, E., A. E. Skutelsky, and M. Wilchek. 1982. The ultrastructural visualization of cell surface glycoconjugates. Methods Enzymol. 83:195-215. 3. Brandt, A. E. 1982. Cell surface saccharides of rat lung alveolar type 1 and type 2 cells. Fed. Proc. Soc. Exp. Biol. 41:755. 4. Campbell, W. G. 1972. Ultrastructure of Pneumocystis in human lung. Life cycle in human pneumocytosis. Arch. Pathol. 93:312-324. 5. Gabius, H. J., S. Bodanowitz, and A. Schauer. 1988. Endogenous sugar binding receptors in human breast tissue and benign and malignant lesions. Cancer 61:1125-1131. 6. Gabius, H. J., P. L. Debbage, R. Engelhardt, R. Osmers, and W. Lange. 1987. Identification of endogenous sugar-binding proteins (lectins) in human placenta by histochemical localization and biochemical characterization. Eur. J. Cell Biol. 44:265-272. 7. Gabius, H. J., and G. A. Nagel (ed.). 1988. Lectins and glycoconjugates in oncology. Springer-Verlag KG, Berlin. 8. Graham, R. C., U. Linholm, and M. J. Karnovsky. 1965. Cytochemical demonstration of peroxidase activity with 3-amino-9-ethyl-carbazole. J. Histochem. Cytochem. 13:150-152.
9. Katsuyama, T., and S. S. Spicer. 1977. A cation retaining layer in the alveolar-capillary membrane. Lab. Invest. 36:428-435. 10. Kieda, C., F. Delmotte, and M. Monsigny. 1977. Preparation and properties of glycosylated cytochemical markers. FEBS Lett. 76:257-261. 11. Kieda, C., A. C. Roche, F. Delmotte, and M. Monsigny. 1979. Lymphocyte membrane lectins. Direct visualization by the use of fluoresceinyl glycosylated cytochemical markers. FEBS Lett. 99:329-332. 12. Kikkawa, Y., H. S. Hahn, S. S. Yang, and J. Berstein. 1970. Mucopolysaccharide in the pulmonary alveolus. II. Electron microscopic observations. Lab. Invest. 22:272-280. 13. Luke, J. L., and S. S. Spicer. 1966. Histochemistry of surface epithelial and pleural mucins in mammalian lung. The demonstration of sialomucin in alveolar cuboidal epithelium. Lab. Invest. 14:2101-2109. 14. Meban, C. 1972. An electronmicroscopic study of the acid mucosubstance lining the alveoli of hamster lung. Histochem. J. 4:1-8. 15. Mirelman, D. (ed.). 1986. Microbial lectins and agglutinins. John Wiley & Sons, Inc., New York. 16. Monsigny, M., C. Kieda, and A. C. Roche. 1979. Membrane lectins. Biol. Cell. 33:289-300. 17. Monsigny, M., C. Kieda, and A. C. Roche. 1983. Membrane glycoproteins, glycolipids and membrane lectins as recognition signals in normal and malignant cells. Biol. Cell. 47:95-110. 18. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherence of
3146
19.
20.
21. 22.
23. 24. 25.
NOTES
Escherichia coli to human mucosal cells mediated by mannose receptors. Nature (London) 265:623-625. Schrevel, J., D. Gros, and M. Monsigny. 1981. Cytochemistry of cell glycoconjugates. Prog. Cytochem. Histochem. 14:1-269. Schrevel, J., C. Kieda, D. Caignaux, D. Gros, F. Delmotte, and M. Monsigny. 1979. Visualization of cell surface carbohydrates by a general two-step lectin technique: lectins and glycosylated cytochemical markers. Biol. Cell. 36:259-266. Sparrow, C. P., H. Leffler, and S. H. Barondes. 1987. Multiple soluble ,-galactoside-binding lectins from human lung. J. Biol. Chem. 262:7383-7390. Tanabe, K., S. Takasaki, J.-I. Watanabe, A. Kobata, A. Egawa, K. Egawa, and Y. Nakamura. 1989. Glycoproteins composed of major surface immunodeterminants of Pneumocystis carinii. Infect. Immun. 57:1363-1368. Uhlenbruck, G. 1987. Bacterial lectins: mediators of adhesion. Zentralbl. Bakteriol. Hyg. A 263:497-508. Vavra, J., and K. Kucera. 1970. Pneumocystis carinii Delanoe, its ultrastructure and ultrastructural affinities. J. Protozool. 17:463-483. Vierbuchen, M., G. Dufhues, G. Uhlenbruck, and R. Fischer. 1988. Endogenous carbohydrate-binding proteins in mouse tissues-a topohistochemical study. J. Clin. Chem. Clin. Bio-
INFECT. IMMUN.
chem. 26:829-830. 26. Vierbuchen, M., P. J. Klein, G. Uhlenbruck, H. 0. Klein, H. E. Schaefer, and R. Fischer. 1980. The significance of lectin receptors in the kidney and renal adenocarcinoma. Rec. Res. Cancer Res. 75:68-75. 27. Vierbuchen, M., G. Peters, M. Ortmann, G. Pulverer, and R. Fischer. 1989. Topographie und Mechanismus der Ahasion uropathogener Escherichia coli Bakterien im menschlichen Nieren- und Nierenbeckengewebe. Verh. Dtsch. Ges. Pathol. 73:264-267. 28. Walzer, D. 1986. Attachment of microbes to host cells: relevance of Pneumocystis carinii. Lab. Invest. 54:589-592. 29. Yoneda, K., and P. D. Walzer. 1980. Interaction of Pneumocystis carinii with host lungs: an ultrastructural study. Infect. Immun. 29:692-703. 30. Yoshida, Y., Y. Matsumoto, M. Yamada, K. Okabayashi, H. Yoshikawa, and M. Nakazawa. 1984. Pneumocystis carinii: electron microscopic investigation on the interaction of trophozoite and alveolar lining cells. Zentralbl. Bakteriol. Hyg. A 256:390-399. 31. Yoshikawa, H., T. Tegoshi, and Y. Yoshida. 1987. Detection of surface carbohydrates on Pneumocystis carinii by fluorescein conjugated lectins. Parasitol. Res. 74:43-49.
INFECTION AND IMMUNITY, Sept. 1990, p. 3143-3146
0019-9567/90/093143-04$02.00/0 Copyright © 1990, American Society for Microbiology
Endogenous Carbohydrate-Binding Proteins in Pneumocystis cariniit MATHIAS VIERBUCHEN,l* MONIKA ORTMANN,'
AND
GERHARD UHLENBRUCK2
Institutes of Pathology1 and Immunobiology,2 University of Cologne, D-5000 Cologne 41, Federal Republic of Germany Received 22 February 1990/Accepted 13 June 1990
By using biotinylated neoglycoproteins, the in situ occurrence of endogenous carbohydrate-binding proteins (lectins) in Pneumocystis carinii has been glycohistochemically demonstrated in lung tissue specimens from acquired immunodeficiency syndrome patients with P. carinii pneumonia. While the parasite possessed only a weak to moderate density of receptors for L-fucose and N-acetylated amino sugars, a strong specific binding of (P-D-galactoside and D-mannoside neoglycoproteins was observed on the cyst surface and within intracystic bodies. It is suggested that these endogenous lectins may be involved in the adhesion of P. carinii to the target.
iv; (vi) localization of peroxidase activity with 3-amino-9ethylcarbazole (8). Control experiments to ascertain the specificity of neoglycoprotein binding have been described elsewhere (6, 25). To test the effect of enzymatic digestion of tissue on neoglycoprotein binding, prior to the staining sequence (step iii), tissue sections were incubated with various proteinases and glycosidases as described elsewhere (26). Incubation step iii of the staining sequence and enzymatic digestion of tissue sections were performed at 37°C for 60 min. All other incubations were performed at room temperature for 30 min. Intense staining of the parasites was observed with P-Dgalactoside and D-mannoside neoglycoproteins (Fig. 1 and 2), whereas the parasite possessed only a moderate to weak binding capacity for the L-fucose- and the amino sugarcontaining neoglycoproteins. Binding of the neoglycoproteins was concentrated to the cell surface and in many instances to the intracystic bodies (Fig. 2). Even in the same tissue sections, the individual parasites showed a variation in the density of endogenous carbohydrate-binding sites ranging from moderate to very strong staining in samples stained by D-galactoside and Dmannoside neoglycoproteins and from negative staining to moderate staining with neoglycoproteins containing amino sugars. However, this intrasample variation did not influence the overall grading of the endogenous carbohydrate-binding capacities of P. carinii for the different neoglycoproteins. Pretreatment of tissue sections with trypsin abolished the binding of neoglycoproteins, while pretreatment of tissue sections with glycosidases exerted no effect on the staining. This indicates that proteins rather than carbohydrates are involved in the binding of the neoglycoproteins. The presence of an excess of the appropriate nonbiotinylated neoglycoprotein completely inhibited specific staining (Fig. 3). In comparison to simple sugars, which also impaired neoglycoprotein binding, the multivalent neoglycoproteins possessed stronger inhibitory activity. These results are in agreement with the observation that lectin binding extends beyond the single monosaccharide, and multivalency of the carbohydrate ligand induces a significant increase in lectin-binding reactivity in comparison to the simple saccharide (bonus effect of multivalence). Although certain studies have been performed on the binding of P. carinii to the alveolar surface, it is unclear
The nonspecific binding of microorganisms to the target is due to mechanisms including electrostatic forces and hydrophobic-hydrophilic interactions. In contrast, specific attachment is mainly mediated by microbial carbohydrate-binding proteins, which may also be called adhesins but are more generally termed lectins (15, 23). According to this concept, the tissue tropism of the infectious agent is mediated by the carbohydrate specificity of the microbial lectin and the presence of the appropriate carbohydrate in the target. The occurrence of endogenous lectins can be detected biologically, biochemically, and cytochemically (10, 11, 16-20). Recently, neoglycoproteins labeled with biotin have been extensively used for the histochemical (in situ) detection of endogenous lectins in paraffin-embedded tissue specimens (5-7, 25, 27). In the present study, this glycohistochemical technique was used for the detection of endogenous carbohydrate-binding proteins of Pneumocystis carinii in lung tissue sections from 20 male acquired immunodeficiency syndrome patients (21 to 53 years old) presenting with an initial P. carinii infection. The lung biopsy specimens were fixed in neutral buffered formaldehyde (6%) for 24 h. Sections (5 p.m thick) cut from the paraffin-embedded samples were used in these glycohistochemical studies. Bovine serum albumin (BSA) covalently linked to several carbohydrates (D-galactose, D-mannose, Lfucose, D-N-acetylglucosamine, and N-acetylgalactosamine) was obtained from Medac (Hamburg, Federal Republic of Germany [F.R.G.]) and Janssen Biochimica (Russelsheim, F.R.G.). Biotinylation of the neoglycoproteins was performed as described elsewhere (2). Before staining, the labeled neoglycoproteins were dissolved in phosphate-buffered saline (PBS) containing 0.2% BSA. Endogenous carbohydrate-binding proteins were detected in histological sections after deparaffination and rehydration as follows: (i) incubation in 1% H202-methanol in order to block endogenous peroxidase activity; (ii) floating with 1% BSA in PBS in order to reduce unspecific background staining; (iii) incubation with the labeled neoglycoprotein (10 ,ug/ml of PBS); (iv) washing in three changes of fresh PBS and incubation with peroxidase-labeled streptavidin (Dakopatts; 5 ,ug/ml of PBS); (v) repeating steps iii and * Corresponding author. t Dedicated to R. Fischer in honor of his 60th birthday.
3143
INFECT. IMMUN.
NOTES
3144
:.4 .S.K *::
A.
.rS *; i..i
N-E
_.0,
Am,
FIG. 1. Lung from a human immunodeficiency virus-positive patient with P. carinii pneumonia. Intraalveolar clusters of numerous cysts of P. carinii labeled with biotinylated and mannosylated BSA along the surface of the cysts. Arrows indicate weak to moderate mannose-binding activity of alveolar macrophages. Magnification, x 1,200.
whether nonspecific factors or specific forces like carbohydrate-lectin interaction are involved in the binding of the parasite (1, 4, 24, 28-30). This glycohistochemical study revealed that P. carinii contained a distinct number of lectin specificities. The pneumocytes have been shown to possess a distinct pattern of complex carbohydrates (9, 12-14). It has been shown that pneumocyte I especially contains galactoseand mannose-type glycoconjugates (3), which may serve as receptors for the microbial lectins occurring at highest density in P. carinii, as shown in this study. On the other hand, multiple soluble lectins have been detected in human lung tissue (21) and P. carinii has been shown to possess a broad spectrum of complex carbohydrates which could act as ligands for these lung lectins (22, 31). As proposed for the adhesion of Escherichia coli (27), a dual recognition mechanism employing a second set of complementary molecules from the parasite (carbohydrate) and the lung (lectin) may be involved in P. carinii binding. FIG. 2. Section of a lung tissue specimen showing a cluster of P. carinii attached to the alveolar surface. Intense binding of galactoseBSA-biotin along the surface of the cysts as well as of intracystic bodies in some cysts. Magnification, x2,400.
VOL. 58, 1990
NOTES
3145
FIG. 3. Lung tissue section from the same sample as shown in Fig. 1 stained in the presence of an excess of unlabeled neoglycoprotein (mannose-BSA) preventing the staining of P. carinii cysts. Magnification, x 1,200. We thank E. Vierkotten for her expert technical assistance. Supported by Deutsche Forschungsgemeinschaft grant Vi 106/1-1. LITERATURE CITED 1. Barton, E. G., and W. G. Campbell. 1969. Pneumocystis carinji in the lungs of rats treated with cortisone acetate. Ultrastructural observations related to the life cycle. Am. J. Pathol. 54:209-236. 2. Bayer, E., A. E. Skutelsky, and M. Wilchek. 1982. The ultrastructural visualization of cell surface glycoconjugates. Methods Enzymol. 83:195-215. 3. Brandt, A. E. 1982. Cell surface saccharides of rat lung alveolar type 1 and type 2 cells. Fed. Proc. Soc. Exp. Biol. 41:755. 4. Campbell, W. G. 1972. Ultrastructure of Pneumocystis in human lung. Life cycle in human pneumocytosis. Arch. Pathol. 93:312-324. 5. Gabius, H. J., S. Bodanowitz, and A. Schauer. 1988. Endogenous sugar binding receptors in human breast tissue and benign and malignant lesions. Cancer 61:1125-1131. 6. Gabius, H. J., P. L. Debbage, R. Engelhardt, R. Osmers, and W. Lange. 1987. Identification of endogenous sugar-binding proteins (lectins) in human placenta by histochemical localization and biochemical characterization. Eur. J. Cell Biol. 44:265-272. 7. Gabius, H. J., and G. A. Nagel (ed.). 1988. Lectins and glycoconjugates in oncology. Springer-Verlag KG, Berlin. 8. Graham, R. C., U. Linholm, and M. J. Karnovsky. 1965. Cytochemical demonstration of peroxidase activity with 3-amino-9-ethyl-carbazole. J. Histochem. Cytochem. 13:150-152.
9. Katsuyama, T., and S. S. Spicer. 1977. A cation retaining layer in the alveolar-capillary membrane. Lab. Invest. 36:428-435. 10. Kieda, C., F. Delmotte, and M. Monsigny. 1977. Preparation and properties of glycosylated cytochemical markers. FEBS Lett. 76:257-261. 11. Kieda, C., A. C. Roche, F. Delmotte, and M. Monsigny. 1979. Lymphocyte membrane lectins. Direct visualization by the use of fluoresceinyl glycosylated cytochemical markers. FEBS Lett. 99:329-332. 12. Kikkawa, Y., H. S. Hahn, S. S. Yang, and J. Berstein. 1970. Mucopolysaccharide in the pulmonary alveolus. II. Electron microscopic observations. Lab. Invest. 22:272-280. 13. Luke, J. L., and S. S. Spicer. 1966. Histochemistry of surface epithelial and pleural mucins in mammalian lung. The demonstration of sialomucin in alveolar cuboidal epithelium. Lab. Invest. 14:2101-2109. 14. Meban, C. 1972. An electronmicroscopic study of the acid mucosubstance lining the alveoli of hamster lung. Histochem. J. 4:1-8. 15. Mirelman, D. (ed.). 1986. Microbial lectins and agglutinins. John Wiley & Sons, Inc., New York. 16. Monsigny, M., C. Kieda, and A. C. Roche. 1979. Membrane lectins. Biol. Cell. 33:289-300. 17. Monsigny, M., C. Kieda, and A. C. Roche. 1983. Membrane glycoproteins, glycolipids and membrane lectins as recognition signals in normal and malignant cells. Biol. Cell. 47:95-110. 18. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherence of
3146
19.
20.
21. 22.
23. 24. 25.
NOTES
Escherichia coli to human mucosal cells mediated by mannose receptors. Nature (London) 265:623-625. Schrevel, J., D. Gros, and M. Monsigny. 1981. Cytochemistry of cell glycoconjugates. Prog. Cytochem. Histochem. 14:1-269. Schrevel, J., C. Kieda, D. Caignaux, D. Gros, F. Delmotte, and M. Monsigny. 1979. Visualization of cell surface carbohydrates by a general two-step lectin technique: lectins and glycosylated cytochemical markers. Biol. Cell. 36:259-266. Sparrow, C. P., H. Leffler, and S. H. Barondes. 1987. Multiple soluble ,-galactoside-binding lectins from human lung. J. Biol. Chem. 262:7383-7390. Tanabe, K., S. Takasaki, J.-I. Watanabe, A. Kobata, A. Egawa, K. Egawa, and Y. Nakamura. 1989. Glycoproteins composed of major surface immunodeterminants of Pneumocystis carinii. Infect. Immun. 57:1363-1368. Uhlenbruck, G. 1987. Bacterial lectins: mediators of adhesion. Zentralbl. Bakteriol. Hyg. A 263:497-508. Vavra, J., and K. Kucera. 1970. Pneumocystis carinii Delanoe, its ultrastructure and ultrastructural affinities. J. Protozool. 17:463-483. Vierbuchen, M., G. Dufhues, G. Uhlenbruck, and R. Fischer. 1988. Endogenous carbohydrate-binding proteins in mouse tissues-a topohistochemical study. J. Clin. Chem. Clin. Bio-
INFECT. IMMUN.
chem. 26:829-830. 26. Vierbuchen, M., P. J. Klein, G. Uhlenbruck, H. 0. Klein, H. E. Schaefer, and R. Fischer. 1980. The significance of lectin receptors in the kidney and renal adenocarcinoma. Rec. Res. Cancer Res. 75:68-75. 27. Vierbuchen, M., G. Peters, M. Ortmann, G. Pulverer, and R. Fischer. 1989. Topographie und Mechanismus der Ahasion uropathogener Escherichia coli Bakterien im menschlichen Nieren- und Nierenbeckengewebe. Verh. Dtsch. Ges. Pathol. 73:264-267. 28. Walzer, D. 1986. Attachment of microbes to host cells: relevance of Pneumocystis carinii. Lab. Invest. 54:589-592. 29. Yoneda, K., and P. D. Walzer. 1980. Interaction of Pneumocystis carinii with host lungs: an ultrastructural study. Infect. Immun. 29:692-703. 30. Yoshida, Y., Y. Matsumoto, M. Yamada, K. Okabayashi, H. Yoshikawa, and M. Nakazawa. 1984. Pneumocystis carinii: electron microscopic investigation on the interaction of trophozoite and alveolar lining cells. Zentralbl. Bakteriol. Hyg. A 256:390-399. 31. Yoshikawa, H., T. Tegoshi, and Y. Yoshida. 1987. Detection of surface carbohydrates on Pneumocystis carinii by fluorescein conjugated lectins. Parasitol. Res. 74:43-49.
