Zbl. Bakt. 274, 359-365 (1990) © Gustav Fischer Verlag, StuttgartlNew York
Biological Properties of Staphylococcal Lipoteichoic Acid and Related Macromolecules)' Y. OHSHIMA 1, H. L. K0 2 , and W. ROSZKOWSKI'
J.
BEUTH 2 , K. ROSZKOWSKI 3 ,
Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 213, Japan 2 Institute of Medical Microbiology and Hygiene, University of Cologne, D-5000 Kaln 41 3 National Research Institute of Tuberculosis and Lung Diseases, Warsaw, Poland 1
Received March 2, 1990 . Accepted March 15, 1990
Summary Lipoteichoic acids (L TAs) and related macromolecules (e.g. cell surface substance, CSS; cell surface antigen, CSA; cell surface complex, CSC) are a group of phosphate-containing polymers associated with the cell walls of Gram-positive bacteria (32). They may be considered as surface-reactive antigens (immunogens, biological response modyfiers) as well as membrane components which mediate the attachment of certain bacteria (S. saprophyticus, S. aureus,group A streptococci) to host cell tissues.
Zusammenfassung Lipoteichonsauren (LTAs) sowie verwandte Maktomolekule (z. B. cell surface substance, CSS; cell surface antigen, CSA; cell surface complex, CSC) geharen zur Gruppe phosphathaltiger Polymere, die in der Regel mit der Zellwand Gram-positiver Bakterien assoziiert sind. Sie fungieren als aktive Oberflachenantigene (Immunogene, Immunomodulatoren) sowie als Mediatoren der bakteriellen Adhasion (z. B. von S. saprophyticus, S. aureus, S. pyogenes) an Organzellen.
Lipoteichoic Acids: Bacterial Antigens During recent decades, lipotelchoic acids (L T As) were realized to be important and presumably essential components of most Gram-positive bacteria. Much of the impetus for studying them, however, has come from their immunological properties. ". Dedicated to Prof. Dr. Dr. h. c. G. Pulverer on the occasion of his 60th birthday.
360
Y.Ohshima, H. L. Ko,
J. Beuth, K. Roszkowski, and W. Roszkowski
Historically it was the group D streptococcus that provided the first example for such an antigen although the group F lactobacillus antigen was the first to be shown to contain the glycolipid component and to be located on the plasma membrane (38). The presence of this glycolipid component also accounts for observations, extending over 20 years, of erythrocyte-sensitizing antigens present in, on the surface of, or in the culture fluid from a variety of Gram-positive bacteria. Apparently, it is the glycolipid component which accounts for the ability of the LTA to sensitize erythrocytes (4, 11, 22). Recent studies suggest, however, that the carbohydrate part is responsible for the antigenicity whereas the lipid fraction mediates the biological activity (25). Lipoteichoic acids (LTAs) are a group of phosphate containing polymers associated with the cell walls and plasma membranes of Gram-positive bacteria (15, 18, 38). However, they do not occur in all species of Gram-positive microorganisms and they apparently are absent from Gram-negative bacteria (14,38). LTAs are composed of a lipid portion covalently linked to a polyglycerophosphate chain and are believed to be adhesins of certain streptococci and staphylococci. Thus, the possible role of LT A in mediating the attachment of Staphylococcus saprophyticus, Staphylococcus aureus, and group A streptococci to host cell tissue was recently demonstrated (3, 6, 24). LTA shares certain biologic properties with lipopolysaccharides (LPS) of Gram-negative bacteria. Both are amphipathic molecules, bind spontaneously to host cell membranes and the binding of each is mediated by ester-linked lipid moieties (14, 38). LPS, however, is more toxic than LT A, probably due to the presence of hydroxacyl esters in the lipid A portion of the molecule (38). Of particular interest is the response of the immune system to these amphipathic molecules. LPS has been shown to serve as a nonspecific mitogen for B-Iymphocytes but not for T-Iymphocytes (2). LTA, however, could be shown to bind to specific receptor sites on T-Iymphocytes to trigger the mitogenic response (3, 38). In the course of investigations lipoteichoic acid was extracted from Staphylococcus saprophyticus (strains S 1 and S 35) as described by Beachey et al. and Moskowitz (5, 22). Some related macromolecules (e.g. cell surface substance, CSS; cell surface antigen, CSA; cell surface complex, CSC) were similarly isolated from coagulase-negative staphylococci and could also be shown to consist of phosphate, protein, fatty acids, and carbohydrates, however, the composition was different for each molecule examined (16, 26, 27). Lipoteichoic Acids: Adhesion-like Molecules Bacterial adherence to epithelial cells is regarded as an initial and necessary step for colonization by both indigenous and pathogenic species (7, 21, 33). The capacity of bacteria to adhere to eukaryotic cells is dependent on several physicochemical factors (e.g. surface charge, hydrophobic interactions) and on the presence of characteristic surface receptors (e.g. carbohydrates) and adhesion molecules (e.g. lectins), respectively (33, 35, 37). Considerable evidence has been accumulated showing that bacterial lectins may be supposed to play the dominant role, at least in the specific adherence process (6, 21). Recently, Iipoteichoic acid (LT A) has been shown to mediate the attachment of staphylococci and streptococci to host cells (5, 6,34). To demonstrate and quantitate LT A-mediated staphylococcal adhesion, human uroepithelial cells (UC) were incubated with Staphylococcus saprophyticus. The total number of adherent bacteria was determined by microscopic examination of 100 UC and the mean value
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was calculated for S. saprophyticus strain S 1 (23.2 per UC) and strain S 35 (24.4 per UC). Preincubation of UC with autologous LT A evidently decreased staphylococcal adhesion (79.3% decrease for strain S 35). Heterologous LTA as well inhibited the adherence of S. saprophyticus to UC, however, to a lesser degree (6). Thus, non-specific inhibition of microbial adherence to UC by LT A from different staphylococcal strains suggest that these molecules obviously are quite similar structurally and functionally. Immunomodulating Activity of Lipoteichoic Acids It has become apparent that the LT A molecule of staphylococci and streptococci is highly reactive, binding spontaneously to a variety of host cell membranes and inducing tissue alterations and injury (6, 23, 34). However, it was of special interest to define the interactions of LT A with cellular elements of the immune system since recently it could be shown that LT A isolated from group A streptococci stimulated mitogenesis of human and murine lymphocytes (3, 38). One of the most intriguing findings was that LTA stimulated T- but not B-lymphocytes which contrasts with the B-lymphocyte mitogenicity of lipopolysaccharides (LPS) from Gram-negative bacteria (2). The difference in the response is most likely due to interacton of each molecule with distinct binding sites on cell membranes that are specific for each molecule. Lucigenin-dependent chemiluminescence may be considered to be a sensitive method to measure the release of oxygen radicals that are believed to process bactericidal properties. Because of their toxicity they obviously have cytotoxic (tumoricidal) effects on transformed cells and tissue damage during inflammation (1). As compared to nontreated control phagocytes, an increased chemiluminescence response of human monocytes and BALB/c-mouse peritoneal macrophages (which was statistically significant) could be induced with LT A preparations of two S. saprophyticus strains (SI and S 35). Mild deacylation procedures, which remove fatty acyl and D-alanyl ester groups and destroy the micellar structures were shown to suppress the biological activity of the LTAs. This finding was in favour of the postulation that the lipid fraction apparently mediates the biological activity (11, 25). Human polymorphonuclear leukocytes (PMNL) did not show a noteworthy chemiluminescence response after incubation with LTA (25). The increasing evidence that macrophages are important in host defense against neoplasms and certain infectious diseases has stimulated interest in agents that can enhance macrophage-mediated destruction of tumor cells and microorganisms. Bacteria and bacterial preparations are known to be highly effective in stimulating the immune system. Lipoteichoic acids (L TAs) and related cell surface macromolecules of staphylococcal origin have been suggested to be potent immunomodifiers stimulating cell populations involved in specific and non-specific resistance (28). The immunomodulating activity of staphylococcal LTAwas established in BALB/c-mice using spleen enlargement as a response measurement. Compared to non-treated mice, LT A administration induced a considerable splenomegaly which was statistically significant. It is generally accepted that there is a close correlation between spleen weight gain and antitumor activity of immunomodifiers. Accordingly, the spleen test can be used as a method for comparative studies on lymphoreticular stimulation properties of different substances as most of them involve the stimulation of the reticuloendothelial system (RES) or stimulation of recruitment and proliferation of competent cells (17). Since in the literature numerous tumor tests report a differing susceptibility
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to immunotherapy, the spleen test and chemiluminescence response of phagocytic cells are considered most suitable for establishing standardized and reproducible results. To evaluate the antineoplastic activity, the effect of staphylococcal LTA on metastatic sarcoma L-1 tumor colonization into the lungs of BALB/c-mice was investigated. Compared to a control group, the number of pulmonary colonies was significantly lower in LTA-treated mice. Appearance of tumor colonies in organs other than lung has never been observed in this model system. However, the possibility that LTA treatment might cause extrapulmonary metastases was routinely checked but no evidence for such a possibility was found (28). Further studies concerning the reactivity of natural killer (NK)-cells and diverse subsets of T-Iymphocytes are in progress and might clarify the immunomodulating effect achieved by LT A treatment. Listeria monocytogenes is a facultative intracellular bacterium which parasitizes host macrophages (12). Specific immunity to Listeria is apparently conferred by a bicellular mechanism in which T -lymphocytes act as inducers and mononuclear phagocytes as expressors of immunity (19). Unspecific host defense mechanisms in early Listeria infection are mainly based on activated macrophages (39). Therefore, stimulation of the mononuclear phagocytic system (MPS) was supposed to enhance resistance to Listeria monocytogenes (8, 12). In a recent study the highly Listeriasensitive BALB/c-mouse strain was investigated. The protective role of activated macrophages after immunostimulation with LTAwas found to significantly reduce organ colonization of Listeria monocytogenes in BALB/c-mice. In vitro, exposure to peritoneal macrophages from LTA-treated mice significantly decreased listerial viability. Direct LTA-macrophage interactions causing a changed bactericidal activity could not be demonstrated (29). Immunoactive LT A-Related Staphylococcal Macromolecules The presence of capsules or slime substances in coagulase-negative staphylococci has already been demonstrated (13). However, knowledge concerning the biochemical and immunological properties of these substances is still limited. We previously found that cell surface substance (CSS) from several encapsulated strains of Staphylococcus epidermidis induced protective activity against those strains. Furtheron, active immunization of mice with CSS induced resistance against experimental infection with homologous staphylococci (13, 30, 36). CSS could be isolated from S. epidermidis (strain ATCC-31432) and fractionated by DEAE Sephadex ion exchange chromatography (30). Biochemical and serological analyses of CSS (sub)fractions resulted in the detection of 1) serologically inactive (sub)fractions 2) serologically active (sub)fractions (so called cell surface antigen, CSA) 3) teichoic acid-like (sub)fractions, as described (31) 4) nucleic acid (sub)fractions. Immunologically active (sub)fractions (CSA) were separated from teichoic acid-like (sub)fractions by ion exchange chromatography as shown for the preparation of CSA from Staphylococcus aureus (40). CSS and CSA were different from the S. epidermidis slime substance with respect to carbohydrate components and serological properties. According to our results, CSA seems to contain a type-specific antigen causing protective activity as well as absorbing activity of protective antibodies. Teichoic acidlike (sub)fractions displayed a lower protective activity in mice than CSA. Recent investigations demonstrated that experimental tumor growth in mice can be drastically reduced (statistically significant) after administration of CSS (26). The in vitro stimula-
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tion of human granulocytes and the increase in immunoglobulin M response suggest antiinfectious (antibacterial) activity of both, CSS and CSA (26, 27). Preliminary studies show that similar effects may be achieved in vivo after experimental infection of mice. The application of CSS and CSA apparently leads to a potent stimulation of the specific and non-specific cellular immune system. Granulocyte Activation by LT A-Related Macromolecules: A Receptor-Mediated Phenomenon Polymorphonuclear leukocytes (PMNL) generally are the first line of defense against extracellular bacterial pathogens. Phagocytosis of particulate material by those cells can be separated experimentally into two steps 1) attachment of the particle to the cell surface and 2) ingestion of the particle (9). The magnitude and quality of the response of the PMNL plasma membrane to a phagocytic stimulus appears to be govered by the number, size and shape of the particles attached to it. However, the zipper mechanism suggests that phagocytosis will occur only if PMNL membrane receptors can bind to the ligands (10). High molecular weight cell surface complex (CSC) was obtained after elution of crude phenol extract from Staphylococcus saprophyticus (strain S 1) from a Sepharose CL-6B column with ammonium acetate. Biochemical studies on this staphylococcal CSC revealed that it consisted of phosphate, protein, fatty acids and carbohydrates, however, the composition was different from lipoteichoic acid (L T A), cell surface substance (CSS), and cell surface antigen (CSA), respectively (16). In further investigations it was of special interest to define the interaction of CSC from S. saprophyticus with cellular elements of the specific and non-specific immune system. Contrary to LT A the CSC of staphylococcal origin can substantially enhance human PMNL chemiluminescence response whereas human monocytes could not be activated. Since heating as well as protease treatment of the CSC abolished its biological activity it was suggested that the protein part of the molecule was responsible for its PMNL activating potency (16). Since it was observed that formylmethionyl pep tides (fMLP) can be generated by invading bacteria (20) and apparently serve as ligands for appropriate receptors on PMNL, our attention was focussed on the fMLP receptor on human PMNL as eventual target for staphylococcal Csc. However, measurement of the PMNL chemiluminescence response to fMLP, CSC, and non-opsonized S. saprophyticus cells prior to and after pretreatment of the phagocytes with fMLP and CSC, respectively, suggested that the fMLP receptor of PMNL is not involved in the interaction with CSc. Evaluation of the bactericidal capacity of PMNL to opsonized S. saprophyticus cells positively correlated to chemiluminescence measurements. Pretreatment of the phagocytes with CSC evidently inhibited their bactericidal activity by interfering with attachment and phagocytosis. Apparently, CSC-mediated PMNL activtion is dependent on immunoglobuline/complement receptors but not on fMLP receptors. These data suggest that PMNL activation by S. saprophyticus is not only mediated by well known ligands like opsonins but may also be related to certain staphylococcal surface complexes such as lectins or cell surface macromolecules.
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References 1. Allen, R. c.: Lucigenin chemiluminescence: a new approach to the study of polymorphonuclear leukocytes redox activity. In: M. A. De Luca and W. D. Mc Elroy (eds.), Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications, pp. 45-53. Academic Press, New York (1982) 2. Andersson, J., F. Me/chers, C. Galanos, and O. Luderitz: The mitogenic effect of lipopolysaccharide on bone marrow-derived mouse lymphocytes. Lipid A as the mitogenic part of the molecule. J. Exp. Med. 137 (1973) 943-953 3. Beachey, E. H., J. B. Dale, A. Ahmed, W. A. Simpson, and I. Ofek: Lymphocyte binding and T-cell mitogenic properties of group A streptococcallipoteichoic acid. J. Immunol. 122 (1979) 189-195 4. Beachey, E. H., J. B. Dale, W. A. Simpson, J. E. Evans, K. W. Knox, I. Ofek, and A. j. Wicken: Erythrocyte binding properties of streptococcal lipoteichoic acid. Infect. Immun. 23 (1979) 618-625 5. Beachey, E. H.: Binding of group A streptococci to human oral mucosal cells by lipoteichoic acid. Trans. Ass. Am. Physic. 88 (1975) 285-292 6. Beuth, j., H. L. Ko, Y. Ohsima, A. Yassin, G. Uhlenbruck, and G. Pulverer: The role of lectins and lipoteichoic acid in adherence of Staphylococcus saprophyticus. Zbl. Bakt. Hyg. A 268 (1988) 357-361 7. Beuth, J., H. L. Ko, G. Uhlenbruck, and G. Pulverer: Lectin-mediated bacterial adherence to human tissue. Eur. J. Clin. Microbiol. 6 (1987) 591-593 8. Beuth, J., H. L. Ko and G. Pulverer: The role of hepatic lectins and the activity of the mononuclear phagocyte system in systemic Listeria monocytogenes infection in BALB/cmice. Med. Microbiol. Immunol. 177 (1988) 47-49 9. Griffin, F. M. and S. C. Silverstein: Segmental response of the macrophage plasma membrane to a phagocytic stimulus. J. Exp. Med. 139 (1974) 323-326 10. Griffin, F. M., J. A. Griffin, J. E. Leider, and S. C. Silverstein: Studies on the mechanism of phagocytosis. 1. Requirements for circumferential attachment of particle bound ligands to specific receptors on the macrophage plasma membrane. J. Exp. Med. 142 (1975) 1263-1282 11. Guthrie, L. A., L. C. Mc Phail, P. M. Henson, and R. B. johnston: Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharides. J. Exp.Med. 160 (1984) 1656-1671 12. Hahn, H.: Antibacterial defense mechanisms. Infection 11 (1983) 112-118 13. Ichiman, Y. and K. Yoshida: The relationship of capsular-type of Staphylococcus epidermidis to virulence and induction of resistance in mice. J. Appl. Bact. 55 (1981) 229-241 14. Iwasaki, H., A. Shimada, K. Yokohama, and E. Ito: Structure and glycosylation of lipoteichoic acids in Bacillus strains. J. Bact. 171 (1989) 424-429 15. Knox, K. W. and A. T. Wicken: Immunological properties of lipoteichoic acids. Bact. Rev. 37 (1973) 215-257 16. Ko, H. L., Y. Ohshima, J. Beuth, P. Quie, and G. Pulverer: Granulocyte activation by a cell surface complex of Staphylococcus saprophyticus: a receptor-mediated phenomenon. Zbl. Bakt. Hyg. A 271 (1989) 104-113 17. Ko, H. L., W. Roszkowski, J. jeljaszewicz, and G. Pulverer: Comparative study on the immunostimulatory potency of different Propionibacterium strains. Med. Microbiol. Immunol. 170 (1981) 1-9 18. Lambert, P. A., I. C. Hancock, and j. Baddiley: Occurrence and function of membrane teichoic acids. Biochem. Biophys. Acta 472 (1977) 1-12 19. Mackaness, G. B.: The immunological basis of acquired cellular resistance. J. Exp. Med. 120 (1964) 105-120 20. Marasco, W. A., S. A. Phan, and H. Krutzsch: Purification and identification of formylmethionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. J. BioI. Chern. 40 (1984) 5430-5439
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21. Mirelman, D. and I. Ofek: Introduction to microbial lectins and agglutinins. In: D. Mirelman (ed.), Microbial Lectins and Agglutinins, pp. 1-20. John Wiley and Sons, New York (1986) 22. Moskowitz, M.: Separation and properties of a red cell sensitizing substance from streptococci. J. Bact. 81 (1966) 2200-2204 23. Nealon, T. J. and S. J. Mattingly: Association of elevated levels of cellular lipoteichoic acids of group B streptococci with human neonatal disease. Infect. Immun. 39 (1983) 1243-1251 24.0fek, I., E. H. Beachey, W. Jefferson, and G. L. Campbell: Cell membrane binding properties of group A streptococcallipoteichoic acid. J. Exp. Med. 141 (1975) 990-998 25.0hshima, Y., J. Beuth, A. Yassin, H. L. Ko, and G. Pulverer: Stimulation of human monocyte chemiluminescence by staphylococcal lipoteichoic acid. Med. Microbiol. Immunol. 177 (1988) 115-121 26. Ohshima, Y., H. L. Ko, J. Beuth, Y. Ichiman, K. Yoshida, and G. Pulverer: Immunostimulating cell surface substance from Staphylococcus epidermidis strain ATCC-31432 prevents metastatic lung colonization in BALB/c-mice. Med. Microbiol. Immunol. 176 (1988) 281-287 27. Ohshima, Y., Y. Ichiman, Y. Usui, H. L. Ko, J. Beuth, G. Pulverer, and K. Yoshida: Cell surface antigen of encapsulated Staphylococcus epidermidis ATCC 31432. J. Clin. Microbiol. 25 (1987) 1338-1340 28. Ohshima, Y., H. L. Ko, J. Beuth, and G. Pulverer: Immunostimulating staphylococcal lipoteichoic acid prevents pulmonary tumor colonization in BALB/c-mice. Zbl. Bakt. Hyg. A 270 (1988) 213-218 29.0hshima, Y., J. Beuth, H. L. Ko, K. Roszkowski, D. Hauck, and G. Pulverer: Immunomodulatory effects of staphylococcallipoteichoic acid in early Listeria monocytogenes infection in BALB/c-mice. Zbl. Bakt. Hyg. A 269 (1988) 251-256 30. Ohshima, Y., Y. Usui, Y. Ichiman, and K. Yoshida: Biochemical characterization of cell surface substance from encapsulated strain of Staphylococcus epidermidis. Zbl. Bakt. Hyg. A Suppl. 14 (1985) 207-212 31. Ohshima, Y., T. Ohtomo, Y. Ichiman, M. Comarat, and K. Yoshida: Comparison of the cell wall teichoic acid fractions isolated from three different encapsulated strains of Staphylococcus epidermidis. Ann. Microbiol. (Paris) 135 A (1984) 207-212 32. Orefici, G., A. Molinary, G. Donelli, S. Paradisi, G. Teti, and G. Arancia: Immunolocation of lipoteichoic acid on group B streptococcal surface. FEMS Microbiol. Lett. 34 (1986) 111-115 33. Sharon, N.: Bacteriallectins. In: I. E. Liener, N. Sharon, and I. J. Goldstein (eds.), The Lectins, pp. 494-522. Academic Press, New York (1986) 34. Teti, G., M. S. Chiafalo, F. Tomasello, F. Fava, and P. Mastroeni: Mediation of Streptococcus saprophyticus adherence to uroepithelial cells by lipoteichoic acid. Infect. Immun. 55 (1987) 839-842 35. Uhlenbruck, G.: Bacteriallectins: mediators of adhesion. Zbl. Bakt. Hyg. A 263 (1987) 497-508 36. Yoshida, K., Y. Ichiman, and T. Ohtomo: Mouse virulent strain of Staphylococcus epidermidis: relation to antiphagocytic activity of the protection-inducing antigen. Jpn. J. Microbiol. 20 (1976) 209-217 37. Wadstrom, T., T.]. Trust, and D. E. Brooks: Bacterial surface lectins. Lectins 3 (1983) 479-494 38. Wicken, A. J. and K. W. Knox: Lipoteichoic acids: a new class of bacterial antigen. Science 187 (1975) 1161-1167 39. Wood, P. and C. Cheers: Activation of non-specific cytotoxic cells in Listeria-susceptible and resistant mouse strains. Immunology 54 (1985) 113-119 40. Wu, T. C. M. and T. J. Park: Chemical characterization of a new surface antigenic polysaccharide from a mutant of Staphylococcus aureus. J. Bact. 108 (1971) 874-884
Biological Properties of Staphylococcal Lipoteichoic Acid and Related Macromolecules)' Y. OHSHIMA 1, H. L. K0 2 , and W. ROSZKOWSKI'
J.
BEUTH 2 , K. ROSZKOWSKI 3 ,
Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 213, Japan 2 Institute of Medical Microbiology and Hygiene, University of Cologne, D-5000 Kaln 41 3 National Research Institute of Tuberculosis and Lung Diseases, Warsaw, Poland 1
Received March 2, 1990 . Accepted March 15, 1990
Summary Lipoteichoic acids (L TAs) and related macromolecules (e.g. cell surface substance, CSS; cell surface antigen, CSA; cell surface complex, CSC) are a group of phosphate-containing polymers associated with the cell walls of Gram-positive bacteria (32). They may be considered as surface-reactive antigens (immunogens, biological response modyfiers) as well as membrane components which mediate the attachment of certain bacteria (S. saprophyticus, S. aureus,group A streptococci) to host cell tissues.
Zusammenfassung Lipoteichonsauren (LTAs) sowie verwandte Maktomolekule (z. B. cell surface substance, CSS; cell surface antigen, CSA; cell surface complex, CSC) geharen zur Gruppe phosphathaltiger Polymere, die in der Regel mit der Zellwand Gram-positiver Bakterien assoziiert sind. Sie fungieren als aktive Oberflachenantigene (Immunogene, Immunomodulatoren) sowie als Mediatoren der bakteriellen Adhasion (z. B. von S. saprophyticus, S. aureus, S. pyogenes) an Organzellen.
Lipoteichoic Acids: Bacterial Antigens During recent decades, lipotelchoic acids (L T As) were realized to be important and presumably essential components of most Gram-positive bacteria. Much of the impetus for studying them, however, has come from their immunological properties. ". Dedicated to Prof. Dr. Dr. h. c. G. Pulverer on the occasion of his 60th birthday.
360
Y.Ohshima, H. L. Ko,
J. Beuth, K. Roszkowski, and W. Roszkowski
Historically it was the group D streptococcus that provided the first example for such an antigen although the group F lactobacillus antigen was the first to be shown to contain the glycolipid component and to be located on the plasma membrane (38). The presence of this glycolipid component also accounts for observations, extending over 20 years, of erythrocyte-sensitizing antigens present in, on the surface of, or in the culture fluid from a variety of Gram-positive bacteria. Apparently, it is the glycolipid component which accounts for the ability of the LTA to sensitize erythrocytes (4, 11, 22). Recent studies suggest, however, that the carbohydrate part is responsible for the antigenicity whereas the lipid fraction mediates the biological activity (25). Lipoteichoic acids (LTAs) are a group of phosphate containing polymers associated with the cell walls and plasma membranes of Gram-positive bacteria (15, 18, 38). However, they do not occur in all species of Gram-positive microorganisms and they apparently are absent from Gram-negative bacteria (14,38). LTAs are composed of a lipid portion covalently linked to a polyglycerophosphate chain and are believed to be adhesins of certain streptococci and staphylococci. Thus, the possible role of LT A in mediating the attachment of Staphylococcus saprophyticus, Staphylococcus aureus, and group A streptococci to host cell tissue was recently demonstrated (3, 6, 24). LTA shares certain biologic properties with lipopolysaccharides (LPS) of Gram-negative bacteria. Both are amphipathic molecules, bind spontaneously to host cell membranes and the binding of each is mediated by ester-linked lipid moieties (14, 38). LPS, however, is more toxic than LT A, probably due to the presence of hydroxacyl esters in the lipid A portion of the molecule (38). Of particular interest is the response of the immune system to these amphipathic molecules. LPS has been shown to serve as a nonspecific mitogen for B-Iymphocytes but not for T-Iymphocytes (2). LTA, however, could be shown to bind to specific receptor sites on T-Iymphocytes to trigger the mitogenic response (3, 38). In the course of investigations lipoteichoic acid was extracted from Staphylococcus saprophyticus (strains S 1 and S 35) as described by Beachey et al. and Moskowitz (5, 22). Some related macromolecules (e.g. cell surface substance, CSS; cell surface antigen, CSA; cell surface complex, CSC) were similarly isolated from coagulase-negative staphylococci and could also be shown to consist of phosphate, protein, fatty acids, and carbohydrates, however, the composition was different for each molecule examined (16, 26, 27). Lipoteichoic Acids: Adhesion-like Molecules Bacterial adherence to epithelial cells is regarded as an initial and necessary step for colonization by both indigenous and pathogenic species (7, 21, 33). The capacity of bacteria to adhere to eukaryotic cells is dependent on several physicochemical factors (e.g. surface charge, hydrophobic interactions) and on the presence of characteristic surface receptors (e.g. carbohydrates) and adhesion molecules (e.g. lectins), respectively (33, 35, 37). Considerable evidence has been accumulated showing that bacterial lectins may be supposed to play the dominant role, at least in the specific adherence process (6, 21). Recently, Iipoteichoic acid (LT A) has been shown to mediate the attachment of staphylococci and streptococci to host cells (5, 6,34). To demonstrate and quantitate LT A-mediated staphylococcal adhesion, human uroepithelial cells (UC) were incubated with Staphylococcus saprophyticus. The total number of adherent bacteria was determined by microscopic examination of 100 UC and the mean value
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361
was calculated for S. saprophyticus strain S 1 (23.2 per UC) and strain S 35 (24.4 per UC). Preincubation of UC with autologous LT A evidently decreased staphylococcal adhesion (79.3% decrease for strain S 35). Heterologous LTA as well inhibited the adherence of S. saprophyticus to UC, however, to a lesser degree (6). Thus, non-specific inhibition of microbial adherence to UC by LT A from different staphylococcal strains suggest that these molecules obviously are quite similar structurally and functionally. Immunomodulating Activity of Lipoteichoic Acids It has become apparent that the LT A molecule of staphylococci and streptococci is highly reactive, binding spontaneously to a variety of host cell membranes and inducing tissue alterations and injury (6, 23, 34). However, it was of special interest to define the interactions of LT A with cellular elements of the immune system since recently it could be shown that LT A isolated from group A streptococci stimulated mitogenesis of human and murine lymphocytes (3, 38). One of the most intriguing findings was that LTA stimulated T- but not B-lymphocytes which contrasts with the B-lymphocyte mitogenicity of lipopolysaccharides (LPS) from Gram-negative bacteria (2). The difference in the response is most likely due to interacton of each molecule with distinct binding sites on cell membranes that are specific for each molecule. Lucigenin-dependent chemiluminescence may be considered to be a sensitive method to measure the release of oxygen radicals that are believed to process bactericidal properties. Because of their toxicity they obviously have cytotoxic (tumoricidal) effects on transformed cells and tissue damage during inflammation (1). As compared to nontreated control phagocytes, an increased chemiluminescence response of human monocytes and BALB/c-mouse peritoneal macrophages (which was statistically significant) could be induced with LT A preparations of two S. saprophyticus strains (SI and S 35). Mild deacylation procedures, which remove fatty acyl and D-alanyl ester groups and destroy the micellar structures were shown to suppress the biological activity of the LTAs. This finding was in favour of the postulation that the lipid fraction apparently mediates the biological activity (11, 25). Human polymorphonuclear leukocytes (PMNL) did not show a noteworthy chemiluminescence response after incubation with LTA (25). The increasing evidence that macrophages are important in host defense against neoplasms and certain infectious diseases has stimulated interest in agents that can enhance macrophage-mediated destruction of tumor cells and microorganisms. Bacteria and bacterial preparations are known to be highly effective in stimulating the immune system. Lipoteichoic acids (L TAs) and related cell surface macromolecules of staphylococcal origin have been suggested to be potent immunomodifiers stimulating cell populations involved in specific and non-specific resistance (28). The immunomodulating activity of staphylococcal LTAwas established in BALB/c-mice using spleen enlargement as a response measurement. Compared to non-treated mice, LT A administration induced a considerable splenomegaly which was statistically significant. It is generally accepted that there is a close correlation between spleen weight gain and antitumor activity of immunomodifiers. Accordingly, the spleen test can be used as a method for comparative studies on lymphoreticular stimulation properties of different substances as most of them involve the stimulation of the reticuloendothelial system (RES) or stimulation of recruitment and proliferation of competent cells (17). Since in the literature numerous tumor tests report a differing susceptibility
362
Y.Ohshima, H. L. Ko,
J. Beuth, K. Roszkowski, and W. Roszkowski
to immunotherapy, the spleen test and chemiluminescence response of phagocytic cells are considered most suitable for establishing standardized and reproducible results. To evaluate the antineoplastic activity, the effect of staphylococcal LTA on metastatic sarcoma L-1 tumor colonization into the lungs of BALB/c-mice was investigated. Compared to a control group, the number of pulmonary colonies was significantly lower in LTA-treated mice. Appearance of tumor colonies in organs other than lung has never been observed in this model system. However, the possibility that LTA treatment might cause extrapulmonary metastases was routinely checked but no evidence for such a possibility was found (28). Further studies concerning the reactivity of natural killer (NK)-cells and diverse subsets of T-Iymphocytes are in progress and might clarify the immunomodulating effect achieved by LT A treatment. Listeria monocytogenes is a facultative intracellular bacterium which parasitizes host macrophages (12). Specific immunity to Listeria is apparently conferred by a bicellular mechanism in which T -lymphocytes act as inducers and mononuclear phagocytes as expressors of immunity (19). Unspecific host defense mechanisms in early Listeria infection are mainly based on activated macrophages (39). Therefore, stimulation of the mononuclear phagocytic system (MPS) was supposed to enhance resistance to Listeria monocytogenes (8, 12). In a recent study the highly Listeriasensitive BALB/c-mouse strain was investigated. The protective role of activated macrophages after immunostimulation with LTAwas found to significantly reduce organ colonization of Listeria monocytogenes in BALB/c-mice. In vitro, exposure to peritoneal macrophages from LTA-treated mice significantly decreased listerial viability. Direct LTA-macrophage interactions causing a changed bactericidal activity could not be demonstrated (29). Immunoactive LT A-Related Staphylococcal Macromolecules The presence of capsules or slime substances in coagulase-negative staphylococci has already been demonstrated (13). However, knowledge concerning the biochemical and immunological properties of these substances is still limited. We previously found that cell surface substance (CSS) from several encapsulated strains of Staphylococcus epidermidis induced protective activity against those strains. Furtheron, active immunization of mice with CSS induced resistance against experimental infection with homologous staphylococci (13, 30, 36). CSS could be isolated from S. epidermidis (strain ATCC-31432) and fractionated by DEAE Sephadex ion exchange chromatography (30). Biochemical and serological analyses of CSS (sub)fractions resulted in the detection of 1) serologically inactive (sub)fractions 2) serologically active (sub)fractions (so called cell surface antigen, CSA) 3) teichoic acid-like (sub)fractions, as described (31) 4) nucleic acid (sub)fractions. Immunologically active (sub)fractions (CSA) were separated from teichoic acid-like (sub)fractions by ion exchange chromatography as shown for the preparation of CSA from Staphylococcus aureus (40). CSS and CSA were different from the S. epidermidis slime substance with respect to carbohydrate components and serological properties. According to our results, CSA seems to contain a type-specific antigen causing protective activity as well as absorbing activity of protective antibodies. Teichoic acidlike (sub)fractions displayed a lower protective activity in mice than CSA. Recent investigations demonstrated that experimental tumor growth in mice can be drastically reduced (statistically significant) after administration of CSS (26). The in vitro stimula-
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tion of human granulocytes and the increase in immunoglobulin M response suggest antiinfectious (antibacterial) activity of both, CSS and CSA (26, 27). Preliminary studies show that similar effects may be achieved in vivo after experimental infection of mice. The application of CSS and CSA apparently leads to a potent stimulation of the specific and non-specific cellular immune system. Granulocyte Activation by LT A-Related Macromolecules: A Receptor-Mediated Phenomenon Polymorphonuclear leukocytes (PMNL) generally are the first line of defense against extracellular bacterial pathogens. Phagocytosis of particulate material by those cells can be separated experimentally into two steps 1) attachment of the particle to the cell surface and 2) ingestion of the particle (9). The magnitude and quality of the response of the PMNL plasma membrane to a phagocytic stimulus appears to be govered by the number, size and shape of the particles attached to it. However, the zipper mechanism suggests that phagocytosis will occur only if PMNL membrane receptors can bind to the ligands (10). High molecular weight cell surface complex (CSC) was obtained after elution of crude phenol extract from Staphylococcus saprophyticus (strain S 1) from a Sepharose CL-6B column with ammonium acetate. Biochemical studies on this staphylococcal CSC revealed that it consisted of phosphate, protein, fatty acids and carbohydrates, however, the composition was different from lipoteichoic acid (L T A), cell surface substance (CSS), and cell surface antigen (CSA), respectively (16). In further investigations it was of special interest to define the interaction of CSC from S. saprophyticus with cellular elements of the specific and non-specific immune system. Contrary to LT A the CSC of staphylococcal origin can substantially enhance human PMNL chemiluminescence response whereas human monocytes could not be activated. Since heating as well as protease treatment of the CSC abolished its biological activity it was suggested that the protein part of the molecule was responsible for its PMNL activating potency (16). Since it was observed that formylmethionyl pep tides (fMLP) can be generated by invading bacteria (20) and apparently serve as ligands for appropriate receptors on PMNL, our attention was focussed on the fMLP receptor on human PMNL as eventual target for staphylococcal Csc. However, measurement of the PMNL chemiluminescence response to fMLP, CSC, and non-opsonized S. saprophyticus cells prior to and after pretreatment of the phagocytes with fMLP and CSC, respectively, suggested that the fMLP receptor of PMNL is not involved in the interaction with CSc. Evaluation of the bactericidal capacity of PMNL to opsonized S. saprophyticus cells positively correlated to chemiluminescence measurements. Pretreatment of the phagocytes with CSC evidently inhibited their bactericidal activity by interfering with attachment and phagocytosis. Apparently, CSC-mediated PMNL activtion is dependent on immunoglobuline/complement receptors but not on fMLP receptors. These data suggest that PMNL activation by S. saprophyticus is not only mediated by well known ligands like opsonins but may also be related to certain staphylococcal surface complexes such as lectins or cell surface macromolecules.
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