Fraenkel-Conrat, H., and H. Olcott. 1948. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups.
INFECTION AND IMMUNITY, Sept. 1987, p. 2047-2051 0019-9567/87/092047-05$02.00/0 Copyright © 1987, American Society for Microbiology
Vol. 55, No. 9
Physical and Morphological Characteristics of Eucaryotic Ribosomes and Lipopolysaccharide Complexes MARSHALL PHILLIPS* AND KIM A. BROGDEN National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 50010 Received 9 February 1987/Accepted 28 May 1987
Lipopolysaccharides (LPS) from Pasteurella multocida or Brucella abortus were complexed with Aspergillus fumigatus ribosomes by mixing and fixation for 3 days in 3.8% formaldehyde. To investigate the nature of their physical association, ribosomes, LPS, and ribosome-LPS complexes were (i) centrifuged in CsCI gradients to determine buoyant densities, (ii) examined by electron microscopy, and (iii) monitored by sodium dodecyl sulfate-pqlyacrylamide gel electrophoresis. Ribosomes were found to bind to LPS from either P. multocida or B. abortus, producing complexes with densities of 1.45 to 1.50 g/ml. The buoyant density of the fixed ribosomes was 1.54 g/ml, and the buoyant densities of the fixed P. multocida and B. abortus LPS were 1.41 and 1.35 g/ml, respectively. Electron microscopy showed that formaldehyde-fixed ribosomes were attached to the LPS. Complexing of ribosomes to LPS may be of importance as a potentiator or carrier for experimental subunit vaccines.
The effectiveness of subunit vaccines depends upon the presence of three components: an immunogen, a carrier, and a potentiator (29). Ribosomes from bacteria or other microorganisms have been shown to be effective carriers or
List Biologicals, Campbell, Calif. Brucella abortus 2308S LPS was obtained by a butanol extraction procedure (19) followed by proteinase K digestion as described by Hitchcock and Brown (12). LPS was assayed for protein concentration (17) before use with bovine serum albumin as the standard. Each preparation was found to contain less than 1.0% protein. Ribosomes. Ribosomes were isolated from Aspergillus fumigatus germlings and purified as previously described (22). Briefly, germlings were disrupted in a mechanical cell homogenizer, and crud/e ribosome preparations were obtained by differential centrifugation. Purified ribosomes were obtained after chromatography over a Sepharose CL-4B column (Pharmacia, Inc., Piscataway, N.J.), followed by centrifugation at 100,000 x g for 5 h. These ribosome preparations were found to be free of contaminating spore material as assessed by chemical determinations, analytical ultracentrifugation, and electron microscopy. Ribosome-LPS mixtures. A. fumigatus ribosomes, LPS from P. multocida or B. abortus, and ribosome-LPS mixtures were fixed in 3.8% formaldehyde for 3 days as previously described (20, 21). Two ribosome-to-LPS ratios were examined; one contained 2.0 mg of ribosomes and 2.0 mg of LPS (1:1 ratio), and the other contained 2.8 mg of ribosomes and 0.7 mg of LPS (4:1 ratio). Ribosomes alone and both P. multocida X-73 LPS and B. abortus LPS alone were fixed in 3.8% formaldehyde similarly. Cesium chloride field-formed gradients. Portions of the above preparations were added to CsCl solutions in 0.05 M Tris hydrochloride at pH 7.8. Field-formed gradients of 40 or 45% CsCl by weight were generated by centrifugation in tubes for the SW55.1 centrifuge for 36 h at 39,000 rpm at 25°C. Density positions in the tubes were determined by refractive indices. Electron microscopy. Samples containing the formed bands were negatively stained with 1.0% uranyl acetate as previously described (5, 6, 22). SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of LPS after fixing with formaldehyde was performed in 12% discontinuous gradients as described by Laemmli (13). Samples containing 3 to 13 jig of LPS were heated at 100°C with 2% SDS and 2% 2-
potentiators for antigens providing protective immunity (10). The mechanism of action of ribosomal vaccines has been controversial. Some investigators (14, 15) have provided evidence that the ribosomes are intrinsically immunogenic and protective. Others, such as Eisenstein and Angerman (4, 7), demonstrated that the lipopolysaccharides (LPS) contained in Salmonella typhimurium ribosomal vaccines provided the protective moiety after experimental infection. Later, Phillips and Rimler added Pasteurella multocida ribosomes to subimmunogenic amounts of P. multocida LPS in vaccines and observed passive hemagglutination titers to LPS and protection in chickens against challenge exposure (25). Formaldehyde has been used as a fixing agent to maintain the integrity of ribosomal particles (28). Fixing of the ribosomes and LPS with formaldehyde was also shown to improve their stability and effectiveness as immunomodulators. Heterologous eucaryotic ribosomes were found to be as effective when substituted (21, 25). Phillips et al. (20) demonstrated that formaldehyde crosslinked complexes of S. typhimurium LPS and heterologous ribosomes could overcome the hyporesponsiveness of C3H/HeJ mice to antibody formation against LPS. The ribosome-LPS complex induced immunoglobulin G antibody, which suggested that complexing with the ribosomes may have converted the LPS to a T-cell-dependent immunogen. This paper reports
(i) the physical characteristics of these complexes in cesium chloride gradients and (ii) the morphology of the ribosome-LPS complexes formed by formaldehyde cross-linking. MATERIALS AND METHODS
LPS. P. multocida X-73 LPS was obtained as a gift from R. B. Rimler and prepared as previously described (26). Salmonella minnesota wild-type LPS was obtained from *
Corresponding author. 2047
2048
PHILLIPS AND BROGDEN
INFECT. IMMUN. 10 I. -.
-1.40
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1.45
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1.54
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1.35
-
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FIG. 2. CsCl gradient bands of fixed B. abortus LPS and fixed A. fumigatus ribosome-B. abortus LPS complex. All samples were fixed with formaldehyde in 40% CsCl gradients.
mercaptoethanol for 2 min. The gels were silver stained as described by Tsai and Frasch (30). Development conditions were controlled to yield only LPS bands. Gel diffusion precipitin test. Monospecific antibody was prepared in chickens against P. multocida LPS (24) and used in the gel diffusion precipitin test described by Phillips and Rimler (11, 21).
complexes were not formed and the ribosomes and LPS banded at their expected densities (data not shown). Formaldehyde-fixed B. abortus LPS buoyed in a single band at 1.35 g/ml (Fig. 2). When ribosomes were complexed with B. abortus LPS at a ratio of 1:1, the density and appearance of LPS were altered after binding to the ribosomes, and discrete bands were not observed. SDS-PAGE. Samples of the P. multocida LPS and ribosomal complexes were electrophoresed on discontinuous SDS-PAGE, and the LPS was identified by silver staining procedures (Fig. 1). Proteins solubilized from the ribosomes by the SDS treatment were observed by Coomassie brilliant blue stain (data not shown). The P. multocida LPS from both ribosome-LPS complexes migrated as double bands which were nearly identical to P. multocida LPS fixed alone and to untreated P. multocida LPS. The faint-appearing highmolecular-weight component in lane 3 is due to a reduced sample concentration of LPS in the preparation (see the legend to Fig. 1). Gel diffusion precipitin test. The gel precipitin activity of P. multocida LPS in the CsCl gradient was determined with antibody directed to the LPS (Fig. 3). Similar precipitin bands were obtained from the unfixed LPS, the fixed LPS, and the LPS in the ribosome-LPS complexes. LPS was not detected in other fractions of the gradient.
RESULTS Density gradient centrifugation. A. fumigatus ribosomes fixed with formaldehyde banded in the CsCI gradient at a density of 1.54 g/ml (Fig. 1). Fixed P. multocida LPS banded in the gradient at a density of 1.41 g/ml (Fig. 1). However, when the ribosomes were mixed with the LPS and fixed in the presence of formaldehyde, a complex was formed that differed in density from either component. Ribosomes fixed with P. multocida LPS at a 1:1 ratio resulted in ribosomeLPS complexes banding at a density of 1.45 g/ml (Fig. 1). When ribosomes were fixed with P. multocida LPS at a ratio of 4:1, ribosome-LPS complexes were also formed and banded at a density of 1.50 g/ml but there was an excess of ribosomes as evidenced by a band at the same density as that of the ribosomes themselves (1.54 g/ml). Separately, fixed ribosomes were added with fixed P. multocida LPS in CsCl and centrifuged under identical conditions. In this case,
FIG. 3. Gel diffusion precipitin pattern of P. multocida fractions from CsCl gradient tubes. Wells: 1 and 2, portions of CsCl gradient without bands; 3, fixed LPS band (Fig. 1, lane 2); 4, fixed ribosome band (Fig. 1, lane 4); 5, fixed ribosome-LPS (4:1) complex (Fig. 1, lane 3); 6, P. multocida LPS (unfixed); A, antisera to P. multocida LPS.
FIG. 1. CsCl gradient bands and SDS-PAGE patterns of A. fumigatus ribosomes, P. multocida LPS, and A. fumigatus ribosome-P. multocida LPS complexes. All samples in 45% CsCl gradients were fixed with formaldehyde. Lanes: 1, fixed ribosomeLPS complex (1:1; 10 p.g of LPS, 10 Rg of ribosomes); 2, fixed LPS (10 p.g); 3, fixed ribosome-LPS complex (4: 1; 3 [±g of LPS, 12 ~Lg of ribosomes); 4, fixed ribosomes (10 [Lg); 5, unfixed LPS (10 jig); 6, unfixed S. minnesota marker (10 ~Lg); 7, protein marker (5 p.g).
RIBOSOME-LPS COMPLEXES
VOL. 55, 1987
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FIG. 4. Electron micrographs showing negative-stained preparations of A. fumigatus ribosomes (a), P. multocida LPS (b), and complexes formed after fixation of ribosomes to LPS (c and d).
Electron microscopy. A. fumigatus ribosomes were approximately 27.76 ± 0.99 nm in size, and formaldehyde fixation retained their integrity (Fig. 4a). The preparation was also free of any extraneous material. P. multocida LPS appeared as a flat ribbon that branched freely (Fig. 4b). Its diameter varied but averaged 16.0 ± 0.6 nm. B. abortus LPS, however, consisted of small, oblong, electron-transparent units in palisade aggregates 0.13 ± 0.01 by 0.06 ± 0.01 ,um in size (Fig. 5b). In the gradient fractions containing the ribosome-LPS complexes, the ribosomes could be seen attached directly to the LPS molecule of either organism. In ribosome-P. multocida LPS complexes, the ribosomes were individually attached periodically along the LPS ribbon (Fig. 4c). High magnification showed that the ribosome was intimately attached firmly enough to prevent it from being stripped off during CsCl gradient centrifugation (Fig. 4d). In ribosomeB. abortus LPS complexes, the ribosomes were attached in clusters to the LPS molecule like an agglutination of ribosomes and LPS (Fig. Sc). DISCUSSION The physical and morphological characteristics of ribosome-LPS complexes were examined in this study to explain the effectiveness of their immunizing properties. Rimler and Phillips found that ribosomes complexed with LPS potentiated the response of chickens to a subimmunogenic dose of
LPS and that the ribosomal protein served as a carrier in the vaccines. Fixing these preparations in formaldehyde served to enhance their stability without compromising their immunologic properties (25). The use of formaldehyde for fixing A. fumigatus ribosomes to LPS from P. multocida or B. abortus yielded bands in CsCl gradients at densities different from those of the individual components. These complexes were formed during the fixing process since fixed ribosomes added separately to fixed P. multocida LPS in the CsCl and centrifuged under identical conditions formed bands only at the densities observed for the ribosomes and LPS, respectively. The buoyant densities of the ribosome-LPS complexes were those expected from a combination of the ribosomes and LPS. Fixing of ribosomes, LPS, or ribosomes and LPS in mixtures did not appear to alter (i) the electrophoretic migration of LPS in SDS-PAGE gels, (ii) the serologic properties of the LPS, and (iii) the morphologic appearance of the ribosomes, LPS, or either component in ribosomeLPS complexes. The immunizing properties of ribosomeLPS mixtures have been shown to be far superior to those of either component alone (25). The ultrastructural morphology of A. fumigatus ribosomes was identical to that reported previously (6, 22), and the morphology of P. multocida LPS was typical of the LPS extracted from Escherichia coli and other bacterial species (1, 5, 16, 27). However, the ultrastructure of B. abortus LPS
IFC.IMN INFECT. IMMUN.
PHILLIPS AND BROGDEN
2050
1.
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.:
5,
to allow attachment of the ribosomes. At a 1:1 ratio of ribosomes to LPS, all of the ribosomes were observed to be attached to the LPS, and these complexes had a density of 1.45 gIml. When the ribosome-to-LPS ratio was increased to 4:1, additional ribosomes were able to attach, increasing the density of the complexes to 1.50 g/ml. In electron micro-
.M.r N. * "A ,
graphs of these preparations, the LPS ribbon
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micrographs showing negative-stained preparafumigatus ribosomes (a), B. abortus LPS (b), and
Electron
of A.
complexes formed after fixation of ribosomes
to LPS
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usually described and may have been by which it was extracted. The method of extraction (1-3, 23) and the ionic composition of the buffer (2, 3) have been shown to influence the morphology of LPS. The ribosomes could be easily observed attached to the LPS of either bacterial species, and the degree of attachment could be related to the morphologic structure of the LPS. The P. multocida LPS was linear, with enough surface areas was
different from that
due to the method
was
virtually
covered with ribosomes and the rest were unattached (data not shown). This explains the presence of complex bands at different buoyant densities (Fig. 1). In contrast, the morphology of the B. abortus LPS was that of a small, flat sheet, and only a limited number of ribosomes could bind. These preparations were difficult to visualize and photograph since they often agglutinated into large, amorphous mats. Insertion of the ribosome into the P. multocida LPS ribbon may result in an orientation that leaves the ribosome moiety responsible for its carrier function exposed in order to modulate a humoral or cellular response to the LPS. Attachment of LPS to ribosomes was influenced more by the conditions of incubation in the presence of formaldehyde than the source of either the A. fumigatus ribosomes or LPS. In previous studies, ribosomes from P. multocida (21), B. abortus (20, 21), or chicken liver (21) have been observed to complex with LPS from P. multocida (21) or S. typhimurium (20). Formation of LPS-ribosome complexes was dependent upon the presence of formaldehyde. Similar complexes were not formed when ribosomes and LPS were combined before incubation and centrifuged under identical conditions. The electron micrographs indicate that the ribosomes are on the surface of the LPS ribbon or LPS aggregates. Since ribosomes contain amino acids, pyrimidines, and purines and LPS contain amino and amide groups, a number of possible inter- or intralacing cross-links may occur (8, 9, 18). The binding of ribosomes to LPS is such that it was not disrupted in the presence of 40 to 45% CsCl. It may also be a matter of conformation of the LPS in the fixing solution. P. multocida LPS had more surface area for ribosomes to attach, whereas B. abortus LPS was more of a sheet and had less surface area for attachment of ribosomes. Whether the morphology of the molecule has any role in the degree of complexing and immunogenicity will have to be determined by alteration of the morphologic structure of the individual LPS molecule with detergents (23), solvents (1, 19), or mechanical disruption, followed by examination of immunogenicity. LITERATURE CITED 1. Amano, K., and K. Fukushi. 1984. Chemical and ultrastructural comparison of endotoxins extracted from Salmonella minnesota wild type and R mutants. Microbiol. Immunol. 28:149-159. 2. Amano, K., and K. Fukushi. 1984. Electron microscopic studies of endotoxins treated with alkaline and acid reagents. Microbiol. Immunol. 28:161-168. 3. Amano, K., T. Sato, and K. Fukushi. 1985. Effect of pH on turbidity and ultrastructure of endotoxins extracted from Salmonella minnesota wild type and Re mutant. Microbiol. Immunol. 29:75-80. 4. Angerman, C. R., and T. K. Eisenstein. 1980. Correlation of the duration and magnitude of protection against Salmonella infection afforded by various vaccines with antibody titers. Infect. Immun. 27:435-443. 5. Brogden, K. A., R. C. Cutlip, and H. D. Lehmkuhl. 1986. Complexing of bacterial lipopolysaccharide with lung surfactant. Infect. Immun. 52:644-649. 6. Brogden, K. A., M. Phillips, J. R. Thurston, and J. L. Richard. 1984. Electron microscopic examination of ribosome preparations from germinated spores of Aspergillus fumigatus. Mycopathologia 77:165-171. 7. Eisenstein, T. K., and C. R. Angerman. 1978. Immunity to
VOL. SS, 1987
8. 9.
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experimental Salmonella infection: studies on the protective capacity and immunogenicity of lipopolysaccharide, acetonekilled cells, and ribosome-rich extracts of Salmonella typhimurium in C3H/HeJ and CD-1 mice. J. Immunol. 121:1010-1014. Feldman, M. Y. 1973. Reactions of nucleic acids and nucleoproteins with formaldehyde. Prog. Nucleic Acid Res. Mol. Biol. 13:1-49. Fraenkel-Conrat, H., and H. Olcott. 1948. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups. J. Am. Chem. Soc. 70:2673-2675. Gregory, R. L. 1986. Microbial ribosomal vaccines. Rev. Infect. Dis. 8:208-217. Heddleston, K. L., J. E. Gallagher, and P. A. Rebers. 1972. Fowl cholera: gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis. 16:925-936. Hitchcock, P. J., and T. M. Brown. 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154:269-277. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Lieberman, M. M., and R. C. Allen. 1986. Opsonic activity of antisera to ribosomal vaccine fractions with live and formalinized Pseudomonas aeruginosa. Can. J. Microbiol. 32:531533. Lieberman, M. M., H. L. Walker, E. Ayala, and I. Chapa. 1986. Active and passive immunization with Pseudomonas aeruginosa ribosomal vaccines and antisera in the burned rat model. J. Surg. Res. 40:138-144. Lopes, J., and W. E. Inniss. 1970. Electron microscopic study of lipopolysaccharide from an avian strain of Escherichia coli 018. J. Bacteriol. 103:238-243. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Moeller, W., J. Rinke, A. Ross, G. Buddle, and R. Brimcombe. 1977. The use of formaldehyde in RNA-protein cross-linked studies with ribosomal subunits from E. coli. Eur. J. Biochem. 76:175-187. Morrison, D. C., and L. Leive. 1975. Fractions of lipopolysaccharide from Escherichia coli O111:B4 prepared by two extrac-
RIBOSOME-LPS COMPLEXES
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tion procedures. J. Biol. Chem. 250:2911-2919. 20. Phillips, M., T. K. Eisenstein, and J. Meissler. 1985. Immunomodulation of the antibody response to lipopolysaccharide in C3H/HeJ mice by complexing with heterologous ribosomes. Infect. Immun. 48:244-247. 21. Phillips, M., and R. B. Rimler. 1984. Protection of chickens by ribosomal vaccines from Pasteurella multocida is dependent upon homologous lipopolysaccharide. Am. J. Vet. Res. 45: 1785-1789. 22. Phillips, M., J. R. Thurston, K. A. Brogden, and J. L. Richard. 1982. Isolation of 80S ribosomes from spores of Aspergillus fumigatus and their antigenicity in rabbits. Mycopathologia 77:165-171. 23. Ribi, E., R. L. Anacker, R. Brown, W. T. Haskins, B. MaImgren, K. C. Milner, and J. A. Rudbach. 1966. Reaction of endotoxin and surfactants. I. Physical and biological properties of endotoxin treated with sodium deoxycholate. J. Bacteriol. 92:1493-1509. 24. Rimler, R. B. 1984. Comparisons of serologic responses of White Leghorn and New Hampshire red chickens to purified lipopolysaccharides of Pasteurella multocida. Avian Dis. 28: 984-989. 25. Rimler, R. B., and M. Phillips. 1986. Fowl cholera: protection against Pasteurella multocida by ribosome-lipopolysaccharide vaccine. Avian Dis. 30:409-415. 26. Rimler, R. B., P. A. Rebers, and M. Phillips. 1984. Lipopolysaccharides of the Heddleston serotypes of Pasteurella multocida. Am. J. Vet. Res. 45:759-763. 27. Shands, J. W., J. A. Graham, and K. Nath. 1967. The morphologic structure of isolated bacterial lipopolysaccharide. J. Mol. Biol. 25:15-21. 28. Spirin, A. S., N. V. Belitsina, and M. I. Lerman. 1965. Use of formaldehyde fixation for studies of ribonucleoprotein particles by caesium chloride density-gradient centrifugation. J. Mol. Biol. 14:611-615. 29. Stewart-Tull, D. E. S. 1983. Immunopotentiating products of bacteria, p. 1-42. In C. S. F. Easmon and J. Jeljaszewicz (ed.), Immunization against bacterial disease. Academic Press, Inc., New York. 30. Tsai, C., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels. Anal. Biochem. 119:115-119.
