(2) ISOLATING HIGHLY PURIFIED SALMONELLA FLAGELLINS. VOL. 22, 1985. TABLE 1. Antigenic characteristics and molecular weights of flagellins from 10 Salmonella serotypes as determined by. SDS-polyacrylamide gel electrophoresis Serotypea. Oranienburg 1,9,12:g,m:Kentucky Waycross Abortus-equi Tennessee 4,12:d:Untypeablec Worthington Lille. Reference type nob H-antigen. Antigen phase. Mol wt. 1254 1267 1285. m,t gm. I I. 56,900 58,400. Z6. 1312 1451 1623 1634 1635 1695 1721. e,n,x Z29 d 1,2. Il I Il I I II. 55,800 47,700 54,600 58,400 57,100 57,100. II I. 57,700 50,700 and 49,700. Z4,Z23. l,w. Z38d. held overnight at 4°C and then centrifuged at i5,000 x g for 15 min at 4°C. The precipitate, which contained polymerized flagellin, was dissolved in approximately 5 ml of distilled water and then transferred to dialysis tubing which had a molecular weight cutoff of 50,000 (Spectrum Medical Industries, Los Angeles, Calif.). Dialysis was carried odt under running tap water initially for 2 h and thên for 18 h at 40C with constant stirring in 4 liters of distilled watet containing 20 g of activated charcoal (Ajax Cherticals, Australia). The dialyzed flagellin preparations were then lyophilized and stored at 4°C in the dark over dried silica gel. Electron microscopy. Samples were negatively stained with 2% (wt/vol) phosphdlùngstic acid neuttalized to pH 7.4 with 1 M KOH. Electron micfoscopy was carried out with a Philips EM300 electron microscope. SDS-polyacrylamide gel electrophoresis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was carried out in 0.1% SDS-pdlyacrylamide slab gels by using the Tris glycine discontinuous buffer system of Laemmli (15) universally supplemented with 0.5 M urea. The stacking and separating gels contained 4 and 10% acrylamide, respectively. Electrophoresis runs were done with a vertical electrophoresis unit (LKB, Bromma, Sweden). Each gel lane was loaded with 20 to 25 p.g of flagellin and was run at a constant current setting of 7.5 mA per gel slab for 18 h at room temperature. A standard molecular weight protein mixture was purchased from Bio-Rad Laboratories, Richmond, Calif. The mixture consisted of six proteins with molecular weights of 14,400, 21,500, 31,000, 45,000, 66,200, and 92,500. Serological methods. Thirty-six New Zealand White female rabbits were used to produce specific flagellin antisera by injection of 50 ,ug of polymeric flagellin in complete Freund adjuvant per rabbit. The antigens were administered on the backs of three to four animals by the multiple intradermal injection method described by Vaitukaitis et al. (21), except that antigen emulsions contained heat-killed Mycobacterium tuberculosis at a final concentration of 0.3 mg/ml and the administration of 0.5 ml of crude Bordetella pertussis vaccine was omitted. The responses to this immunization were monitored regularly over a period of 200 days by determining salmonella H agglutination titers in tubes. H agglutination was done by incubating 0.5 ml of twofold serial dilutions of. antisera and 0.5 ml of 18-h Salmonella cultures (in brain heart infusion broth) at 50°C for 2 h. O agglutination was done the same way by using boiled (10 min) salmonella cell suspensions and incubating the antigen-antiserum mixtures at 50°C for 4 h. RESULTS Growth of Salmonella serotypes and flagellin yields. The growth of Salmonella serotypes in the modified medium of Clark and Maal0e (5) was similar to that in the more complex brain heart infusion broth. This result was established by determinihg the final absorbance of the cultures in each medium. The pH. of the modified medium used to produce cell biomass for the isolation of flagellin averaged 4.75 + 0.18 at the end of the incubation period. This indicated that the buffering capacity of the medium was sufficient to prevent a breakdown of flagella, since these are known to dissociate into subunits below pH 3.0.. Electron-microscopic examination of Salmonella serotypes indicated that the synthesis of flagella in the modified diedium was generally similar to that in brain heart infusion broth in terms of shape, length, and density on cells. The yield of flagellin preparations varied considerably. In general, 5 g of wet cell mass of each of the 10 serotypes produced 28.7 ± 17.4 mg of freeze-dried flagellin. Purity of flagellins. Electron-tnicroscopic examination of all flagellin preparations revealed a high degree of purity, as only polymeric flagellin units could be observed (Fig. 1). Only one protein band was observed after SDSpolyacrylamide gel electrophoresis with each of the flagellins from serotypes worthington (phase l,w), kentucky (phase Z6), 1,4,5,12:-:1,2, and 4,12:d:- (Fig. 2). Flagellin from serotype lille repeatedly showed two major protein bands with estimated molecular weights of 49,700 and 50,700. Flagellins from serotypes 1,9,12:g,m:-, waycross, and abortus-equi showed one major band as well as one or two barely visible bands (Fig. 2). The remaining flagellins, from serotypes tennessee and oranienbufg, showed one major band each às well as two or five minor bands, respectively. These minor bands were also apparent after electrophoresis of flagellitis that had been incubated at pH 7 for 2 h at 37°C in the presence of an equal mass of the trypsinlike enzyme inhibitor aprotinin (Boehringer GmbH, Mannheim, Federal Republic of Germany). Aprotinin incubations were done directly with polymeric flagellins as well as after monomerization of polymers by boiling. The estimated molecular weights of the major protein bands of all flagellins ranged from 47,700 to 58,400 (Table 1). The immune response of rabbits to each flagellin was apparent after less than 30 days from the time of immunization. High-titered H-agglutinating antisera were found using the tube agglutination test, and these were specific in that each antiserum was capable of agglutinating only the Salmonella serotype from which the immunogen was isolated. Substantial differences in the immune response to each flagellin as well as between animals immunized with the same immunogen were observed. During the postimmunization period (200 days), the H agglutination titer varied but generally remained fairly high. The maximum H agglutination titers of the antisera from the 10 groups of animals ranged from 10,000 to 1,280,000. By contrast, the O agglutination titers of antisera, which were regularly monitored during the entire postimmunization period, were <10 with nine of the flagellin preparations. The remaining preparation,. Downloaded from http://jcm.asm.org/ on April 10, 2020 by guest. a All serotypes were monophasic except for kentucky and worthington; in these two cases, the unlisted phases (ie., i and z, respectively) were eliminated before flagellin isolation. b From the Salmonella Reference Laboratory. c 1,4,5,12:-:1,2 d Two major protein bands were observed.. 1041.
(3) 1042. IBRAHIM ET AL.. J. CLIN. MICROBIOL.. from serotype waycross, produced an O agglutination titer of up to 320. DISCUSSION. Compared with other published methods (2-4, 6, 10, 12), the method described in this report for the isolation of Salmonella flagellins is simple, does not require sophisticated technical equipment, and is straightforward. Isolation requires only 3 days to accomplish from the time of medium inoculation until the end of flagellin dialysis. The isolated flagellins are of such a high purity that further purification is not necessary. Previous investigators concerned with the production of flagellins cultured their bacterial cells in complex media. -. (c),. waycross. (d),. containing high levels of proteinaceous nutrients (4, 6, 14, 20). Such extraneous proteins unnecessarily complicate flagellin purification and may readily carry over to contaminate the final product. To avoid this problem, we cultured salmonella in a chemically defined medium supplemented with only trace amounts of yeast extract to stimulate a high degree of growth and high yields of flagellin. Electronmicroscopic examination revealed no apparent differences between flagellum lengths or density on cells grown in either this medium or the more conventional brain heart infusion broth. The dissociation of flagella from salmonella cells was achieved by reducing the pH to 2 with HCl for only 30 min. This resulted in a complete detachment and breakdown of. Downloaded from http://jcm.asm.org/ on April 10, 2020 by guest. FIG. 1. Electron-microscopic observation of Salmonella flagellins from serotypes fille (a), tennessee (b), 1,9,12:g,m: kentucky (e), and 4,12:d:- (f). Bars, 200 nm..
(4) ISOLATING HIGHLY PURIFIED SALMONELLA FLAGELLINS. VOL. 22, 1985 A. qu. B. -. C. «M. D E. F. G. «. t-. 1. H. J. K<. L. _ -. of flagellin. to contamination with proteolytic enzymes. These bands could be an intrinsic part of flagellin subunits or could represent contamination with nonflagellar proteins. However, assuming that these minor bands do represent contamination, then the magnitude of such contamination is quite insignificant. Densitometric scans of the polyacrylamide gels indicated that these contaminants represented less than 1% of the total protein mass tested. The estimated molecular weights of the major protein bands of all flagellin preparations ranged from 47,700 to 58,400. This result is similar to the findings of Kondoh and Hotani (14), who reported that the molecular weights of flagellin preparations from seven Salmonella serotypes were 51,000 to 57,000. Immunization of rabbits with our flagellin preparations produced antisera with very high H agglutination titers. By contrast, the O agglutination titers were < 10 for nine flagellin preparations and between 10 and 320 for the remaining flagellin preparation. This result demonstrates that the flagellins produced had a high degree of freedom from contaminating O antigens, which are known to have adjuvant properties and are thus highly immunogenic. ACKNOWLEDGMENT. flagella into the monomeric form and, ap art from this, salmonella cells so treated appeared to be nmorphologically normal when examined with the electron m microscope. The characteristic dissociation of flagella in HC I at pH values below 3 has been known since 1945 (1). Fey (6) dissociated flagella from salmonella cells at pH 1.5 overriight at 4°C but reported the presence of O antigens in his fl2agellin preparations. Such antigens could not be completely separated from flagellin after adsorption with activated cha]rcoal, repeated O centrifugation and washing, anionic exchange ~, masking by 0 antibodies, and lectin adsorption. Fey (6), however, succeeded in separating the O antigens by pr eparative zone electrophoresis. It is conceivable that the prolonged fiagellum dissociation step used by Fey (6) acccounted for the 0-antigen contamination. Many other workedrs have dissociated flagella from bacterial cells by mechaniical means (16, 17, 19, 20). We considered this approach undesirable because of its possible fragmenting of the bactaerial cells. For example, terminal hooks have been obse rved in many flagellin preparations obtained from mechani ically disrupted cells (1, 11, 17, 19). Contamination of flagell in preparations with RNA, O antigens, or O lipopolysacchl arides has also been reported (16, 17). The dissociation of flagella at pH 2 into the monomeric form made flagellin no longer centrifugable at 100,000 x g and, consequently, this property was explo cited to remove pH 2.0-insoluble contaminants. Further piurification was achieved by ammonium sulfate precipitati(on of flagellin. This approach has been used previously by ot ther workers (6, 13, 17). Our final purification step was done tby dialysis with membrane tubing (molecular weight cutoff, 50,000) in the presence of activated charcoal. Electron-microscopic examinations of all flagellin preparations revealed a high degree of purity. No terminal hooks or other contaminating materials were observ'ed. Also, SDSpolyacrylamide gel electrophoresis results sh owed only major protein bands with five flagellin preparations. Other flagellin preparations showed, apart from major bands, location ofa a barely visible or minor bands. The ass mociation proteolytic enzyme with bacterial flagellin pireparations has been reported (M. Farquhar and H. Koffier, Bacteriol. Proc., p. 30, 1968). However, the presence (of minor bands (Fig. 2) with our flagellin preparations appearscd not to be due. 'asking. )ne wofrlaellin.. This research was supported by a grant from the Australian Dairy Research Committee. LITERATURE CITED 1. 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A 245:55-56. 7. Fey, H., and H. P. Wetzstein. 1975. Production of potent salmonella H antisera by immunization with flagellae, isolated by immunosorption. J. Med. Microbiol. Immunol. 161:73-78. 8. Kagawa, H., S. Aizawa, S. Yamaguchi, and J. Ishizu. 1979. Isolation and characterization of bacterial flagellar hook proteins from salmonella and Escherichia coli. J. Bacteriol. 138:235-240. 9. Kagawa, H., H. Morishita, and M. Enomoto. 1981. Reconstitution in vitro of flagellar filaments onto hook structures attached to bacterial cells. J. Mol. Biol. 153:465-470. 10. Kagawa, H., K. Owaribe, S. Asakura, and N. Takahashi. 1976. Flagellar hook protein from Salmonella SJ25. J. Bacteriol. 125:68-73. 11. Kerridge, D., R. W. Horne, and A. M. Glauert. 1962. Structural components of flagella from Salmonella typhimurium. J. Mol. Biol. 4:227-238. 12. Kobayashi, T., J. N. Rinker, and H. Koffler. 1959. Purification and chemical properties of flagellin. Arch. Biochem. Biophys. 84:342-362. 13. Koffler, H., G. E. Mallett, and J. Adye. 1957. Molecular basis of biological stability to high temperatures. Proc. Natl. Acad. Sci. USA 43:464-477. 14. Kondoh, H., and H. Hotani. 1974. Flagellin from Escherichia. Downloaded from http://jcm.asm.org/ on April 10, 2020 by guest. FIG. 2. SDS-polyacrylamide gel electrophoresis preprations from 10 Salmonella serotypes. Lanes: A, kentucky; C, 1,4,5,12:-:1,2; D, 4,12:d:-; E, stamndard molecular weight protein mixture; F, 1,9,12:g,m:-; G, way(cross; H, lille; I, tennessee; J, abortus-equi; K, standard molecula r weight protein mixture; L, oranienburg.. 1043.
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