Method for the isolation of highly purified Salmonella flagellins

(1)JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1985, p. 1040-1044 0095-1137/85/121040-05$02.00/0 Copyright © 1985, American Society for Microbiology. Vol. 22, No. 6. Method for the Isolation of Highly Purified Salmonella Flagellins GEORGE F. IBRAHIM,l* GRAHAM H. FLEET,2 MARY J. LYONS,l AND RETA A. WALKER' New South Wales Department of Agriculture, Hawkesbury Agricultural Research Unit, Richmond,' and School of Food Technology, University of New South Wales, Kensington,2 New South Wales, Australia Received 13 May 1985/Accepted 26 August 1985. as. immunogens.. Bacterial flagella have been the subject of considerable physical and chemical characterization (1, 4, 9, 12, 14, 17, 22) and examination for their immunogenic properties (2, 7, 18, 20). The isolation of bacterial flagellin has been reported previously by a number of workers. Procedures generally involved the propagation of bacteria in complex media, followed by detachment of flagella by mechanical means with omni mixers, blenders, and other apparatus that produce violent agitation. Laborious steps of differential centrifugations and washings were then applied to isolate the flagella (2-4, 10, 12). Fey (6) recognized that the mechanical detachment of flagella may result in considerable disintegration of bacterial cells, thus liberating 0-antigen contaminants. Also, bacterial flagella detached by shearing from intact cells have been shown to consist of two parts, a helical filament and a proximal hook, eacb of which has entirely different antigenic specificities (8). Further purification of flagella isolated from Spirillum, Proteus, and Bacillus species has been achieved through the use of ion-exchange chromatography (16). Fey (6) applied preparative zone electrophoresis to separate salmonella flagellins from O antigens. Petersen et al. (19) eluted Treponema reiteri flagella from a DEAE-cellulose column, and after ultrafiltration, the flagella were purified by gel filtration. The development of immunoassay systems for the detection of salmonella (e.g., in foods) requires a supply of specific flagellar antisera of high titers. Highly purified flagellin preparations are a prerequisite for such antiserum production. This communication reports a method for the isolation of highly purified flagellins from Salmonella serotypes. The method is simple and reproducible and produces high yields.. infusion broth (BBL Microbiology Systems, Cockeysville, Md.) and later inoculated into flasks containing a modified version of the chemically defined medium described by Clark and Maal0e (5). This modified medium was prepared as follows. Solution A contained the following constituents in distilled water at the concentrations indicated (grams per liter): (NH4)2SO4, 10.0; Na2HPO4, 30.0; KH2PO4, 15.0; NaCl, 15.0; and Na2SO4, 0.055. Solution B consisted of the following in grams per liter (in distilled water): MgCl2, 0.25; CaCl2, 0.013; FeCl3 * 7H20, 0.0006; yeast extract, 0.125; and 0.31 of each of the amino acids DL-tryptophan, L-histidine, L-proline, L-threonine, L-arginine, glycine, DL-a-alanine, and L-methionine. After being adjusted to pH 7.2, solutions A and B were separately autoclaved; 100 ml of solution A was added to 400 ml of solution B. This mixture was supplemented with 10 ml of 25% (wt/vol) sterilized glucose. Isolation of flagellins. Highly motile isolates of each Salmonella serotype were obtained after several passages on 0.5% swarm agar in petri plates which had been incubated at 37°C for 16 h. Biphasic serotypes (kentucky and worthington) were rendered monophasic by elimination of the unwanted phases (i.e., i and z, respectively) by immobilization with homologous salmonella H-agglutinating serum (Wellcome Diagnostics, Rosabery, N.S.W. Australia). Each isolate was then inoculated into a tube containing 10 ml of the modified medium described above. After incubation, the tubes were used to inoculate five 500-ml quantities per isolate of the modified medium into 1,000-ml Erlenmeyer flasks. The flasks were incubated at 35 2°C in an orbital shaker incubator (Gallenkamp, England) at 80 rpm for 16 h. Salmonella cells were harvested by centrifugation at 5,000 g for 30 min and then mixed with saline solution to form a moderately thick suspension. The suspension was then adjusted to pH 2.0 with 1 M HCl and maintained at that pH under constant stirring for 30 min at room temperature. The bacterial cells, which were now devoid of flagella, were separated by centrifugation at 5,000 x g for 30 min. The supernatant, which contained detached flagellin in monomeric form, was further centrifuged at 100,000 x g for 1 h at 4°C to sedimnent the pH 2.0-insoluble material. The pH of the supernat4nt was adjusted to 7.2 with 1 M NaOH. Ammonium sulfate was added slowly with vigorous stirring to achieve two-thirds saturation (2.67 M). The mixture was ±. X. MATERIALS AND METHODS Organisms and growth media. Ten Salmonella serotypes. obtained from the Salmonella Reference Laboratory, Adelaide, South Australia. These serotypes, their reference numbers, and their antigen types 4nd phases are shown in Table 1. The serotypes were initially grown in brain heart were. *. Corresponding author. 1040. Downloaded from http://jcm.asm.org/ on April 10, 2020 by guest. Ten different Salmonella serotypes were grown in a chemically defined medium supplemented with 0.01% yeast extract. After sedimentation of the cells by centrifugation, flagella were detached by exposure to pH 2 for 30 min at room temperature. The flagellaless cells were removed by centrifugation, and the flagellin in the supernatant was further purified by high-speed centrifugation, ammonium sulfate precipitation, and dialysis in 50,000-molecular-weight-cutoff tubing. The 10 flagellin preparations were of a high degree of purity, as demonstrated by electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of salmonella H and O agglutination titers of antisera raised in rabbits with the flagellin preparations.

(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. Abram, D., and H. Koffler. 1964. In vitro formation of flagellinlike filaments and other structures from flagellin. J. Mol. Biol.. 9:168-185. 2. Ada, G. L., G. J. V. Nossal, J. Pye, and A. Abbot. 1963. Behaviour of active bacterial antigens during the induction of the immune response. Nature (London) 199:1257-1262. 3. Ansorg, R., G. Fraatz, and D. Strempel. 1980. Isolation, depolymerization and repolymerization of flagella of Pseudomonas aeruginosa. Zentralbl. Bakteriol. Mikrobiol. Hyg. 1 Abt. A 246:363-372. 4. Asakura, S., G. Eguchi, and T. lino. 1966. Salmonella flagella: in vitro reconstruction and over-all shapes of flagella filaments. J. Mol. Biol. 16:302-316. 5. Clark, D. J., and O. Maal0e. 1967. DNA replication and the division cycle in Escherichia coli. J. Mol. Biol. 23:99-112. method for the production of salmonella 6. Fey, H. 1979. A novel of H flagellar antigen. Il. Further purification for the preparation antisera. Zentralbl. Bakteriol. Mikrobiol. Hyg. 1 Abt. Orig. 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.

(5) 1044. 15.. 16. 17.. 18.. IBRAHIM ET AL.. coli K12: polymerization and molecular weight in comparison with Salmonella flagellins. Biochim. Biophys. Acta 36:117-139. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Martinez, R. J. 1963. A method for the purification of bacterial flagella by ion exchange chromatography. J. Gen. Microbiol. 33:115-120. McDonough, M. W. 1965. Amino acid composition of antigenically distinct Salmonella flagellar proteins. J. Mol. Biol. 12:342-355. Nossal, G. J. V., G. L. Ada, and C. M. Austin. 1964. Antigens in immunity. Il. Immunogenic properties of flagella, polymerized flagellin and flagellin in the primary response. Aust. J. Exp.. J. CLIN. MICROBIOL.. Biol. Med. Sci. 42:283-294. 19. Petersen, C. S., N. Strandberg Petersen, and N. H. Axelsen. 1981. A simple method for the isolation of flagella from Treponema reiter. Acta Pathol. Microbiol. Scand. Sect. C 89:379-385.. 20. Pitt, T. L. 1981. Preparation of agglutinating antisera specific for the flagellar antigens of Pseudomonas aeruginosa. J. Med. Microbiol. 14:251-260. 21. Vaitukaitis, J., J. B. Robbins, E. Neischlag, and G. T. Ross. 1971. A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. 33:988-991. 22. Vegotsky, A., F. Lim, J. F. Foster, and H. Koffler. 1965. Disintegration of flagella by acid. Arch. Biochem. Biophys. 111:296-307.. Downloaded from http://jcm.asm.org/ on April 10, 2020 by guest.

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