Fractions from Salmonella typhimurium - Infection and Immunity

Vol. 24, No. 3

INFECTION AND IMMUNITY, June 1978, p. 808-816 0019-9567/79/06-0808/09$02.00/0

Identification of Protective Cell Surface Proteins in Ribosomal Fractions from Salmonella typhimurium MICHAEL L. MISFELDTt AND WILLIAM JOHNSON* Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received for publication 28 December 1979

Cell surface antigen preparations from Salmonella typhimurium SR-il prepared by either trichloroacetic acid extraction or boiling in sodium dodecyl sulfate were able to protect C3H/HeJ, C3H/HeDub, and A/J mice. Some of the proteins found in these preparations were shown to exist in the protective ribosomal fraction isolated from S. typhimurium SR-il. Passage of ribosomes isolated from S. typhimurium SR-il and 6707 through a Sepharose 2B column removed the protective immunogen from 6707 ribosomes but did not completely remove it from SR-11 ribosomes. Immunity to salmonella infection in C3H/HeJ mice induced by ribosomal vaccines may be dependent on the presence of cell surface proteins in the ribosomal fraction.

Although Salmonella ribosomal fractions have been shown to be protective against Salmonella infection (8, 12, 14, 21, 22, 28, 30, 49), identification of the protective immunogen of Salmonella typhimurium has not been clearly established. Many divergent conclusions as to the protective substance of the ribosomal fraction have emerged. Evidence has been presented suggesting that the ribonucleic acid (RNA) (49), protein (22, 32), or contaminating substances including 0 antigens- (8, 12-14, 18, 19, 24) may be responsible for the protection associated with Salmonella ribosomal vaccines. Previous investigations with inbred mice have shown that individual inbred strains may vary in their ability to be protected by Salmonella antigens (30-32, 36). C3H/HeJ mice which are low responders to bacterial lipopolysaccharides (LPS) (10, 17, 41, 45, 48), were not protected against Salmonella infection by immunization with either purified endotoxin (9, 13, 31) or lipid A (32). In addition, purified Salmonella ribosomal RNA did not protect C3H/HeJ mice against a lethal challenge (32). But, C3H/HeJ mice were protected by ribosomes and protein extracted from the Salmonella ribosomal fraction (32). In contrast, A/J and C3H/HeDub mice were significantly protected by immunization with purified ribosomal RNA (32) or LPS

(31).

Earlier experiments have shown that the ribosomal preparations were contaminated with cell surface material (8, 12, 13, 24, 31). Although some investigators presume that the protective t Present address: National Institute of Child Health and Human Development, Bethesda, MD 20014.

antigen of Salmonella must be a cell surface component, several investigators have shown that both subcellular or ribosomal fractions (8, 12, 18, 30, 42, 49) and protein extracts from whole cells (3-7) can protect mice against a lethal Salmonella challenge. This study was undertaken to examine cell surface antigens for their protective ability and any possible association with the protective ribosomal fraction. MATERLALS AND METHODS Animals. The following inbred mouse strains were used: C3H/HeTex (Texas Inbred, Houston, Tex.); A/ J, C57BL/6J, and C3H/HeJ mice (Jackson Laboratory, Bar Harbor, Maine); and C3H/HeDub (Flow Laboratories, Dublin, Va.). Adult male mice weighing 16 to 24 g were used in all experiments. Mice were housed 10/cage during the experiments and given mouse chow and water ad libitum. Organisms. S. typhimurium strain SR-li was supplied by L. J. Berry, University of Texas, Austin. S. typhimurium strain Keller was obtained from Steven I. Vas, McGill University, Montreal, Canada. S. typhimurium strain 6707 was obtained from Nancy J. Bigley, Wright State University, Dayton, Ohio. S. typhimurium strain 6707 was originally isolated by Bruce N. Ames, University of California, Berkeley. Strain 6707 was initially described by Ames et al. (1) as TA1538, a defective deep rough mutant of S. typhimurium LT2. Salmonella enteritidis and Salmonella choleraesuis bioserotype Kunzendorf were obtained from the State Hygienic Laboratory, University of Iowa. Cultures were maintained on brain heart infusion agar (Difco). The 50% lethal dose (LD5o) was determined by the method of Reed and Muench (35) for each organism in every mouse strain employed. Survival was measured over 30 days. The LDr0 of the SR-il, Keller, and S. enteriditis strains was less than 10 organisms for all of the strains of mice used. The

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VOL. 24, 1979

SURFACE PROTEINS IN RIBOSOMAL FRACTIONS

LD50 for S. cholerasuis was approximately 1,000 organisms for all mouse strains. Preparation of cultures. Eight-hour cultures of S. typhimurium SR-li or 6707 were centrifuged in a Sorvall RC-2B centrifuge equipped with an SZ-14 continuous-flow rotor, and the cells were washed as previously described (30). Isolation of ribosomes. Ribosomes were prepared from the Salmonella cell suspensions as described previously (30). Bovin-type endotoxin. Endotoxin was prepared from SR-il bacterial cells by the trichloroacetic acid extraction method as described by Staub (43). Fifty grams of bacterial cells was suspended in five times its weight of 4VC water. An equal volume of 0.5 N trichloroacetic acid was added, and the mixture was allowed to incubate for 3 h at 40C. The mixture was then warmed to room temperature and centrifuged at 15,000 x g for 15 min. The supernatant fluid was neutralized to pH 6.5 with sodium hydroxide. Two volumes of -100C ethanol was added to the neutralized supernatant fluid. The precipitate which formed overnight was centrifuged at 40C at 15,000 x g for 15 min. The precipitate was dissolved in 1/10th the original volume of distilled water, neutralized, and dialyzed for 2 days against tap water and 2 days against sterile distilled water. The dialysate was centrifuged at 27,000 x g for 15 min and stored at 40C. Endotoxin preparation. Endotoxin was prepared from SR-11 as described previously (31, 51). The phenol layer of the endotoxin extraction was flash evaporated to remove any remaining aqueous layer. The phenol phase of the endotoxin extraction (PPE) was dialyzed against sterile distilled water and concentrated by lyophilization until the volume was 50 to 100 ml. It was then stored at 40C until further use. Purified endotoxin from the smooth strain of S. typhimurium LT2 was provided by Otto Luderitz (Max Plank Institute, Freibrug, West Germany). Preparation of SR-li bacterial cell envelope proteins. Bacterial cell envelope proteins were prepared from SR-il bacterial cells by the method of Wu and Heath (52). Fifty grams of bacterial cells was suspended in 250 ml of tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.0, containing 20,ug of both deoxyribonuclease and ribonuclease per ml. The suspension was sonicated in 40-ml amounts for two periods of 5 min each. The sonicated suspension was incubated at room temperature (25°C) for 1 h. The suspension was centrifuged at 1,000 x g for 15 min to remove any whole bacterial cells. The supernatant fluid containing the cell envelope particulate fraction was sedimented by centrifugation at 78,000 x g for 2 h. The supernatant fluid was discarded, and the pellet was suspended in 250 ml of 1% sodium dodecyl sulfate and placed in a boiling-water bath for 5 min. The suspension was centrifuged at 40,000 x g for 30 min. The supernatant fluid which contained the cell envelope proteins was sterilized by filtration and frozen at -20°C for further use. Ribosomal RNA. Ribosomal RNA from SR-u l was prepared as previously described (32). The phenol layer of the RNA extraction was flash evaporated to remove any contaminating aqueous layer. The phenol

809

phase of the RNA extraction (PPR) was dialyzed and concentrated as described above for the phenol phase of the endotoxin extraction. It was stored at 4°C. Gel filtration column chromatography of ribosomes. Sepharose 2B-300 was suspended in 0.01 M Tris buffer containing 0.01 M MgCl2 and 0.01 M NH4HCO3 (pH 7.8). The Sepharose was deaerated before pouring into the column. A column (50 by 2.5 cm) was filled with Sepharose until the bed volume was 35 cm high. The column was equilibrated with starting buffer by pumping 2 volumes of buffer through the column. The sample, containing either SR-li ribosomes or 6707 ribosomes incubated for 2 h at 37°C in starting buffer and 1% sodium dodecyl sulfate, was pumped onto the column by using reverse flow at a flow rate of 3 ml/5 min. Three-milliliter fractions were collected in a LKB UltroRac 7000 fraction collector. Fractions were read in a Perkin-Elmer model 165 spectrophotometer at 240, 260, and 280 nm. Immunizations and challenge. Groups of 10 mice were immunized intraperitoneally with 0.2 ml of the appropriate vaccine preparation. Each mouse received two injections of equal concentration of antigen 14 days apart. Mice were challenged with 100 LD5o's 14 days after immunization. Rabbits were immunized with the various vaccine preparations as described previously (30). Antiserum to the LT2 endotoxin was prepared in rabbits by intravenous injection of endotoxin on days 1 (10 ,ug), 4 (200 jig), 7 (300 jig), and 11 (500 pg). Immunodiffusion. Ouchterlony double immunodiffusion was done in 1% agarose made up in Trisbuffered saline containing 1% Triton X-100 by the method of Raynaud et al. (34). Three milliliters of melted agar was layered on a clean microscope slide (15 by 75 mm), and wells were punched in the solidified agar with an LKB gel punch. Twenty microliters of antisera or antigen was added to the wells with Drummond Wiretrols, and the slides were incubated at room temperature in a humidity chamber for 24 to 48 h. Immunoelectrophoresis. Ionagar was dissolved in distilled water to a concentration of 3%. Five parts of the ionagar was added to eight parts of Veronal buffer for electrophoresis and mixed thoroughly. Eight milliliters of melted ionagar in Veronal buffer was poured on a level, clean glass slide (70 by 90 mm). After the agar solidified, the holes and troughs were cut with an LKB gel cutter. Five to ten microliters of antigen was added to the wells. The slides were electrophoresed in a Buchler electrophoresis chamber. Contact between the slide and the electrode was made by using 1-cm-wide filter paper strips that had been soaked in Veronal buffer. Electrophoresis of the samples was done at 30 A or 150 to 200 V/slide for 1 h. After electrophoresis, the agar was removed from the troughs, and 40 yA of antiserum was added. The slides were then incubated at room temperature in a humidity chamber for 24 to 48 h. Acrylamide gel electrophoresis. Sodium dodecyl sulfate-slab gel electrophoresis was performed by the method of Laemmli (23). Biochemical analysis of subcellular fractions. Protein was determined by the method of Lowry et al. (25) with bovine serum albumin fraction V (Pentex

810

INFECT. IMMUN.

MISFELDT AND JOHNSON

Inc., Kankakee, Ill.) as a standard. RNA was measured by the orcinol method (11), and deoxyribonucleic acid was measured by the method of Ashwell (2). Yeast RNA and pancreatic deoxyribonucleic acid (Sigma Chemical Co., St. Louis, Mo.) served as standards. LPS was estimated by the carbocyanine assay described by Janda and Work (20). 2-Keto-3-deoxy sugars were assayed by the method of Waravdekar and Saslaw (50). LPS from S. typhimurium LT2 and 2-keto-3-deoxy sugar which served as standards were the gift of Otto Luderitz, Max Planck Institute, Freiburg, West Germany. Statistical evaluation. Significance levels for protection were determined by the Fisher exact probability test by the method of Siegel (39). Buffers. TM buffer contained 0.01 M Tris-hydrochloride, pH 7.5, containing 0.01 M MgCl2. TMA buffer consisted of TM buffer containing 1 M NH4Cl.

RESULTS washing of SR-11 chloride Ammonium ribosomes. To determine whether the immunogen was intrinsic to the ribosomes, SR- 11 ribosomes were prepared as shown in the flow diagram (Fig. 1). Each fraction was tested for its protective ability in A/J and C3H/HeTex mice. The results are presented in Table 1. All of the fractions tested in mice had protective activity. SR-11 ribosomes washed with 1 M NH4C1 induced significant levels of protection. In addition, the supernatant fluid from the washed ribosomes was also able to protect the mice from 100. CELLS BREAK 10 MINUTES IN BRAUN MSK CELL HOMOGENIZER CFG 27,000 X s 30 MINUTES I CFG 27,00M x

DlT*D

30 MINUTES DMG

FRAr'TN

CFG

125,000 X

a

I

(ULTRACENTR IFUGE CFG 125,000 X 3 HOURS

PELLET)

FRACTION II (RIBOSOMAL SUPERNATANT FLUID)

FRAr4N III (RIB OMES)

RESUSPIDED

1/f2 OF

VOLUME IN TMA CFG

125,000

X

3 HURS IV (SUPERNATANT FLUID FROM NH4CL WASHED RIBOSOMES)

FRACTION V (NH4CL WASHED

FRACTION

RIBOSOMES) FIG.

1.

Flow

diagram outlining the preparation of

S. typhimurium lysate fractions.

TABLE 1. Comparative immunogenicity of S. typhimurium SR-i1 lysate fractions

Immunizing material

Fraction I

Fraction II Fraction III Fraction IV Fraction V

Controls

% Survival"

Dose

([Lg of protein) 200 100

A/J

90b ND"

C3H/HeTex

80b ND

200 100

l0b

200

100"

80b

100

100"

88.89h

200 100

ND

200 100

100'

80" gob 88.89

0

77.78b ND

gob

70" 0

"Mice were challenged with 100 LD50 of SR-11 14 days after immunization. 'P C 0.005; significantly different from sham-immunized controls. 'ND, Not determined. a lethal Salmonella challenge. Polyacrylamide gel electrophoresis of the fractions is shown in Fig. 2. The electropherogram shows that NH4Clwashed ribosomes contain many of the same bands found in the other fractions. However, differences are also observed which indicate that some components are removed by NH4Cl washing. Column chromatography of ribosomal preparations. Molecular sieve chromatography of the ribosomal preparations was undertaken to determine whether the protective immunogen could be removed by chromatography. Romanowska (26, 37) reported that gel filtration of a mixture of LPS and RNA on a Sepharose 2B or 4B column could separate the two components from each other. Therefore, ribosomal preparations from S. typhimurium SR-11 and 6707 were chromatographed on a Sepharose 2B column to determine whether LPS or some other contaminating substance could be removed from the ribosomes and what effect this would have on the protective ability of the ribosomes. As shown in Fig. 3, both SR-11 and 6707 ribosomes chromatographed into two peaks. Those fractions which had a ratio of absorbance at 260 to 280 nm of 1.10 or less (fractions 15 to 25) were combined for peak 1, and those fractions with an absorbance ratio at 260 to 280 nm of 1.70 and above (fractions 43 to 52) were combined for peak 2. The two peaks along with the starting ribosomal preparations were injected into mice to determine their protective ability. Groups of

01r-vTz

VOL. 24, 1979

5


1

-2~

_

. . .^

811

-by all of the fractions tested. C3H/HeJ mice were protected by SR-11 ribosomes, trichloroacetic acid-extracted endotoxin, and cell envelope proteins but not by PPE or PPR. However, PPE and PPR prolonged survival for almost 2 weeks inwss the C3H/HeJ mice (personal observation).

Sr is

FIG. 2. SDS-polyacrylamide gel electrophoresis of

NH4C_-washed SR-Il lysate fractions. Symbols: 1, fr-action I; 2, fr-action 1I; 3, fr-action III; 4, fr-action IV; 5, fraction V.

4 mice were immunized with either 50 Atg of peak 1 material or 200 jig of peak 2 material. The dataB AI NS obtained from this experiment are presented in Table 2. SR-il ribosomes and the two peaks obtained from chromatography of SR-il ribosomes were equally protective in both the A/J and C3H/HeDub mice. 6707 ribosomes and 1 from the chromatographed 6707 ribosomes B were protective for the A/J and C3H/HeDub mice. However, peak 2 of the chromatographed 1. 6707 ribosomes could not induce significant proX tection in either the A/J mice or the C3H/ Os HeDub mice. Protective ability of S. typhimurium SR11 cell wall preparations. The previous experiments had shown that some of the protective I immunogen could be removed either by highsalt wash or by column chromatography. Since ribosomal fractions had been shown to be contaminated with cell surface materials (8, 12, 13, 18, 31), various preparations of cell wall antigens were tested for their immunogenicity in mice. Cell envelope proteins, bovin-type endotoxin containing protein (trichloroacetic acid extraction), protein in PPE, and protein in PPR were FRACTION .MNSER used to immunize mice. The results are preFIG. 3. Elution pattern of S. typhimurium SR-11 sented in Table 3. A/J mice were protected by and 6707 ribosomes on a Sepharose 2B column. The all of the immunogens except the trichloroacetic buffer used was TM containing 0.01 M NaHCO.3. The acid-extracted endotoxin which was toxic to the numbers refer to the peaks eluting offthe column. (A) A/J mice at doses of 100 or 50 jig of protein. Absorbance at 260 nm; (B) absorbance at 240 nm. C3H/HeDub mice were significantly protected Symbols: 0, 6707 ribosomes; 0, SR-il ribosomes.

peak

812

INFECT. IMMUN.


TABLE 2. Protective ability of Sepharose 2B column fractions % Survival Fraction

SR-11 ribosomes Peak 1 Peak 2 6707 ribosomes Peak 1 Peak 2

A/J

C3H/HeDub

gob

100b

Loob 70b 80"

gob gob 70b 60"

0

30

loob

10 0 Controls Mice were challenged with 100 LDr0 of SR-11 14 days after immunization. b p _ 0.01; significantly different from sham-immunized controls.

TABLE 3. Immunogenicity of SR-1I cell wall preparations and ribosomes % Survival' Dose' Immunogen (n)

(pg) A/J C3H/HeDub C3H/HeJ 200 100' 100' 100' SR-11 ribosomes 70' 88.89' 200 100' SR-11 cell envelope proteins ND' 100' 80' 100 TCA-extracted endotoxin' 0 200 90' 60' Phenol-phase endotoxin (PPE) 9WI 0 200 70c Phenol-phase RNA (PPR) 0 0 0 Controls a Micrograms of protein as determined by method of Lowry

(25).b

Mice were challenged with 100 LDr, SR- 1114 days after final immunization. P c 0.01; significantly different from sham-immunized controls. " ND, Not done, toxic to mice. TCA, trichloroacetic acid.

lope proteins were injected into mice. The immunized mice were challenged with 100 LD5o of either S. typhimurium SR-11, S. typhimurium Keller, S. enteritidis, or S. choleraesuis. The protection observed is shown in Table 4. SR-11 ribosomes were able to significantly protect both the A/J and C3H/HeDub mice from challenge with SR-11 and Keller but not against challenge with S. enteritidis or S. choleraesuis. SR-11 cell envelope proteins gave the same protection as the SR-11 ribosomes with the exception that they were able to protect the immunized mice against a lethal challenge of S. choleraesuis. DISCUSSION Although the identification and location of the protective antigen of S. typhimurium have been the subject of intensive investigation, the complexity of the antigenic makeup of Salmonella has made the elucidation of the protective immunogen difficult. Because of the close association of somatic proteins with the polysaccharide fraction of the 0 antigen, the role of 0 antigens of LPS in the immunogenicity of the ribosomal vaccines has not yet been resolved. Several investigators (12, 13, 24) have shown that protective ribosomes isolated from smooth strains of Salmonella are contaminated with 0 antigens, whereas ribosomes isolated from mutants lacking the 0 antigen are not protective. This implies that 0-antigen contamination of the ribosomes

A

I

6-0

I

Immunodiffusion analysis of these preparations is shown in Fig. 4. With rabbit anti-SR-11 ribosomes, it could be shown that precipitating antibody could be found against all of the fractions with the exception of PPE and PPR. Immunoelectrophoresis of these fractions shown in Fig. 5 and 6 illustrates that rabbit antiserum to SR11 ribosomes formed precipitin lines with SR-il ribosomes cell envelope proteins, trichloroacetic acid-extracted endotoxin, ribosomal protein, and PPR. Mouse antiserum, prepared by immunizing C3H/HeJ mice with SR-11 ribosomes, was able to form precipitin lines with those antigens which could protect the mice from a lethal challenge. FIG. 4. Immunodiffusion studies of SR-11 cell wall Specificity of protection induced by SR- preparations. (A) Rabbit anti-SR-1l ribosomes. Sym11 ribosomes or cell envelope proteins. To bols: ASR, anti-SR-11 ribosomes; 1, SR-I I ribosomes; determine the specificity of the protection ob- 2, trichloroacetic acid-extracted endotoxin; 3, PPE; served, SR-l1 ribosomes and SR-il cell enve- 4, SR-il ribosomes; 5, cell envelope proteins; 6, PPR.

5

ASR

43

i2


VOL. 24, 1979 ASR

ALT2

ASR

ALT2

ASR

ALT2

FIG. 5. Immunoelectrophoretic analysis of Salmonella subcellular fractions. Symbols: ASR, rabbit anti-SR-Il ribosomes; ALT2, rabbit anti-LT2 endotoxin; 1, 6707 ribosomes; 2, SR-lI ribosomes; 3, SR11 cell envelope proteins; 4, protein extracted from SR-Il ribosomes; 5, LT2 endotoxin.

0

FIG. 6. Immunoelectrophoretic analysis of Salmonella protein fractions. Symbols: ASR, rabbit antiSR-Il ribosomes; MASR, C3H/HeJ mouse anti-SR11 ribosomes; 1, PPR; 2, SR-lI ribosomes; 3, SR-Il trichloroacetic acid-extracted endotoxin; 4, SR-I1 cell envelope proteins.

813

It is possible that ribosome preparations may contain multiple protective antigens. Eisenstein (12) has presented evidence to suggest that ribosome preparations isolated from S. adelaide contain a heat-stable non-O antigen in addition to 0 antigen. In a previous study (32) our laboratory used the C3H/HeJ mice to attempt to resolve the role of endotoxin in the immunity induced by the salmonella ribosomal vaccines since these mice are unresponsive to endotoxin. Immunization of these mice with ribosomal preparations from S. typhimurium protected against lethal challenge, and no antibody to 0 antigens was detected in mice immunized with ribosome preparations. This suggested that although the ribosomes may be contaminated with 0 antigens, the presence of 0 antigens could not account for the immunity observed in C3H/HeJ mice. In contrast, a recent study by Eisenstein and Angerman (13) showed that, although C3H/HeJ mice were unresponsive to purified endotoxin, immunization of these mice with ribosome preparations isolated from S. typhimurium induced antibody to 0 antigens. The reasons from this discrepancy have not yet been resolved. Barber and Eylan (6) have contended that the main protein antigens of Salmonella have been overlooked because most of the classical techniques used to extract bacterial antigens destroy TABLE 4. Specificity ofprotection induced by SR-i1 ribosomes or cell envelope proteins % Survival' Mouse Material injected

challenge

A/J

SR-il ribosomes'

S. typhimurium SR-il S. typhimurium Keller S. enteritidis S. chokraesuis S. typhimurium SR-li S. typhimurium Keller S. enteritidis S. choleraesuis S. typhimurium

100' 100C

C3H/HeDub

80c is necessary for protective activity. An alternative explanation suggested by the protection ob80' NDd served with the cell envelope proteins in C3H/ 40' HeJ mice (Table 3) is that the 0 mutants may 50' 50c either have lost or have decreased amounts of SR-il cell enve100' 70' certain cell envelope proteins. If these cell enlope proteins' velope proteins, either alone or completed with 100' 87.5c 0 antigens, were necessary for immunity, then ND 11.11' ribosomes isolated from mutants lacking 0 an90C 85.7' tigens would not be protective. Lin and Berry Controls 10 0 (24) showed that ribosomes isolated from a muSR-li 0 0 S. typhimurium tant of S. typhimurium requiring the addition of Keller galactose for expression of 0 antigen gave conS. enteritidis ND 10 sistently lower protection in mice than did ri30 S. choleraesuis 0 bosomes isolated from the smooth parent strain 'Mice were with 100 LD,0 of the appropriate challenged when the mutant was grown in the presence of organism 14 days after final immunization. b galactose. This suggests that the mutant even Mice injected with 200 itg of protein of appropriate vacwhen grown in the presence of galactose may cine. ' P c 0.025; significantly different from sham-immunized either produce less 0 antigen or may be deficient in certain envelope proteins necessary for full controls. d ND, Not determined. expression of immunity when associated with ' P 0.05; not significantly different from sham-immunized controls. the ribosomes.

814


these proteins. Barber and Eylan (3-5, 7) have shown that proteins isolated from whole Salmonella cells could protect mice against infection both against the homologous organism and heterologous strains. Work in this laboratory confirms that protein isolated from the ribosomal fraction could protect mice against infection (22, 32). These observations provided the basis for examining any possible relationship between somatic proteins and proteins isolated from the ribosomal fraction. High-salt washing of ribosomes was unable to remove enough of the immunogen to cause the washed ribosomes to lose their immunogenicity. But, since the supernatant wash was also able to protect mice from a lethal challenge, it would suggest that one of the protective immunogens is extrinsic to the ribosomes. This is consistent with the work of Hoops et al. (18). The findings observed in the Sepharose 2B studies indicate that the quantity of the extrinsic antigen may vary depending on the chemotype of the organism used. 6707 ribosomes, prepared from an Re mutant, lost the immunogen when they were passed through the column, whereas SR-li ribosomes retained their protective capacity for mice after passage through the column. It is possible that there may be multiple protective antigens in the ribosome preparation and that 6707 ribosomes only contain the antigen which is found in peak 1 (Fig. 3). Work by Severtsova and Stanislovsky (38) is in agreement with our work with SR-il ribosomes. They found that protective activity was associated with both a high polymeric (molecular weight 2 2 x 106) and a low-molecular-weight compound (molecular weight, 15,000 to 20,000) which had been isolated from the S. typhimurium antigenic complex passed through Sepharose 2B. In view of the fact that most protective antigens are usually surface antigens (44), our observations would support that concept. Many investigators have shown that ribosomes are contaminated with cell surface material (8, 12, 18, 24, 31). Studies with membrane fractions and other surface components have shown these fractions may protect against Salmonella infection (15, 16, 47). Mates and Yosipovici (27) have stated that the Salmonella protective antigen is located on the surface of the bacteria. Our preparations of cell envelope proteins and trichloroacetic acidextracted endotoxin which can protect C3H/HeJ mice from Salmonella challenge indicate that cell surface proteins are protective in mice. The observation that these preparations formed precipitin lines with rabbit antiserum prepared against SR-11 ribosomes provides evidence that

INFECT. IMMUN.

ribosomes are either contaminated with some of these cell surface components or they have similar antigenic determinants. The protective ability of the PPE and PPR fractions in the A/J and C3H/HeDub mice suggests that a lipoprotein or a protein similar to the one described by Melchers et al. (29), Morrison et al. (33) and Sultzer and Goodman (46) is present in the ribosome preparations and may be necessary for the immunogenicity of the ribosome preparations. Additional evidence to support a role for a lipoprotein is suggested by the fact that the trichloroacetic acid-extracted endotoxin and cell envelope protein fractions which would contain the lipoprotein are protective for the C3H/HeJ mice. Skidmore et al. (40) have shown that the responsiveness of C3H/HeJ mouse spleen cells to LPS is dependent on the method used to extract the LPS. Whether protective ability is also dependent on the method of extraction is not known. The search for the protective immunogen in trichloroacetic acid-extracted endotoxin and cell envelope proteins and its relationship to the "endotoxin protein" or lipoprotein may finally locate the long-sought protective antigen of S. typhimurium. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant AI10449 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Ames, B. N., F. D. Lee, and W. Dueston. 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 70:782-786. 2. Ashwell, G. 1957. Colorimetric analysis of sugars. Meth-

ods Enzymol. 3:73-105. 3. Barber, C., and E. Eylan. 1974. The proteins from S. enteriddis: their protective role in mice and the antibodies induced during infection with the homologous strain. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 226:331-335. 4. Barber, C., and E. Eylan. 1975. Heterologous protections in experimental salmonellosis. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 230:452-457. 5. Barber, C., and E. Eylan. 1975. Confirmation of the protective role of proteins from S. typhimurium in infection of mice with their natural pathogen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 230:461-465. 6. Barber, C., and E. Eylan. 1976. The unfortunate role of precedent in bacteriology. L The main antigens of Salmonellae: the proteins. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 234:5359. 7. Barber, C., E. Eylan, and R. Heiber. 1972. The protective role of proteins from SalnoneUa typhimurium in infection of mice with their natural pathogen. Rev. Immunol. 36:77-84. 8. Berry, L. J., G. N. Douglas, P. Hoops, and N. E. Prather. 1975. The background of immunization against salmonellosis, p. 388-398. In E. Neter and F.

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