FEMS Microbiology Immunology 47 (1989) 485-490 485 Published by Elsevier

FEMSIM 00077

Immunogenic endotoxin associated protein from a rough strain of Salmonella

Marshal l Phill ips 1, R a y m o n d Cas tagna 2, Ba rne t M. Sul tzer 2 an d T o b y K. Eisenste in 3

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, 1,4, U.S.A., 2 Department of Microbiology and Immunology, State University of New York, Downstate Medical Center, Brooklyn, New York, NY, U.S.A.

and 3 Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA, U.S.A.

Received 10 April 1989 Accepted 2 June 1989

Key words: Endotoxin protein; Salmonella; Rough strain

1. SUMMARY 2. I N T R O D U C T I O N

A multimolecular complex of polypeptides found associated with the lipopolysaccharide en- dotoxin in Salmonella, referred to as endotoxin- associated protein (EP), has been extracted from a rough strain of Salmonella typhimurium which does not synthesize 0 antigens. Since standard methods of extraction applicable to smooth strains of Salmonella were not successful for this rough strain, two modified procedures were developed. The resulting products were similar to smooth EP in terms of their biochemical, physical and mito- genic properties. When the immunogenicity of the rough EP was characterized by a protection assay in mice challenged with virulent Salmonella, it was found that the rough EP preparations were protec- tive; however, they were not as active as the EP from a smooth strain of S. typhimurium.

Correspondence to: Toby K. Eisenstein, Department of Micro- biology and Immunology, Temple University School of Medi- cine, 3400 N. Broad St., Philadelphia, PA 19140, U.S.A.

There is continuing interest in defining the nature of the protective antigens on Salmonella, both from a theoretical point of view and for vaccine development [1]. In mouse models of Salmonella infection, it is firmly established that 0 antigens can induce protective antibodies [2-4]. Preparations of outer membrane proteins of Salmonella have also been shown to be protective in mouse studies [5-7], and anti-porin antibodies have been shown to be present in human sera after infection with Salmonella typhi [8] or other Gram-negative organisms [9].

Endotoxin protein (EP) is a protein complex closely associated with lipopolysaccharide (LPS) in the outer membrane of most Gram-negative bacteria [10,11]. It is a multimolecular composite of three to five hydrophobic polypeptides which vary in number and concentration between differ- ent genera and species [11]. EP stimulates B-cell proliferation, activates polyclonal antibody syn- thesis and acts as a protein adjuvant for T cell- dependent and T cell-independent antigens

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[10-14]. When smooth organisms are used as the EP source, EP co-purifies with LPS extracted by mild procedures such as trichloroacetic acid (TCA) or butanol [10,14,16]. The protein fraction can be separated from the TCA-LPS by subsequent ex- traction with phenol water [10]. That EP consists of outer membrane proteins when TCA extraction is used has been verified by Goldman et al. [17].

We have shown previously that EP extracted from smooth strains of Salmonella can protect innately Salmonella-resistant strains of mice against challenge with virulent Salmonella [15,18]. In CD-1 mice EP induces protection against 500 LDs0 doses [15]. Whether or not EP from rough strains of Salmonella would also be immunogenic, however, has not been addressed. It is conceivable that their antigenic characteristics might be al- tered. Additionally we considered the possibility that although 2-keto-3-deoxyoctonate (KDO) was not detectable in the smooth EP preparations, indicating intact LPS was absent, small amounts of free 0 antigen might have contaminated the EP. Given the potent adjuvant activity of EP, it was possible that the protective activity we observed was due in part to this combined effect.

Consequently, we set about to extract EP from a rough strain of Salmonella typhimurium (TA1659, Rc Type) which does not synthesize 0 antigen due to the absence of galactose 4-epimerase [19]. This report describes two methods for obtaining EP from the rough strain, and compares the capacity of these fractions with EP obtained from a smooth strain of S. typhimurium in the protection of mice against challenge with virulent Salmonella.

3. MATERIALS AND METHODS

3.1. Bacterial strains S. typhimurium, strain TA1659, was obtained

from Dr. Bruce A.D. Stocker (Dept. of Medical Microbiology, Stanford University School of Medicine). It was originally isolated by Dr. Bruce Ames (Dept. of Biochemistry, University of Cali- fornia at Berkeley), and has a deletion of the gal gene duster, rendering it incapable of synthesizing 0 antigens. S. typhimurium Wl18-2 is a smooth, virulent strain (0:4, 12) used extensively in previ-

ous work in our laboratory [18,20]. Organisms used to prepare EP were grown for 18 h at 37°C in trypticase soy broth (Difco Laboratories, De- troit, Mich). Cells were harvested by centrifuga- tion, washed, and frozen at - 2 0 ° C.

3.2. EP extraction EP was prepared from the smooth strain,

Wl18-2, as previously described [10]. Briefly, harvested cells were directly extracted with TCA, and the TCA-LPS obtained was further treated with phenol water. The EP fraction was recovered from the phenol phase, and designated WEP. When this procedure was applied to the rough strain, TA1659, there was no yield of material following the TCA extraction. Butanol extraction [14] also yielded little material. Further, pre- liminary studies showed that there was minimal mitogenic activity in the TCA, butanol, or water phases when the rough organism was used. There- fore, two methods were developed, both of which yielded EP from the rough strain,

3.3. Preparation of interface EP (IEP) In the first method 75 g (wet weight) of cells of

TA1659 were extracted with a mixture of 325 ml of water and 325 ml of water-saturated butanol [16]. Extractions were carried out with gentle stir- ring at 25°C for 15 min and the phases were partitioned by low-speed centrifugation at 4 ° C into an aqueous, a butanol, and an interface frac- tion. The recovered butanol phase was re-ex- tracted with 150 ml of water, and the recovered aqueous phase was added to the original water phase. The water phase was concentrated and dialysed through an Amicon PM-30 membrane, and the butanol phase was dialysed against water and then lyophilized. Neither of these fractions contained significant amounts of protein, nor were they mitogenic. However, when the interface frac- tion was extracted with 90% phenol at 68 °C for 15 rain EP was recovered from the phenol phase by precipitation with 70% ethanol at - 2 0 ° C . This EP fraction, after washing four times with ethanol, was lyophilized and designated IEP.

3.4. Direct extraction of EP In the second procedure 75 g (wet weight) of

TA1659 was mixed with 75 ml of phenol water

and heated at 68°C for 15 min [21]. However, following the separation of the phenol and water phases by centrifugation, three volumes of 70% ethanol were added to the clarified phenol phase at - 2 0 °C to directly precipitate the solubilized protein. Subsequently, the precipitate was washed four times with ethanol and lyophilized. This frac- tion was designated direct EP (DEP).

3.5. S. typhimurium mitogenic (STM) protein STM, a protein-rich fraction prepared from

cells of a Re mutant of S. typhimurium, was purchased from Ribi Immunochemicals (Ham- ilton, Mont.).

3.6. Fraction characterization To assess the composition of each fraction,

protein was estimated by the method of Lowry et al. [22], using bovine serum albumin as standard. Total carbohydrate was analysed by the method of Dubois et al. [23], and KDO was determined by the method of Karkanis et al. [24]. Nucleic acid was estimated by absorption at 259 nm. Discon- tinuous sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method described by Laemmli [25]. 12%-Gels were used and stained with Coomassie blue. The slab gel was run overnight at 30 V with 75/xg of protein applied for each sample. Densitometer tracings were done with a Quick Scan Jr. (Helena Labs. Corp., Beaumont, Tex.).

3. 7. Protection Female CD-1 mice, purchased from Charles

River Laboratory, were given 25 #g of the desired immunogen i.p. in 0.5 ml of saline, and 2 weeks later a booster of 50 #g. Three weeks after the booster, all animals were challenged i.p. with viru- lent S. typhimurium Wl18-2 in saline, and mortal- ity was scored for 30 days.

4. RESULTS

4.1. Characteristics of EP fractions IEP prepared from TA1659 was found to con-

sist of 84.8% ± 1.8% protein and 4.5% 5: 1.6% carbohydrate. DEP contained 81.5% ± 1.9% pro-

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I 2 3

-68K

45K

O

Fig. 1. SDS-PAGE and densitometer tracing of WEP (lane 1), IEP (lane 2), and DEP (lane 3). - - WEP; . . . . . IEP,

. . . . . . DEP.

tein and 5.6% + 1.7% carbohydrate. Both prepara- tions had < 1.0% KDO and < 1.0% nucleic acid. These values are in agreement with those for EP from smooth S. typhimurium, S. typhi and E. coli [10,12]. Comparison of the band patterns of EP extracted from the rough strains obtained by the two different methods with that of EP from the smooth strain (WEP) is shown in Fig. 1. All three preparations gave two prominent bands between 25 and 45 kDa, which are typical of EP from Salmonella [12,15]. In addition, all three extracts had a distinct band between 45 and 68 kDa. The smooth strain showed a diffuse area of Coomassie blue between 12 and 25 kDa, probably a result of degraded higher weight polypeptides, whereas the rough strains showed multiple faint bands above 45 kDa. STM showed two low-molecular-weight polypeptide bands, one at approximately 12 kDa and the other at about 15 kDa (not shown).

4.2. Mitogenicity When the IEP and DEP preparations were

compared with those of WEP for mitogenic activ- ity [26] using C 3 H / H e J splenocytes (Table 1), all three extracts were found to stimulate [3H]thymi- dine incorporation. At 10 /~g, however, the smooth-strain EP (WEP) gave significantly greater stimulation than either extract of the rough strain.

4.3. Protective capacity The two rough EP preparations, plus a com-

mercially available protein-rich fraction from S.

488

typhimurium, were further compared with EP from the smooth strain for abil i ty to protect mice against challenge with virulent Salmonella (Table 2). The results show that bo th methods of prepar ing EP from the rough strain (IEP and DEP) yielded fractions tha t were protective against challenge doses up to 27 LDs0s. However, nei ther of these fractions was as effective as EP from the smooth strain, which gave 70% and 90% survivors against

challenge doses of 170 or 300 LDs0s, respectively. The STM was not protective against the lowest challenge dose used in these experiments (17

LDs0s). To determine whether the EP extracted from

the rough strain had induced anti-0 antibodies, pooled sera were collected from three addi t ional mice of each of the groups used in the second protect ion exper iment (see Table 2, footnote c).

These animals were bled from the retroorbi tal plexus before immuniza t ion , and 3 weeks after the booster enzyme- l inked i m m u n o s o r b e n t assays (ELISAs) were carried out using wells coated with phenol-water LPS extracted f rom the smooth s t rain of S. typhimurium W l 1 8 - 2 [27]. The LPS used as ant igen in this assay had less than 1% associated pro te in and was inactive as a mi togen for C 3 H / H e J mouse spleen cells. Nei ther IEP, DEP, W E P nor STM was found to induce anti-0 anti- body when tested at our s tandard star t ing serum

Table 1

Mitogenic activity for C3H/HeJ spleen cells of EP extracted from rough and smooth S. typhimurium

Fraction /~g/ml Mean net cpm S.I. b of incorporation o f [ 3 H ] t h y m i d i n e a

Control 1925 + 104

IEP 1 29 906 -I- 1663 15.5 10 31926 + 1227 16.6

DEP 1 22065+ 368 11.5 10 30669+ 462 15.9

WEP 1 30295+ 617 15.7 10 53 585 + 1886 27.8

a All cultures were harvested after 48 h of incubation. The mean of triplicate cultures is shown + the standard error.

b S.I. = stimulation index. The mean cpm of the stimulated cultures divided by the mean cpm of the control cultures.

Table 2

Protection by EP fractions extracted from rough and smooth Salmonella

Mouse Survivors~total b,c

immunized Challenge dose (LDs0 doses) d with a

12 17 27 60 85 170 300

WEP e (smooth) 10/10 10/10 9/10 10/10 10/10 7/10 9/10

DEP f (rough) - 4/10 4/10 - 0/10 0/10 -

IEP g (rough) 6/10 8/10 6/10 0/10 0/10 0/10 0/10

STM h _ 0/10 1/10 -- 0/10 0/10 - Saline

or PBS 0/10 0/10 0/10 0/10 0/10 0/10 0/10

a Mice received two i.p. injections spaced 2 weeks apart of 50 /tg and 25 ttg, respectively, of the desired EP preparation suspended in 0.5 ml of PBS. Controls received saline or PBS.

b Challenge was i.p. three weeks post-booster. Survivors scored at 30 days post-challenge.

c Data were collected from three experiments. Experiment 1, challenge doses of 12, 60, and 300 LDs0s; experiment 2, challenge doses of 17, 85 and 170 LDs0s; experiment 3, 27 LDs0 dose.

d 1 LDs0 of S. typhimurium Wl18-2 is 1 × 104 cells, i.p. e Extracted from S. typhimurium strain Wl18-2. f Extracted from S. typhimurium strain TA1659 directly by

phenol. g Extracted from S. typhimurium strain TA1659 by butanol

foUowed by phenol extraction of interface material. h Purchased from Ribi Immunochemicals, Hamilton, MT.

d i lu t ion of 1 : 200. The same groups of pooled sera were also tested using an ELISA in which EP from the smooth strain was the test antigen. Both the rough and the smooth EP prepara t ions induced signif icant an t i -EP ant ibodies (titer < 10 000), but the titers were 4- to 8-fold lower in mice receiving IEP or D E P as compared with WEP.

5. D I S C U S S I O N

The results show that two different extract ion methods yielded mater ia l f rom a rough strain of S. typhimurium with biochemical , physical and mito- genic activities s imilar to those of EP obta ined from a smooth strain. Both IEP and D EP were able to induce ant ibodies that reacted with smooth-s t ra in-der ived EP, and both of these pre-

parations protected against moderate challenge doses of virulent Salmonella. However, the EP prepared from the smooth strain was significantly more protective than either extract from the rough strain and induced higher levels of anti-EP anti- body. In our previously published experiments, the protective capacity of EP from smooth Wl18-2 was titrated and found to give 90% survivors against a challenge of 500 LDs0 doses, and 40% survivors against 1000 LDs0 doses [15]. Thus it can be concluded that EP extracted from a rough Salmonella strain that does not produce 0 antigens can protect mice against a significant challenge dose with virulent Salmonella, but the protection is at least 10-fold less than that afforded by the EP obtained from the smooth strain.

Another consideration is that core polysac- charide material might be present in the EP ex- tracts, and could contribute to the protection in- duced by these fractions. Others have shown core oligosaccharide material to be present in outer membrane protein extracts of Salmonella of the Rb 2 type made by methods different from those used in this present work; however, based on absorption of passively transferred immune serum and on assay of antibodies to core antigens in immunized mice, these oligosaccharides were con- cluded not to be protective immunogens [6,7].

Of more direct concern perhaps is that the concentration of the polypeptides between 25 and 45 kDa in the smooth WEP is noticeably greater than that found in both the DEP and IEP, which correlates with the higher levels of anti-EP anti- body produced by the WEP. In addition, the mitogenic activity of the WEP at an optimal con- centration is also significantly greater than both the DEP and IEP. As previously shown, the mito- genic activity of the individual proteins of the smooth S. typhimurium EP is additive [11]. There- fore, it is possible that the greater activity ex- hibited by the WEP may be the result of a higher concentration of immunogenic polypeptides, but at this time a conclusive argument cannot be made, since the immunogenicity of the purified individual components from the rough and smooth EPs has yet to be determined.

In conclusion, these studies present two meth- ods for extraction of EP from a rough strain of

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Salmonella. The results of the protection studies show that 0 antigens are not essential for EP to induce protection; however, E P from a smooth strain is more active as an immunogen than the EP from a rough strain.

A C K N O W L E D G E M E N T S

We thank Mr. Joseph Meissler for excellent technical assistance, and Dr. Loran Killar for help in carrying out some of the protection experi- ments. This work was supported by Public Health Service grants AI15613 and AI16782 from the National Institutes of Health.

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