FEMS Microbiology Letters 28 (1985) 317-321 Published by Elsevier

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FEM 02151

Galactose epimeraseless (GalE) mutant G30 of Salmonella typhimurium is a good potential live oral vaccine cartier for

fimbrial antigens

(K88; Pili; adhesins; E. coli)

Gordon Stevenson and Paul A. Manning *

Department of Microbiology and Immunology, The Unioersity of Adelaide, Adelaide, S.A. 5000, Australia

Received 27 April 1985 Accepted 29 April 1985

1. SUMMARY •

The plasmid pFM205 encoding the cloned K88ab gene cluster from an enterotoxigenic Escherichia coli has been introduced into Salmonella typhimurium Strain G30. The G30/pFM205 derivative has been shown to effi- ciently express the K88 pilusand this strain has been used for immunizing mice. Mice were im- munized orally with live bacteria, intraperitoneally with formalin-killed bacteria, or orally with live bacteria followed by an intraperitoneal booster with killed organisms. All immunizations resulted in good antibody responses to K88 in the serum, however, only those immunizations involving an oral dose produced a significant antibody response in the gut. These results suggest that S. typhimurium strain G30 is a suitable carrier for producing intestinal antibodies to fimbrial anti- gens by oral immunization with live organisms.

2. INTRODUCTION

Intestinal pathogens such as the enterotoxigenic E. coil cause disease by secreting a toxin into the

* To whom all correspondence should be sent.

gut. The organisms themselves are non-invasive and a poor immune response is mounted against them by the host. Salmonella species, however, have the capacity to invade and in doing so the initial sites of infection are the Peyer's patch lymphoid follicles. Here the bacteria can effec- tively stimulate the immune response.

Avirulent Salmonellae have been shown to be capable of stimulating the production of protective antibodies [1]. However, the critical determinant as to whether any particular avirulent strain is capa- ble of generating immunity is its ability to invade the Peyer's patches [2].

Hybrid Saimonella/E. coil strains have been constructed [3] and some such strains are good immunogens. In fact one such hybrid strain 885 which is a S. typhimurium expressing the 0-8 antigen of the parent E. coil 08 strain both pro- tects against Salmonella and leads to high levels of antibodies to the E. coli 08 determinant. These antibodies are secreted into the gut and so would be assumed to be protective against an E. coil 08 infection.

Scours in swine and other domestic stock is often due to the presence of enterotoxigenic E. coil expressing particular surface antigens. These anti- gens, which are fimbrial, provide the organism

0378-1097/85/$03.30 © 1985 Federation of European Microbiological Societies

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with the ability to adhere to the gut epithelium and so resist peristalsis. At the same time these E. coli elicit their toxin(s) which results in often fatal diarrhoea. Antibodies to these fimbriae will pro- tect the host by inhibiting the adherence and col- onization of the organism, and as a result no diarrhoea ensues [4].

If avirulent Salmonella strains capable of gener- ating immunity could also express such fimbrial antigens as K88, would they also induce antibod- ies to these fimbriae? If so, could these antibodies be induced in the gut to provide the relevant protection? We have set out to answer these ques- tions.

The avirulent S. typhimurium strain we have chosen is the galactose-epimeraseless mutant strain G30 [5]. This strain has been shown to produce excellent immunity to Salmonella [3]. As a protein antigen we have used the K88ab fimbrial antigen. This was feasible since the genetic locus determin- ing production of the K88ab antigen, which is plasmid encoded, has been cloned into the plasmid vector pBR322 to give the recombinant plasmid pFM205 [6].

In this paper we report the results of these experiments.

3. MATERIALS AND METHODS

3.1. Bacterial strains and plasmids S. typhimurium strain G30 (a gale mutant of

strain LT2) [5] was obtained from M.J. Osborne, and strain LB5010 (LT2; hsdReT, hsdRsA, hsdRsB, galE) from L.R. Bullas [7]. Strains containing either plasmid pFM205 or pRI8801 [6] were ob- tained from F. de Graaf. Plasmid pBR322 is as described in [8].

All bacterial strains were maintained as frozen cultures in 1% bactopeptone (DIFCO) containing 15% glycerol at -20°C for routine use and at -70°C in glycerol or lyophilized for long-term storage.

3.2. Media All cultures were grown double strength in

nutrient broth (DIFCO) made up at with 5 g per litre of NaC1 added. Strains containing plasmids

were grown in the presence of the appropriate antibiotic.

3.3. Plasmid DNA isolation Plasmid DNA was prepared by centrifugation

on CsCl-ethidium bromide gradients of cleared lysates as described [9], but using 0.5% Triton X-100. Two centrifugations were used: the first spin was for 18 h in a Beckman 60Ti rotor in an L8-80 Beckman Ultracentrifuge after which the plasmid band was removed and recentrifuged for 18 h in an 80Ti rotor.

3.4. Plasmid transformation CsCl-purified DNA was used for transforma-

tion using CaC12-treated cells as described by Brown et al. [10].

3.5. Purification of K88 pili The procedure used for purification of K88 pili

was based on the method of Evans et al. [11]. One ml of an overnight culture of the strain was

spread on each of four 30 c m x 30 cm plates containing 500 ml of nutrient agar. The plates were incubated overnight at 37°C and the cells harvested into 200 ml of 0.1 M phosphate buffer pH 7.2 containing 0.02% sodium azide. The cell suspension, held on ice, was then homogenized using a DuPont Omnimixer (Du Pont Industries; Newtown, CT) at full speed for 2 min to shear off loosely associated surface structures. The bacterial cells were removed by centrifugation 7000 rev./min for 20 min at 4°C in a Sorvall GSA rotor.

The supernatant was then placed in dialysis tubing and dialysed overnight at 4°C against 3 litres of 0.1 M acetic acid. This resulted in the formation of a precipitate which was recovered by centrifugation at 10000 rev./min for 20 min at 4°C.

The precipitate was then resuspended in 14 ml of 0.1 M phosphate buffer pH 7.2 and CsC1 added to a final concentration of 33%. This suspension was then split into two aliquots and centrifuged at 34000 rev./min for 21 h at 15°C in a Beckman SW41Ti rotor in a Beckman L8-80 ultracentrifuge. Two bands were present after centrifugation and these were removed with a syringe by puncturing the side of the tube. This material was dialysed

against 1000 vols. of 0.1 M phosphate buffer pH 7.2. The purity was examined by SDS-poly- acrylamide gel electrophoresis.

3.6. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)

SDS-PAGE was performed on either linear 11% or 11-20% polyacrylamide gradients using a mod- ification of the procedure of Lugtenberg et al. [12] as described previously [13].

3. 7. Immunization experiments Plasmids pBR322 and pFM205 were trans-

formed into strain G30. G30 harbouring either pBR322 or pFM205 was

grown in nutrient broth containing ampicillin (to select for maintenance of the plasmid) to late logarithmic phase (7-9 × 108 cells/ml) and the bacteria harvested by centrifugation (7000 rev./min for 15 min at 4°C in Sorvall GSA rotor). For some experiments these cells were subse- quently treated with 0.1% formalin for 30 min at room temperature.

Groups of 10 mice were immunized as follows: mice in the first group were fed 108 live bacteria in 0.2 ml saline after having first been fed 0.2 ml of 50% saturated NaHCO 3. This was repeated 9 and 16 days later. The second group mice were injected intraperitoneally with 5 × 107 formalin-killed bacteria and again 9 and 16 days later. The third group was fed initially as the first group but the two subsequent immunizations were as for the second group.

Five days after the last immunization the mice were bled and the sera of the individual groups pooled. The mice were then killed, the intestines removed and washed With 1 ml of 0.9% saline per mouse. PMSF (a protease inhibitor) was then ad- ded to the washings to a final concentration of 2 mM.

3.8. ELISA assays Microtiter trays were coated with 100/~1 of 10

#g of purified K88 antigen per ml in TSA buffer (25 mM Tris-HCl, 0.02% NaN 3 in saline pH 7.5). The plates were allowed to stand for 30 min after which the excess antigen was shaken out; the trays were washed three times with TSA buffer on a

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Titertek Microplate washer. 80 /~1 of BT buffer (0.15 M NaC1, 0.0125 M triethanolamine pH 7.6, 0.05% Tween-20, 0.05% NaN 3, 0.2 mg/ml bovine serum albumin) and 2-fold serial dilutions of the fluid (either serum or gut washings)under test were made in the wells and incubated for 4 h at room temperature. The contents Of the tray were shaken out and the tray washed four times. The antibody-conjugate was then added (80 #1 of goat anti-mouse IgG coupled with alkaline phosphatase and diluted 1 in 1000 in buffer (8 g NaC1, 50 ml 0.25 M triethanolamine pH 7.6, 10 ml of 100 mM MgCI 2 + 250 btM ZnC12, 10 ml bovine serum al- bumin (20 mg/ml), 10 ml 10% NaN 3 made to 1 liter with distilled water) and the tray incubated a further 18 h. The contents were again shaken out, the tray was washed three times and 100 ~1 of phosphatase substrate (1 mg/ml in 10.5% di- ethanolamine, 1 mM MgC12, pH 9.8) added. After 2 h incubation at 37°C the absorbance was mea- sured at 405 nm on a Titertek Multiskan MC.

4. RESULTS

4.1. Expression of K88ab pili by G30 Plasmid pFM205 was introduced into S.

typhimurium G30 by first transforming strain LB5010, reisolating plasmid DNA and then trans- forming strain G30. This was to overcome the problem of restriction of the transforming DNA in strain G30. The resulting strain G30/pFM205 was agglutinable with K88 antiserum and can be shown to produce K88 pili. A crude pili preparation of this strain, demonstrating the production of large amounts of pilin, is shown in Fig. 1. It can be seen that material corresponding to the K88ab pilin represents a major component of the loosely asso- ciated surface proteins of the strain G30/pFM205 (Fig. 1). The other major component of this crude material is flagellin of about 50 000 Da.

Material similar to that shown in Fig. 1 was also obtained from E. coli C600 harbouring pRI8801, but was further purified by CsCI gradi- ent centrifugation. This yielded highly purified material (Fig. 2). This purified material was used for coating microtiter trays used in detecting specific antibodies to K88 in the following im- munization experiments.

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67K : , I ~

FLAGELLIN

530K 2 ,TK 1(88

ltlK

Fig. 1. Crude pili preparation from Salmonella typhimurium strain G30 harbouring the plasmid pEM205. Crude pill (right track) were prepared as far as acetic acid precipitation (see METHODS) and the precipitate was analyzed by SDS-PAGE on an 11-20% polyacrylamide gradient. The M r s tandards (left track) were: bovine serum albumin ( M r 67000) carbonic anhydrase ( M r 30000) chymotrypsinogen ( M r 25700) and ribonuclease ( M r 14 000).

Table 1

ELISA titer to purified K88 ab antigen

Strain Immunizat ion ELISA titer

Serum Gut washings

Experiment 1 G30/pFM205

Normal mouse serum

Experiment 2 G30

G30 /pFM205

Normal mouse serum

Oral 8192 N D a IP 65536 ' N D a Ora l / IP 131072 N D a

< 256

Oral < 256 < 2 5 6 IP < 256 < 256 O r a l / I P < 256 < 256 Oral 16384 4096 IP 262144 ,< 256 Ora l / IP 524 288 8192

< 6 4

a ND, Not done.

Fig. 2. Purified K88 pili. K88 pili (fight track) were prepared from strain C600 containing the wild-type K88 plasmid pRI8801. This material was analyzed by SDS-PAGE on an 11% polyacrylamide gel. M r standards (left track) are as in Fig. 1.

4.2. Immunization with G30 and G30/pFM205 S. typhimurium G30 with and without plasrnid

pFM205 has been used for immunizing mice by one of three regimes: orally, intraperitoneally, and orally followed by an intraperitoneal booster.

The results of these experiments are sum- marized in Table 1 where we have analysed for the presence of antibodies to the purified K88 antigen.

As can be seen the oral immunization with G30/pFM205 results in levels of serum antibody lower than when the organisms are given in- traperitoneally orally followed by intraperitoneal boosting. However, oral immunization does result in a vast increase in the presence of specific anti- bodies to K88 in the gut. The intraperitoneal boosting following the oral challenge enhances the levels of antibody in both cases.

5. DISCUSSION

S. typhimurium G30 is a galactose-epimeraseless (galE) mutant rendering it avirulent. The galE

mutation, as its prime effect, results in a defective lipopolysaceharide (LPS). However, this strain still maintains its capacity to invade the Peyer's patch lymphoid follicles of the small intestine and pro- liferate there for a short while. These properties enable it to induce an excellent local immune response to at least its surface components.

In this paper we have shown that by introduc- ing cloned surface antigens into S. typhimurium G30, it may be possible to generate antibodies to those surface antigens. The example we have used is the adhesin produced by certain strains of en- terotoxigenic E. coil, namely the K88 pilus or fimbria. The genes for this structure have been cloned [6] to give the recombinant plasmid pFM205, which has been introduced into strain G30 and is expressed efficiently. This strain we have used in immunization experiments.

It was demonstrated that not only does the oral immunization with G30/pFM205 result in a good antibody response in the serum but it also pro- duces a good response in the gut. Thus, it is reasonable to expect that such a response could be protective against infection by enterotoxigenic E. coli K88 strains.

Given suitable Salmonella strains appropriate for the host, then it should be possible to produce hybrid strains of the type described here for im- munizatiori in different animals. The requirements to be met are that the antigen to be carried by the Salmonella must be expressed on the surface and it must be immunogenic.

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D. Rowley for many useful discussions. This work was supported in part by a grant to

P.A.M. from the National Health and Me.dical Research Council of Australia.

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A CK NOW LEDGEMENTS

The authors wish to thank Dr. F. de Graaf for generously supplying plasmid pFM205 and Prof.