identification of afipia felis antigens in culture medium: reaction with human sera

Copyrighr 0 A P M I S IY97

I S S N OYO3-4641

Identification of Afipia felis antigens in culture medium: reaction with human sera

KRRSTEN ENGBRK. LARS OTTO UTTENTHAL and CLAUS KOCH

Department of Immunological Research and Development, Immunology Division, Statens Seruminstitut, Copenhagen, Denmark

Engbzk, K., Uttenthal, L. 0. & Koch, C. Identification of Afipia.fe1i.s antigens in culture medium: reaction with human sera. APMIS 105: 199-206, 1997.

Fourteen protein antigens were identified on SDS-PAGE of Ajpiafelis culture supernatant. Immuno- blotting against 10 monoclonal antibodies obtained from mice infected with live A . ,felis showed that 4 antibodies reacted with a 56 kDa band and 3 with both 56 kDa and 62 kDa bands. Compared with A . ,felis sonicate, the reacting proteins in culture supernatant showed an increase in molecular mass of 2-3 kDa, suggesting that they were more glycosylated. Purified antigen obtained by affinity chromatography of culture supernatant on the seven immobilized antibodies was tested against antibodies reacting with the 56 kDa and 62 kDa bands. All eluates contained both components, suggesting that the antibodies were directed against different epitopes of a double antigen held together during the affinity chromatography but cleaved by reduction and SDS-PAGE. The molecular size of the uncleaved protein in culture supernatant was determined by size-exclusion chromatography as > 1000 kDa. Testing of pre- and post-infection rabbit sera in immunoblotting against culture supernatant demonstrated that the 56 kDa and 62 kDa components gave the most prominent specific reactions with post-infection sera. One of fifty human sera submitted for testing for cat-scratch disease and I of 50 sera from healthy blood donors reacted with several bands in A. felis culture supernatant, including the 56 kDa and 62 kDa bands.

Key words: Afipio ,felis; cat-scratch disease; soluble antigens; secreted antigens; exoproteins; monoclonal antibodies.

K. Engbzk, Department of Immunological Research and Development, Immunology Division, Statens Seruminstitut, Artillerivej 5 , DK-2300 Copenhagen S, Denmark.

Many bacterial products of immunological im- portance are secreted extracellularly. These products have been variously termed soluble antigens, secreted antigens or exoproteins. Among the more important are those which function as toxins o r extracellular enzymes. In- tracellular components may also be liberated extracellularly by cell autolysis, if bacterial growth is extended beyond the exponential phase. Some bacteria secrete only a limited number of soluble antigens, whereas Staphylo-

Received August 29, 1996. Accepted November 18, 1996.

coccus aweus and Pseudornonas aeruginosa secrete many. In many instances these antigens have been shown to play an important role in the pathogenesis of the infection (14).

Recently, a new group of soluble superantigens has been discovered, which without being processed by antigen-presenting cells bind and stimulate T-lymphocyte clones independently of the antigenic specificity of the T cell. This stimulation leads to a massive release of inflamma- tory cytokines, such as interleukin-1 a, interleukin-2, and tumour necrosis factor. At times, autoreactive T cells are also stimulated, which may lead to an attack on healthy tissues. These antigens are considered to be the underlying

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mechanism of severe disorders such as scarlet fever, toxic shock syndrome, food poisoning, diphtherial myocarditis and polyneuritis, and possibly rheumatic fever and post-streptococcal acute nephritis (3, 6, 9).

Apart from these specific antigens, immune responses to the general bacterial components that enter the culture medium have not been much studied. This is remarkable as the bacterial antigens immediately accessible to the immune defences of the host are those located on the surface or released by the pathogen into sur- rounding host tissues, including the blood- stream. Another reason for studying bacterial products in the culture medium is the presence of large numbers of components in lysates of whole organisms, many of which do not pro- voke the formation of antibodies in the host. If the number of irrelevant components is reduced, the interpretation of experimental results may be facilitated.

We have previously studied the antigens in sonicated whole Afipia felis organisms (1) and characterized a series of monoclonal antibodies (MAbs) from mice infected with live A . felis in terms of their reactivities with these components. In the present study we report on the soluble antigens of A . felis that enter the liquid culture medium during bacterial growth.

Our purpose was twofold: 1) to focus on the antigens most likely to be relevant to early hu- moral immunity, and 2) to study material free from the possible artifacts of sonication and the batch-to-batch variability observed in sonicates from both solid and liquid cultures.

MATERIALS AND METHODS

Bacterial strain and antigen preparation AJipia ,felis reference strain ATCC 53690 was cul-

tured at 27°C for 4 days in buffered-yeast extract broth (13) in tissue culture flasks (Nunc 156502). After routine incubation for 4 days at 27"C, half of the flasks were incubated for 3 h at 37°C. The bacteria were separated from the culture supernatant by centrifugation at 20,OOOXg for 30 min. The supernatant was filtered through a 22 pm pore-size Milli- pore filter and concentrated 100 times by means of Pellicon tangential flow filtration (Millipore, Bedford, MA) and Omegacell ultrafiltration systems (Filtron Technology, Northborough, MA) with a molecular cut-off of approximately 10 kDa. Protease inhibitors

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(1 mM EDTA, 50 pM phenylmethylsulphonylfluori- de, 8 pM E-64, 6 pM phosphoramidon and 32 pM bestatin) were added to the concentrated culture supernatant (CCS), which was divided into 1 ml samples and stored at -80°C.

Post-infection rabbit sera Live A . jelis suspension (McFarland turbidity stan-

dard no. 3, 1.5 x lo9 CFUiml) was injected intracut- aneously, at a dose of 0.2 ml, into two female rabbits. Blood was drawn before and at weekly intervals after the inoculation. Six weeks after the initial inoculation, the rabbits were rechallenged with the same dose, and they were again bled weekly for three weeks.

Immunoelectrophoresis Crossed immunoelectrophoresis (XIE) and

crossed-line immunoelectrophoresis (XLIE) were carried out as described in (1). For XIE, 20 pl A. j e t is sonicate or 35 p1 A . fe1i.s CCS were placed in circular wells in an agarose gel and separated electrophor- etically at a potential gradient of 10 V cm-'. Lanes with separated antigens were cut out and transferred to 5 x 7 cm glass plates. A 1 S X 5 cm intermediate gel and a 4 x 5 cm top gel containing rabbit anti-A. jelis serum were poured onto each plate, and electrophoresis was carried out at right angles to the first direction at a potential gradient of 2 V cm-' for 18 h.

The procedure for XLIE followed that described for XIE, except that 200 p1 A. felis CCS was added to the intermediate gel.

Polyacrylumide gel electrophoresis (PAGE) For SDS-PAGE, samples of CCS were boiled for 4

min with an equal volume of reducing sample buffer (62.5 mM Tris-HC1, pH 6.8, containing 2% wiv sodium dodecylsulphate (SDS), 20% viv glycerol, 5% v/v 2-mercaptoethanol, 0.0125% wiv bromphenol blue) and subjected to electrophoresis on a 10% polyacrylamide gel (SDS-PAGE) in the presence of 0.1 %I w/ v SDS, as described by Laenzmli (12). Protein bands were silver stained (5) or stained with Coomassie Bril- liant Blue R-250 and the relative protein densities determined with an Ultrascan I1 XL Laser Densitometer (LKB, Bromma, Sweden). For native PAGE, samples of CCS were diluted in an equal volume of native sample buffer (250 mM Tris-HC1, pH 6.8,20'%1 v/v glycerol, 0.0125% w/v bromphenol blue) and subjected to electrophoresis on an 8% polyacrylamide gel containing 0.37 M Tris-HC1 buffer, pH 8.8 (10).

Imm unoblo t ting Proteins from SDS-PAGE gels were electroblotted

onto Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore) as described (2). Unoccupied protein binding sites on membranes were blocked with 5% w/v defatted dried milk and 0.5'5, v/v Tween 20 in 10 mM phosphate buffer, pH 7.2, containing

SECRETORY ANTIGENS OF A . FELIS

130 mM NaCl (PBS) for 30 min at room temperature, with gentle agitation. Strips of membranes ( 5 mm) were incubated overnight with dilutions of rabbit or human sera, or MAb-containing culture supernatants. Bound antibodies were demonstrated by means of the appropriate peroxidase-labelled second antibodies as described (1).

Molecular yliuss detrrminution The following methods were used: 1) native and

SDS-PAGE (10,16) of affinity-purified antigen with standard protein markers (Mark 12. Novex, San Di- ego, CA), and 2) size-exclusion chromatography of CCS on a prepacked (1.0 x 30 cm) column of Su- perose 6 (Pharmacia, Uppsala, Sweden). Chromato- graphy was performed in a fast-phase liquid chromatography system (Pharmacia). The column was equilibrated with two volumes (50 ml) of 50 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl at a flow rate of 0.5 ml/min. A 100 p1 sample of CCS or affinity-purified antigen (630 pg/ml) was applied to the column and eluted at 0.2 ml/min in the same buffer while the absorbance at 280 nm was monitored. Fractions (0.5 ml) were subsequently ana- lysed by EIA. For calibration, solutions of molecular size markers ranging from 29 to 2000 kDa (Sigma MWGF-1000) were applied to the column and eluted independently under identical conditions.

Sus fuce iodinut ion The surface proteins of live A. jelis bacilli were

radioiodinated using the iodogen method described by Frnker & Speck (4) . Iodogen (1.3,4,6-tetrachloro- 3cc,6a-diphenylglycouril, 20 p1 of a 1 mgiml solution in dichloromethane) was dried onto the interior of a glass ampoule. A 100 pl sample of washed A. frlis suspension (OD6,,,, 1.5) in PBS was added, followed by 5 p1 I2’I- (0.5 mCi of IMS.30, Amersham, Buck- inghamshire, UK). The suspension was incubated in a water bath for 30 niin at 3 5 T , washed five times in PBS, resuspended in 1 ml PBS and ultrasonicated for 5 min on ice. The sonicated suspension was centri- fuged at 45,OOOXg for 60 min. The supernatant was reduced and subjected to SDS-PAGE or XIE as described above, the gels being stained with Coomassie Brilliant Blue R-250. The gels were dried between sheets of cellophane, placed on a sheet of x-ray film (Hyperfilm RPN 1675 with Hyperscreen RPN 1662, Amersham) in a metal cassette and exposed for 36 h at -70°C. The films were then allowed to warm to room temperature and were developed according to the manufacturer’s instructions.

Antibody purijicution MAbs were purified from hybridonya culture

supernatants on columns (1.6 x 6 cm) of protein A Sepharose 4 fast flow (Pharmacia). Columns were washed with five volumes of PBS and equilibrated with five volumes of binding buffer (0.15 M glycine-

NaOH, pH 8.5, containing 3.3 M NaCl). Culture supernatant (100 ml) was made 3.3 M in NaCl and 0.15 M in glycine, the pH was adjusted to 8.5, and the supernatant was applied to the column. The column was then washed with binding buffer until

of the eluate reached baseline. Bound antibody was eluted with 0.1 M sodium citrate buffer, pH 4.5, reversing the flow and collecting 2 ml fractions in tubes containing 0.2 ml 1 M Tris-HCI, pH 7.5. Frac- tions of OD2*,, >O. 1 were pooled and dialysed against three changes of PBS.

Immunouffinity chromatography CNBr-activated Sepharose 4B (Pharmacia) was

prepared according to the manufacturer’s instructions and reacted with protein-A-purified MAb (2 mgiml gel) for 2 h at 4°C. Excess reactive sites on the matrix were blocked with one volume 0.1 M ethanolamine, and the gel was extensively washed with binding buffer (10 mM phosphate buffer, pH 7.2, containing 0.5 M NaCI), eluant (1 M propionic acid), and PBS. Antibody-conjugated gels were packed into 2 ml columns which were equilibrated with at least three volumes of binding buffer, and A. jelis sonicate ( 1 ml diluted with 3 rnl binding buffer) was applied at 0.5 ml/min. The column was washed with three volumes of binding buffer followed by two volumes of PBS to remove excess salt, and antibody-bound components were eluted with three column volumes of 1 M propionic acid. Fractions (2 ml) were collected in tubes containing 100 p1 of 1 M Tris-HC1, pH 7.5. Pooled eluate was dialysed against three changes of PBS and concentrated by lyophilization.

RESULTS

SDS-PAGE of A. felis culture supernutant Analysis of reduced A . felis CCS by SDS-

PAGE (Fig. I , C) identified 14 silver-stained bands in positions corresponding to molecular masses ranging from 35.5 to 97 kDa. None of the bands was detected in a similar concentrate of uninoculated culture medium (Fig. 1 ,B). Two bands were prominent (56 and 62 kDa), three were of intermediate density (48, 45.5 and 35.5 kDa), and the remaining bands were faint.

Comparison of densitometer tracings showed that cultures that had been exposed to 37°C for 3 h displayed increased relative and absolute densities of the bands at 85, 65, 62, 56, 42.5 and 35.5 kDa.

Crossed immunoelectrophoresis of A. felis culture supernatant

At least 12 precipitin lines could be demonstrated by XIE of A. felis CCS against rabbit

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ENGBEK et al

Fig. 1. SDS-PAGE and immunoblots of Ajipiu felis concentrated culture supernatant (CCS) incubated with monoclonal antibodies 168-1 to 168-10; (A) molecular mass markers (kDa); (B) uninoculated culture medium; (C) A. felis CCS, silver stained;

(H) 168-7; (I) 168-9; and (J) 168-10. (D) 168-1; (E) 168-3; (F) 168-4; (G) 168-6;

anti-A. felis hyperimmune serum (Fig. 2, A). The identity of these antigens was studied in XLIE of A. felis sonicate against rabbit anti-A. jelis serum with A . felis CCS incorporated in the intermediate gel (Fig. 2, C). All precipitin lines in the XLIE were raised and fused with the horizontal lines, demonstrating that the antigens in the culture supernatant were immuno- logically identical with the antigens present in the A . filis sonicate.

Reactivity with monoclonal antibodies Ten MAbs (AF 168-1 to AF 168-10) derived

from mice infected with live A. fezis were tested by immunoblotting with A. felis CCS. MAbs 168-4, -6, -7 and -10 reacted with a single 56 kDa band, whereas MAbs 168-1, -3 and -9 reacted with both 56 kDa and 62 kDa bands. The remaining three MAbs were unreactive (Fig. 1, D-J).

Relation between 56 kDa and 62 kDa antigens All seven reactive MAbs reacted with a com-

ponent of 56 kDa. To examine whether this was

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the same protein for all these MAbs, and to in- vestigate its relationship to the 62 kDa component with which MAbs 168-1, -3 and -9 also reacted, CCS was subjected to immunoaffinity chromatography on each of the seven immobilized antibodies. The eluates from these affinity columns were tested by immunoblotting against the seven MAbs. All the eluates contained both 56 kDa and 62 kDa components as demonstrated by their reaction with MAbs 168-1, -3 and -9. However, MAbs 168-4, -6, -7 and -10 reacted only with the 56 kDa band. These results show that the MAbs react with different epitopes of a double antigen, whose two coni- ponents are bound to each other during affinity chromatography, but which are cleaved by reduction and SDS-PAGE into two separate components. These are evidently interrelated, in that they both carry epitopes for MAbs 168-1, -3 and -9, but differ in that the 56 kDa component carries one or more specific epitopes that react with the remaining antibodies.

The same pattern of reactivity was seen in immunoblots of sonicate of whole A . felis organisms (2). Here, however, the reactive bands cor- responded to molecular masses of 53 kDa and 60 kDa, respectively, and the bands were nar- rower. The small differences in molecular mass of the components in CCS and sonicate were confirmed by SDS-PAGE of the two antigen preparations on the same gel.

Molecular mass determination of uncleaved antigen

In conventional PAGE, the immunoaffinity- purified component appeared as two major bands, with minor bands of lesser mobility that might correspond to oligomeric forms, whereas only two major bands of 56 kDa and 62 kDa were demonstrated when the antigen was reduced and subjected to SDS-PAGE. In size-exclusion chromatography on an analytical Su- perose 6 column (Fig. 3 ) , the antigen was eluted at the void volume as a skewed peak with a shal- low trailing edge, corresponding to a molecular size equivalent to >lo00 kDa for a globular protein. Identical elusion profiles were obtained irrespective of whether immunoaffinity-purified antigen or material recovered from the top or trailing edge of the peak was applied to the column. A slight shoulder on the trailing edge of the peak did not become more prominent when


Fig. 2. Crossed immunoelectrophoresis and crossed-line immunoelectrophoresis of A. felis concentrated culture supernatant (CCS). (A) 35 pl A. .felis CCS against 40 p1 rabbit antiserum cm-2 with a blank intermediate gel; (B) 18 pg A. felis sonicate against 40 p1 rabbit antiserum cm-' with a blank intermediate gel; (C) 18 pg A. f e h sonicate against 40 p1 rabbit antiserum cm-' with 200 p1 A. f d i s CCS in the intermediate gel. Electrophoresis was carried out at 10 V cm-' for 18 h in the second dimension with the anode at the top. Major precipitin arcs are indicated by their reference numbers from (1).

material from this region was rechromatograph- ed. Taken together these findings indicate that the antigen is a multimeric, non-covalently self- associating complex.

A. felis surface antigens XIE and SDS-PAGE patterns of soluble sur-

face-iodinated A . jelis antigens were examined by autoradiography. In XIE, three precipitin arcs were radiolabelled, corresponding to precipitin arcs nos. 16, 20 and 36 in the A . felis reference diagram (1). On SDS-PAGE, nine components in positions corresponding to molecular masses ranging from 87 kDA to 28 kDa were radiolabelled. The most prominent band was a 39.5 kDa component. No surface-labelled component was seen in the 56 kDa region, whereas a faint band was evident in the 62 kDa region.

Immunoblotting with pre- and post-infection rabbit sera and human sera

In order to identify specific antigens in A . felis CCS that could be used in the diagnosis of A . ftlis infections, sera from rabbits taken before and after infection with live A. felis bacilli were tested in immunoblotting against CCS. Repre-

sentative immunoblot patterns obtained with two pairs of pre- and post-infection rabbit sera are shown in Fig. 4. Apart from bands seen with both pre- and post-infection sera, two bands at 56 kDa and 62 kDa, respectively, were particu- larly evident in both of the post-infection rabbit sera, but were not seen in the corresponding pre-infection sera.

To evaluate the occurrence of reactivity against these 2 antigens in human sera, 50 serum specimens submitted for testing for cat- scratch disease and 50 specimens from healthy blood donors were tested in immunoblotting against A. felis CCS. One patient and one blood donor had antibodies against both these antigens, while another patient had antibodies against the 56 kDa component (Fig. 4)

DISCUSSION

SDS-PAGE of A . felis CCS identified 14 silver- stained bands. None of these bands derived from uninoculated culture medium. After ex- posure of the cultures for 3 h at 37"C, 6 of the 14 bands showed an increased density, but the number of bands was not increased. This sug-

203

5

4 aJ u

3 3 L4

8 , 0

4 H w 1

vo 1 2345 6 0 o o o o o o

0 5 10 15 2 0 25 30 ml

Fig. 3. Size-exclusion chromatography on a Superose 6 column ( 1 X 30 cm) of A . j d i s immunoaffinity-purified antigen (-!3), fraction 16 corresponding to the peak (- -0- -), and fraction 24 corresponding to a small shoulder on the trailing edge of the peak (...O...) . Fractions (0.5 ml) were tested in EIA against MAbs 168-9, and molecular markers (0) were: 1 ) thyroglobulin 669 kDa, 2) apoferritin 443 kDa, 3) P-amylase 200 kDa, 4) alcohol dehydrogen- ase 150 kDa, 5) albumin 66 kDa, and 6) carbonic anhydrase 29 kDa. Vo represents the elusion position of Blue Dextran 2000.

gests that the six bands might represent heat- shock proteins. Increased lysis of the cells would have liberated a larger number of proteins and increased the number of bands in the electrophoresis. However, to prove that the proteins are heat-shock proteins, an increase in their syn- thesis at elevated temperatures has to be demonstrated. These investigations are in progress.

Ten MAbs from mice infected with live A. felis organisms were investigated in immunoblotting against A. felis CCS. Four of the antibodies reacted with a single 56 kDa band, and three with both a 56 kDa and a 62 kDa band. Three MAbs did not react with any of the bands in A . .felis CCS, presumably because they were directed against antigens that were not secreted into the culture medium.

Determination of the molecular mass of the reacting bands in immunoblots of A. felis sonicate and A. felis CCS separated on the same SDS-PAGE gel showed that the bands in CCS had increased in molecular mass from 53 kDa to 56 kDa and from 60 kDa to 62 kDa compared with the sonicate. In addition, the bands

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Fig. 4. Immunoblots of A. felis culture supernatant with pre- and post-infection rabbit sera and human sera; position of molecular mass markers shown in kDa; (A) pre-infection rabbit 2906 serum; (B) post- infection rabbit 2906 serum; (C) pre-infection rabbit 2907 serum; (D) post-infection rabbit 2907 serum; (E) patient 1 serum; (F) patient 2 serum: and (G) serum from a blood donor.

had become broader, implying some micro- heterogeneity in their structure. This is compat- ible with a variable degree of additional glycos- ylation of the secreted proteins, which, together with the fact that culture supernatant contains far fewer components, and different ratios of these components than sonicate of whole organisms, suggests that the supernatant proteins are specifically and actively secreted into the medium.

The SDS-PAGE pattern of radio-iodinated A. felis surface components demonstrated nine bands, including a minor component in the 62 kDa region. In contrast to EIA experiments in a previous communication (2), in which MAbs reacting with the 56 kDa band also reacted with whole-cell preparations, no surface iodination of components in the 56 kDa region was observed. Either the 56 kDa antigen is deficient in surface-exposed tyrosine or histidine residues that can be iodinated, or, more probably, the fixation of the cells exposed an epitope that is not available for the surface iodination when the organism is in suspension.


Affinity chromatography of CCS with the seven immobilized antibodies and testing of the eluates in immunoblotting against the same antibodies demonstrated that all seven eluates contained both the 56 kDa and 62 kDa components. These results show that the MAbs react with different epitopes of a double antigen, whose two components are bound together during affinity chromatography but cleaved by reduction and electrophoresis. However, when this antigen is subjected to size-exclusion chromatography it emerges as a skewed peak at the void volume, corresponding to a molecular size >I000 kDa, which implies that the antigen is a multimeric complex. The identical location and appearance of the peak, irrespective of whether the sample is immunoaffinity-purified antigen or fractions recovered from the top or trailing edge of the peak, suggests that the antigen forms a non-covalently self-associating complex in which there is an equilibrium between higher and lower oligomers.

Previous experiments have shown that epitopes of the 62 kDa antigen are also present in antigens of other bacterial species, whereas no cross-reacting epitopes have been demonstrated in the 56 kDa component (2).

These results may be taken to indicate that the double antigen belongs to the group of common protein antigens previously described for numerous Gram-negative and Gram-positive bacteria and spirochaetes (7, 8, 11, 15). How- ever, there are structural and immunological differences between the A . , felis double antigen and the common protein antigen. The common protein antigen in its native form exists as a 250-900 kDa homopolymer composed of 60-62 kDa units ( 1 5), whereas the present antigen has a > 1000 kDa heteropolymeric structure composed of 53-56 kDa and 60-62 kDa units. Fur- thermore, the A . j d i s double antigen does not react with rabbit antiserum against Pseudomotz- ( I S aeruginosa common protein antigen (data not shown). This demonstrates that the present antigen does not belong to the common protein antigen group.

Testing of pre- and post-infection rabbit sera in immunoblotting against A . $elis CCS demonstrated several bands reacting with post-infection sera that were not seen with pre-infection sera. The most prominent of these bands were those at 56 kDa and 62 kDa positions. Examin-

ation of 50 human sera submitted for testing for cat-scratch disease and 50 sera from healthy blood donors revealed one patient serum and one blood donor serum that reacted with several bands in A . felis CCS, including the 56 kDa and 62 kDa bands, while another patient serum reacted with the 56 kDa band. None of the sera had antibodies against Bartonella (Rochdi- maea) henselae. These preliminary results sug- gest that while infections with A. felis occur in the Danish population, they are apparently in- frequent and may not be recognized. Improved methods of serological diagnosis will help to identify clinical manifestations that could be as- cribed to A . felis infection.

The authors acknowledge the excellent technical as- sistance of B. Ruasthoj and C. D. Madsen, and many helpful suggestions from G. Houen.

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