Immunology 1978 34 41

Immunological aspects of cell membrane adhesion demonstrated by porcine enteropathogenic Escherichia coli S. H. PARRY & P. PORTER Unilever Research Laboratory, Immunology Department, Colworth House, Sharnbrook, Bedford

Received 17 March 1977; accepted for publication 2 June 1977

the specificity of adhesion of K88ab to chicken erythrocytes does not reside in this determinant.

Summary. The K88ab antigen of porcine enteropathogenic strains of Escherichia coli exhibited specific affinity for the chicken erythrocyte membrane which correlated with its adhesion characteristics for piglet enterocytes. Thus the chicken erythrocyte provided a useful model for defining the underlying mechanisms of interaction between K88 bearing E. coli and host cell membranes. This affinity of enteropathogenic E. coli for chicken erythrocytes and piglet enterocytes has been used in an investigation of the role of antibody against the a, b and c determinants of K88 in blocking microbial adhesion to cell membranes. Antisera raised against K88ab and K88ac inhibited the adhesion of both K88ab and K88ac bearing organisms to the microvilli of piglet enterocytes. Antisera specific for the K88a determinant also blocked adhesion of both K88 organisms to the enterocyte indicating the potential of a broad spectrum of activity against porcine enteropathogens. Antibodies against K88b and K88c determinants showed appropriate selectivity for homologous strains. The selectivity of antibodies specific for the b and c determinants of K88 was also apparent in the chicken erythrocyte haemagglutination inhibition (HI) assay. However, only low inhibitory activity was demonstrated by anti-K88a sera indicating that

INTRODUCTION The phenomenon of bacterial adhesion has considerable biological significance (Duguid & Collee, 1959). While many bacteria have a capsule which has a repellent and dispersive effect, some species possess adhesive characteristics which enable them to stick to a particular substrate. The bacteria may benefit from their adhesive ability in two ways: by anchorage in a favourable habitat, from which they would be otherwise swept away, and by contact with surfaces where nutrients are being released or absorbed. Surface antigen characteristics of porcine enteropathogens have attracted considerable attention in recent years. It is recognized that porcine neonatal diarrhoea is characterized by proliferation of certain serotypes of E. coli in the small intestine, many of which bear on their surface the common K88 antigen (Sojka, 1973). The ability of these strains of E. coli to proliferate and cause disease has been attributed to their adherent properties for the piglet's intestinal membrane. (Arbuckle, 1970; Drees & Waxler, 1970; Bertschinger, Moon & Whipp, 1972.) Unlike other K antigens K88 is a proteinaceous component forming fine filaments on the surface of the bacterium (Stirm, Orskov & Orskov, 1 967a;

Correspondence: Dr S. H. Parry, Microbiology Department, The Medical School, The University, Newcastleupon-Tyne.

41

S. H. Parry & P. Porter

42

Stirm, Orskov, Orskov & Birch-Andersen, 1966). The antigen is the product of an episomal gene which can be transferred (Orskov & Orskov, 1966), a feature which facilitated the conclusion of Smith & Linggood (1971) that the virulence of E. coli for piglets is associated with this factor. The consistent features of this form of piglet enteritis provide an interesting basis for a model system to investigate the interactions between bacteria and host cell membranes. Antibodies eliminate E. coli adhesion in vivo (Rutter & Jones, 1973) and in vitro (Wilson & Hohmann, 1974; Sellwood et al., 1975) and lend themselves to the investigation of such interactions. In the present studies chicken erythrocytes have been identified as having common receptor characteristics with pig enterocytes for the adhesion of enteropathogenic E. coli carrying K88ab. This specific affinity has been used in an investigation of the role of antibody against the a, b and c determinants of K88 in blocking microbial adhesion to cell membranes. MATERIALS AND METHODS

E. coli strains The following enteropathogenic K88-positive strains were used in adhesion studies: 08: K87(B), K88a, b (L); 0138: K81(B), K88a, c (L); 0141 : K85a, b (B), K88a, b (L); 0147: K89(B), K88a, c (L); 0149: K91(B), K88a, c (L). In addition K88-negative strains of 08, 0138, 0141 and 0149 serotypes were used as controls. All cultures were maintained on nutrient agar slopes, at 40, from which they were subcultured onto nutrient agar plates or Roux flasks for use in adhesions tests.

Preparation of K88 The K88ab adhesion factor was prepared from the 08 strain following generally the method of Stirm, Orskov & Orskov (1966). Nutrient agar (Oxoid) slopes in Roux flasks were heavily inoculated with an overnight broth culture of 08, and incubated at 370 for 24 h. The confluent surface growth was aseptically harvested in 0 01 M pH 7-1 phosphate buffered saline (PBS) and the resulting bacterial suspension was heated at 600 for 30 min to release the K88 antigen from the cell surface. Cellular material was removed by centrifugation at 3000g for 10 min and the resulting supernate was stored at

40

in the presence of 0-2 % sodium azide, for a minimum period of 3 days. During this time some impurities settled out and centrifugation at 3000g was repeated to clarify the preparation. The isoelectric point of K88 is between pH 4-5 and 5 5. Thus dilute acetic acid was added to the continuously stirred bacterial supernate bringing the pH to 5 0. The precipitated material was collected by centrifugation after standing for 24 h at 40 and washed twice with saline brought to pH 5 0 with acetic acid and then finally redissolved in PBS pH 7-1. To obtain a pure product this material was subjected to two more precipitations. The K88ac antigen was prepared in a similar way from the 0149, 'Abbotstown' strain but the K88 was released from the bacteria by homogenization in a Waring blendor for 3 min as it seemed to be more heat labile than the K88ab antigen. Further purification was carried out using isoelectric focusing with an LKB 8101 column, capacity 110 ml, and carrier ampholytes (LKB Producteur, Bromma, Sweden). The column was loaded with 5 mg of protein and was run in a gradient of pH 3-10 for 24 h maintaining a power consumption of 2-3 W. After the run 5 ml fractions were collected, their pH was checked and they were finally dialysed overnight at 40 against PBS pH 7-1.

Collection of erythrocytes Whole blood from chicken, pigeon, turkey, quail, duck, guinea-pig, mouse, rat, pig, calf and sheep was collected using sodium citrate (3 -8 % w/v in distilled water) as anticoagulant. Cells were washed 3 times in PBS, pH 7-1 before use in bacterial adhesion or haemagglutination tests. Preparation of enterocytes The method was based on that described by Evans et al. (1971). Piglets from the age of 1-4 weeks were killed by intravenous injection with pentobarbitone solium. The intestines were removed immediately and the upper jejunum was separated and washed through with 0 3 M sucrose. The lumen was then half filled with EDTA buffer pH 6-8* (Evans et al., 1971), tied off and incubated for 15 min in 0 3 M sucrose. This buffer was drained and the lumen again half filled with phosphate sucrose buffer, pH 7 4t * NaCI, 96 mM; KH2PO4, 8 mM; Na2HPO4, 5-6 mM; KCI, 1 5 mM; EDTA, 10 mM. t Na2HPO4, 76 mM; KH2PO4, 19 mM; sucrose to bring to 485 ideal m osmoles.

Immunological aspects of cell membrane adhesion (Evans et al., 1971). After the ends

were

tied, the

enterocytes were detached from the villi by gently

rubbing the intestine between the fingers. The cell suspension was drained and spun at 80 g for 2 min. The cells were washed in sucrose phosphate buffer several times until the contamination of the supernatant with bacteria and cell debris was minimal. The resulting epithelial cell preparation was either used fresh or stored in Spooner's medium in liquid nitrogen. Haemagglutination assay for K88 The K88ab adhesion factor was assayed by taking advantage of its specific haemagglutinating activity for chicken erythrocytes, which occurred at room temperature. Samples were serially diluted by adding 0-05 ml to an equal volume of PBS, pH 7-1 and carrying out doubling dilutions in microtitre plates. To each dilution was added 0.05 ml of a I % suspension of chicken erythrocytes in PBS, the plates were briefly shaken on an orbital mixer and allowed to settle at room temperature. Plates were read at b1- h later but they remained stable for a number of days. The end point was taken as the last well showing agglutination and the titre was expressed as a reciprocal of this dilution. The K88ac antigen was assayed by the same procedure using guinea-pig cells, the test being carried out at 00 (Jones, 1972).

Haemagglutination inhibition (HI)

test

A standard K88ab preparation was assayed as described above and was diluted to a concentration 4 times its haemagglutination end point. The test antiserum was prepared in serial dilutions of PBS diluent (0 05 ml), an equal volume of the diluted K88 was added to each well and allowed to stand at room temperature for I h. Finally 0 05 ml of a 1 % suspension of washed chicken erythrocytes was added to each well and the test was read j-1 h later. The end point was expressed as the dilution of

43

antiserum which completely inhibited the red cell agglutination.

In-vitro adhesion neutralization test Both pig enterocytes and chicken erythrocytes were used to assess the inhibitory effect of antisera on the adhesive properties of whole organisms. The assay procedure consisted of mixing 0-1 ml of a 108/ml suspension of the bacterium under test with an equal volume of a dilution of test serum. After incubation for 15 min at 370, 0 1 ml of a 106/ml suspension of enterocytes or erythrocytes was added and incubated for a further 15 min. Samples were then examined using phase microscopy. In the case of enterocytes anti-adhesive activity of antisera was assayed by enumerating the number of bacteria sticking to the brush-border of 20 randomly selected enterocytes in each test. Preparation ofantisera Specific hyperimmune sera were raised in rabbits and pigs against purified preparations of K88ab and in rabbits only using K88ac. Animals were injected intramuscularly and subcutaneously with K88 preparations in Freund's incomplete adjuvant in the multiple emulsion form (Herbert, 1967) and bled 3-6 weeks later. Antisera were also produced by immunization of pigs with erythrocytes coated with K88ab. The vaccine was prepared by adding 5 ml of a 1/1000 dilution of a K88 preparation with an HA titre of 8000 to each 1 ml of packed washed sheep erythrocytes. The suspension was incubated for 30 min at 370 and then washed twice in PBS. Pigs were injected intramuscularly with 1 ml of packed sensitized cells suspended in 2 ml of PBS. This was followed by a further injection 2 weeks later and the pigs were then bled within 7-10 days.

Absorption of antisera The absorption procedures and antiserum specificities are summarized in Table 1. Antibodies specific

Table 1. Absorption procedures used to derive specific antisera to K88 determinants

Original specificity of antiserum

Species

K88ab K88ab K88ac K88ac

Pig Pig Rabbit Rabbit

Treatment Abs. 0149 (K88ac) Abs. onto 0149 (K88ac) and eluted at low pH Abs. 08 (K88ab) Abs. onto 08 (K88ab) and eluted at low pH

Final specificity

K88b K88a K88c K88a

44

S. H. Parry & P. Porter Table 2. Bacterial adhesion and haemagglutination characteristics of K88ab and K88ac for a variety of erythrocyte membranes No. of animals positive

Free K88 HA

Chicken* Pigeon* Turkey Quail Duck

Guinea-pigt Mouse Rat Pig Calf Sheep Human A Human B Human AB Human O

20 1 2 5 2 10 10 10 20 5 5 3 3 3 3

Bacterial adhesion to membrane

K88ab(08)

K88ac(0149)

08(K88ab)

0149(K88ac)

20 1 0 0 0 10 0 0 0 0 0 0 0 0 0

0 0 0 0 0 10 0 0 0 0 0 0 0 0 0

20 1 0 0 0 10 0 0 0 0 0 0 0 0 0

0 0 0 0 0 10 0 0 0 0 0 0 0 0 0

* Non-eluting haemagglutination at room temperature.

t Eluting haemagglutination at room temperature, non-eluting at 4°.

for the K88b determinants were produced by absorption of anti-K88ab serum with 0149 (K88ac) and for the K88c determinant by absorption of antiK88ac serum with 08 (K88ab). Two ml aliquots of antisera were absorbed twice with 0-5 ml of packed organisms. Absorptions were performed at 370 for 1 h then cells were centrifuged at 5000 g for 30 min and washed three times in PBS pH 7-1. To provide antibodies with K88a specificity the cells were suspended in a glycine-HCl buffer pH 2-2 and left for 30 min at room temperature. The eluted antibody preparation was centrifuged at 5000 g for 30 min and dialysed immediately against PBS pH 7 1 at 4°. The preparation was passed through a Millipore bacterial filter and stored at 4°. RESULTS Haemagglutination of erythrocytes of different species A large number of different types of erythrocyte were tested for their HA activity with free K88ab and K88ac preparations and for adhesion of whole organisms of the K88ab and K88ac types (Table 2). The most interesting feature was the powerful and consistent affinity of K88ab for the chicken erythro-

cyte membrane which had a sensitivity and specificity not normally evident in other species. The adhesion was stable at room temperature and was not affected by the addition of 0-5-2 % mannose. Pigeon erythrocytes showed a similar specificity for K88ab but the sensitivity of the test was considerably less. Organisms possessing the K88ac antigen failed to adhere to the chicken erythrocyte membrane and free K88ac preparations did not cause haemagglutination. Guinea-pig cells were agglutinated by both types of K88 and adhesion occurred with both K88ab and K88ac organisms. However, the agglutination and adhesion was unstable at room temperature and all tests had to be carried out at 00. Furthermore in our hands the sensitivity of the adhesion was variable and therefore lacked reproducibility. Organisms lacking the K88 antigen failed to adhere to any species of erythrocyte.

Correlation of erythrocyte adhesion with that of enterocytes E. coli of all serotypes tested possessing the K88 antigens adhered to enterocytes isolated from the duodenum or jejunum of the young piglet intectine

Immunological aspects of cell membrane adhesion

45

Figure 1. Adhesion of enteropathogenic E. coli (08: K87(B), K88a, b(L)) to an enterocyte isolated from piglet jejunum shown by interference contrast microscopy (original magnification x 1400). Note adhesion of the majority of bacteria to the microvilli. Figure 2. Adhesion of enteropathogenic E. coli (08: K87(B), K88a, b(L)) to the cell membrane of chicken erythrocytes shown by interference contrast microscopy (original magnification x 1400).

(Fig. 1). It can be seen from the figure that a few organisms adhere to the basement and cell membrane but the majority of organisms are seen to adhere, tightly packed, along the brush border. Tests with the same strains confirmed the results of Table 2 showing that erythrocyte adhesion of K88ab strains correlated with enterocyte adhesion whereas that of K88ac did not. Bacterial adhesion to chicken erythrocytes is shown in Fig. 2. The organisms tend to adhere to the edges of the erythrocyte with a number of organisms adhering to the upper and lower surfaces. The density of organisms is low compared to that seen on the microvilli of enterocytes.

This correlation between adhesion to enterocytes and chicken erythrocytes was further emphasized by absorption of a free K88ab preparation with piglet enterocytes. This totally removed the HA activity of K88ab for chicken enterocytes. Purification of K88 adhesive factor for adhesion inhibition studies A K88ab preparation from the 08 strain was subjected to isoelectric focusing to provide a highly purified preparation for adhesion inhibition studies with K88 antisera. The fractionation profile is shown in Fig. 3. The major protein peak eluted in

46

S. H. Parry & P. Porter I 0*10

10 0 90 8.0

0-09 0-08

10*0

_ 0 07

9-0

E

'a

0.06

8-0

0-05 0 "'-

_

-

0*04

6-0

003

50

0-02

40

e

-'

IX

6.0 2 50

70X

C *

7 0 ^N

h

.-W -

4*0

30 S 2-0

0-01

3-0 1*0 a I a A a- I 0 0 2 4 6 8 10 12 14 16 18 2022 Tube number Figure 3. Isoelectric focusing of K88ab (5 mg) using a pH gradient of 3-10. Five ml fractions were collected and analysed for 0) and haemagglutination of chicken erythrocytes (-----). The bar (tube numbers 6-8) denotes protein content (0 detection of K88 by double diffusion with a precipitating antiserum. a

-1

I

tube number 5 with an isoelectric point of pH 4 1. K88 was detected in tubes 5-7 by a precipitating antiserum. As expected the distribution of K88 adhesion factor as detected by haemagglutination of chicken erythrocytes generally correlated with the protein and precipitin results, the main peak occurring between fractions 4 and 7. However the highest titre in tube 7 did not correlate with the peak protein tube suggesting the possibility of some differentiation of the protein and adhesion components. In this regard it is interesting to note that there are still relatively high levels of haemagglutinating activity in fractions 9-16 but very little protein content. Neutralization of adhesion using K88 antisera The specific affinity of K88ab for the chicken erythrocyte provided a useful system for investigating the inhibitory properties of antisera produced against the K88 adhesion determinants. Antisera raised in rabbits and pigs against purified preparations of K88 provided evidence for a broad specificity of antibody activity using the piglet enterocyte adhesion inhibition assay. The results (Table 3) showed that all antisera whether against K88ab or K88ac totally inhibited adhesion of organisms

Table 3. Inhibition of 08 K88ab adhesion to piglet enterocyte microvilli using various antisera Mean bacterial count/enterocyte*

Antiserum specificity

Species

1/10

K88ab K88ab K88ab K88ab(ChRBC) K88ac Control (saline)

Sheep Rabbit Pig Pig Rabbit

0 0 0 0 0 85

-

Antiserum dilution 1/40 1/320 0-6 04 1-4 1 5 3-5 7-6

25 40 4-3 4-5 55 8-3

* Count per enterocyte-mean of number of bacteria adhering to the microvilli of 20 randomly selected enterocytes.

to enterocyte microvilli at a dilution of 1/10 and even at a dilution of 1/320 a considerable degree of inhibition was evident compared to controls. Using antisera to K88ab the agglutination of

erythrocytes by free K88ab and the adhesion of organisms possessing the K88ab antigen was readily inhibited as in the enterocyte assay. However, antisera to K88ac gave very poor inhibition of free K88ab agglutination or whole organism adhesion.

Immunological aspects of cell membrane adhesion

47

Table 4 Specificity of antisera as determined by slide agglutination

Agglutination of organisms Antiserum

specificity

Species

08(K88ab)+

08(K88)-

0149(K88ac)+

K88ab K88ac K88a* K88at K88b K88c

Pig Rabbit Pig Rabbit Pig Rabbit

+ + + + +

-

+ +

-

-

08(K88)-

+ +

+

-

* Anti K88a of K88ab origin. t Anti K88a of K88ac origin.

Inhibitory activity of antisera specific for the individual antigenic determinants of K88 Antisera specific for the individual determinants of K88 were produced by cross absorption with K88 organisms and elution of absorbed antibodies. Details of sera and absorptions are shown in Table 1. The specificity of antisera was critically tested using slide agglutination, the results of which are shown in Table 4. This confirmed that K88b and K88c antisera specifically agglutinated bacteria with the homologous antigen and that antiK88a serum of both K88ab and K88ac origin agglutinated both K88 organisms. These specific antisera were then examined for their ability to inhibit adhesion and haemagglutination. Table 5 shows the effect of the antisera on the adhesion of

K88ab and K88ac organisms. Antisera to K88ab and K88ac totally inhibited adhesion of both K88ab and K88ac organisms. Furthermore the specificities shown by slide agglutination were also reflected in their ability to inhibit bacterial adhesion. Thus antiK88b only inhibited K88ab, anti-K88c only inhibited K88ac while anti-K88a inhibited both organisms.

The same antisera were also used to study the inhibition of erythrocyte agglutination by pure K88 preparations using the chicken erythrocyte HI test for K88ab and the guinea-pig HI test for K88ac. The results are given in Table 6. Using whole antiserum raised against free K88ab and K88ab bound on the erythrocyte membrane, inhibition of a similar magnitude was achieved for both types of K88. Furthermore antisera to K88b and K88c were

Table 5. Inhibition of adhesion of E. coli 08(K88ab) and 0149(K88ac) to piglet enterocytes with specific K88 antiserum Mean bacterial count/enterocyte*

Antiserum

specificity

Species

08(K88ab)

0149(K88ac)

K88ab K88ac K88a, K88 K88b K88c Control

Pig Rabbit Pig Rabbit Pig Rabbit

0 0 2-4 0 0 > 10 > 10

0 0 5-3 0 > 10 03 > 10

* Count per enterocyte-mean of number of bacteria adhering to the microvilli of 20 randomly selected enterocyte after incubation with a 1/10 dilution of antiserum. Control was incubated in

saline only.

S. H. Parry & P. Porter

48

Table 6. Inhibition of erythrocyte agglutination by pure K88 preparations using specific K88 antisera HI titre

Antiserum specificity K88ab K88ab(RBC)

K88at

K88a(RBC)t K88b K88b(RBC) K88ac K88a§ K88c

Species Pig Pig Pig Pig Pig

Pig Rabbit Rabbit Rabbit

K88ab(08)*

K88ac(0149)t

2048 2048 16 8 256 128 32 8