BAILLIE, ANN (1987). J . appl. Bact. 30 ( l ) , 230-238.

Antigens of Bacillus cereus: A Comparison of a Parent Strain, an Asporogenic Variant and Cell Fractions

ANN BAILLIE? Department of Bacteriology, University of GYmgow, Scotland

(Received 15 August, 1966)

SUMMARY. The antigenic structure of a stable asporogenic variant of the M8 strain of Bacillua cereu~ has been compared with that of the parent strain. Ultrasonic extracts of cells of both parent strain and variant harvested at different ages have been analysed by immunoelectrophoresis against antisera prepared by injecting such extracts into rabbits.

Disintegrates of cells of the asporogenic variant were antigenically identical with disintegrates of vegetative cells of the parent strain. Disintegrates of cells in later stages of sporulation and of mature spores of the parent strain contained thermostable antigens which were never detected in the variant. Antigens of isolated cell walls, protoplasts and flagella were also studied.

Examination of esterase and catalase content of the two strains showed that although the variant had the same enzymes as the young vegetative cells of the parent strain it never developed the thermostable catalase found in disintegrated spores. Protein com- ponents of the two strains at different stages of growthandof theisolated cellfractionswere studied by electrophoresis in polyacrylamide gels.

ANTIQENIC AND ENZYMIC changes occurring during spore formation in Bacillus cereus have been described elsewhere (Baillie & Norris, 1963, 1964). This work has now been extended to the study of an asporogenic variant and of isolated cell fractions of the parent strain, to determine the location of the macromolecules which change during sporulation.

Materials and Methods Organisms

The strain of B. cereus (strain M8) used throughout this work was the same as that in the earlier work (Baillie & Norris, 1964) and was isolated in the first place from Egyptian soil (Mahmoud, 1955).

During the induction of motility in the culture by serial passage through semi- solid agar, (containing 0.3% of agar) the organism was plated on Lab-Lemco agar (Oxoid) as a routine purity check. After incubation for 16 h the plate appeared normal, but after 32 h many of the colonies had undergone a marked change. Instead of being a creamy white they were transparent. On examination by phase contrast microscopy the organisms in these colonies were evidently undergoing lysis, leaving empty cell malls and a few swollen distorted cells. The variant was isolated in pure culture and has since been maintained on nutrient agar. No tendency to revert to the parent form has been noted, the variant remaining stable and asporogenic.

t Present atltlress: Wellcome Rcsearch Laboratories, Reckenham, Kent, England.

[2301

Antigens of B. cereus 231

Culturw in liquid media These were grown in liquid sporulation medium as described by Baillie & Norris (1963).

Cultures on solid media These were grown in Roux bottles containing 150 ml of Lab-Lemco agar.

All cultures were incubated a t 30".

Preparation of cell fractions Flagella

As the parent strain was not actively motile and possessed few detectable flagella, the organism was passed through semisolid agar in order to stimulate motility. Plates of this agar were given a single central inoculum, and after 24-48 h growth had permeated the entire plate. A loopful of growth from the periphery was used to inoculate the centre of a second plate. After 6 passages in this way, the culture was actively motile, composed of peritrichously flagellate rods. Cultures of these motile rods were grown in sporulation medium in shaken culture. The method of isolation of flagella was essentially that of Gard (1945). Cells mere harvested by centrifugation, washed 3 times in distilled water and resuspended in a minimal volume of distilled water. The resulting suspension was transferred to a conical flask and shaken vigor- ously for 2 h on a Microid flask shaker (Griffin & Tatlock Ltd, London). Microscopic examination of such preparations treated with Kirkpatrick's stain showed that the majority of the flagella had become detached from the cells, the latter being removed by centrifugation a t 1000 r/min for 1 h. The clear supernatant was spun a t 18,000 r/min (25,000 g) for 1 h to give a gelatinous precipitate which was resuspended in a small volume of distilled water. Three short runs (10 min) a t lo00 r/min removed residual cells. The flagella were washed twice, and finally resuspended in a small volume of distilled water. For electrophoretic examination the suspension was exposed to ultrasonic vibration for 5 min using the Mullard Ultrasonic Disintegrator (M.S.E. Ltd., Spencer St., London, SWl).

Cell walk Cultures of the variant were grown for 16 h on Lab-Lemco agar in Roux bottles.

The growth was harvested, washed in distilled water and cell walls isolated by the method of Salton (1953). The cells were shaken with an equal volume of Ballotini beads (no. 14) in a Mickle Tissue Disintegrator, the beads and residual cells being removed by centrifugation a t 1000 r/niin for 20 min. The cell walls were washed 4 times in M-NaCl, which rapidly removed cytoplasmic debris. This treatment was followed by 15 washes with distilled water. Phase contrast observation showed that the cell walls were free from intracellular granules. Purity of the preparation was checked under the electron microscope and by chromatographic analyses of hydrolysates. The cell walls were disintegrated by ultrasonic vibration prior to electrophoresis.

Protoplasts The protoplast preparation was made as described by Dark & Strange (1957).

Autolytic enzymes prepared from sporulating cultures of B. cereus (Strange & Dark, 1057) n'wc used to treat vcgetatire cells suspended in buffered sucrose solution.

232 Ann Baillie

Within 60 min a few spherical forms were detected by phase contrast microscopy and these increased in number until after 24 h there was 60-70% conversion. The suspension was centrifuged a t 3000 r/min for 20 min and the protoplast-cell mixture washed twice in buffered sucrose solution. Sedimented cells and protoplasts were suspended in a minimal volume of distilled water, and this resulted in complete lysis of the protoplasts. Residual rods and protoplast membranes were removed by centrifugation.

Preparation of disintegrates Ultrasonic disintegrates of cells and spores were prepared and standardized by the

method of Baillie & Norris (1964). All extract concentrations were adjusted to contain 0.5 mg of amino nitrogen/ml.

Antisera These were prepared in rabbits as described by Baillie & Norris (1964).

Heat resistance Thermostability of enzymes, antigens and proteins were tested by heating a t 80" for 10 min; the antigens were tested immediately prior to electrophoresis.

Electrophoresis Disintegrates were examined by electrophoresis in agar, starch and polyacrylamide gels.

For antigenic studies, agar gel electrophoresis was carried out as described by Baillie & Norris (1964). Polysaccharide-containing antigens were detected by the method of Stewart-Tull (1965) and appeared as deep pink precipitin bands against a paler pink background.

Starch gel electrophoresis and the subsequent detection of esterase and catalase was done by the method of Baillie & Norris (1963).

Polyacrylamide gel electrophoresis for the study of proteins was a modification of the method of Raymond & Weintraub (1959). A 7% (w/v) solution of Cyanogum 41 (B.D.H. Ltd., Poole, Dorset) in Tris-citrate buffer (as used in starch gels) was poly- merized by the addition of 0.01 % of ammonium persulphate and 0.01 % of dimethyl- aminoethyl cyanide. Gels were electrophoresed with a potential gradient of 10 v/cm for c. 4 h. On completion of electrophoresis, gels were sliced horizontally and stained to detect the presence of the separated protein components by immersing a slice in a saturated solution of 10% aqueous acetic acid for 10 min. Decolourization was effected by washing the gel repeatedly in frequent changes of a 5% aqueous acetic acid solution. Protein bands stained dark blue against the clear colourless background of the gel.

Results Four stages of growth and sporulation of the parent strain have already been examined for esterase, catalase and antigen conbent and the results described by Baillie & Norris (1963, 1964). For completeness, a brief summary of these results is included. The stages previously examined were : (i) vegetative cells which appeared uniformly

Antigens of B . cereus 23 3

dense under phase contrast ; (ii) vegetative cells which showed distinct granulation of the cytoplasm ; (iii) sporulating cells and (iv) mature spores released from the sporangia and washed free of vegetative debris.

In the present investigation 5 stages of growth of the asporogenic variant were studied: (a) 24 h cultures in liquid medium, similar in appearance to (i); (b) 48 h cultures in liquid medium, similar in appearance to (ii); (c) 24 h cultures on agar, cells showing granulation and some pleomorphic forms; (d) 48 h cultures on agar, with many swollen and distorted cells some of which uere undergoing lysis to leave cell walls; (e) 72 h cultures on agar, the majority of cells of which had lysed to give large numbers of empty shells, leaving a few pleomorphic rods.

Cells from broth cultures of the asporogenic variant appeared physiologically younger than did the corresponding cells on agar culture. At no point did the variant progress beyond the cytological appearance of stage (ii) of the parent strain.

Antigens The antigenic composition of the cell extracts, cell walls, protoplasts and flagella was studied by immunoelectrophoresis in agar and the results obtained with antisera to stages (i) and (iv) are reported in detail. Confirmatory results were obtained with sera prepared against other stages.

Stage (i) cells had a t least 15 separate antigens which could be detected with serum 1 (the homologous serum) and only 3-5 which could be detected with serum to stage (iv) (serum 4). Stage (ii) cells showed similar properties. Cells of stage (iii) showed 15 precipitin bands with serum 1 and almost as many (12) with serum 4, while stage (iv) cells possessed 9 antigens which reacted with serum 1 and 12 which reacted with the homologous serum. The precipitin patterns of the asporogenic variant closely re- sembled those of stage (i) cells.

TABLE 1 Antigens of Bacillus cereus

No. of antigens detected with Occurrence of heat resistant antigen

- r , Origin of antigens serum 1 serum 2 a b c t d t e t f t g t h k t lt m t n t o t p q

15 3-5 + + + + + + + - - - - - - - - (ii) 15 3-5 + + + + + + + + - - - - - - - (iii) 15 12 + + + + + + + - + - + + + - + (iv) 9 12 _ _ + + + + - - - + + + + + -

14 4 - + + + + + + - - - - - - - - 14 4 - + + + + + + - - - - - - - -

4 - + + + + + + - - - - - - - - 4 - + + + + + + - - - - - - - -

11 4 - - + + + + + - - - - - - - -

Cell walls 5 3 - + + + + - + - - - - - - - - Protoplasts 11 3 - - + + + + + - - - - - - - - Flagella 0 0

{ ( j )

Parent strain

Asporogenic (b)

(d) variant

l ( e )

1::: 10 11

- - - - - - _ - _ _ _ - _ _ _ t Antigens giving a positive result when stained for polysaccharide.

234 Ann Baillie

"I;

Cell wall extracts showed 5 antigens, but none was demonstrated in flagella. Protoplasts (prepared from stage (ii) cells) had 11 antigens demonstrable with serum 1, as compared with the 16 of untreated cells, and three with serum 4 (3-6 in untreated cells).

d e

t

Reaction developed with serum 1 by antigen

'(i) ( C ) (d ) (e l ' (ii)

t

( 1 1 1 )

0 b

d e f

C

k 9

b

d

f

9 'T Reaction developed with serum 4 by antigen

r ( iv) ( a ) (b) (C) ( d ) ( e l * ( i ) (ii) (iii)

C

d e

k" 9 0

n

P

n O

0

Fig. 1. Diagrammatic representation of immunoelectrophoretic analyses of heat resistant antigens of Bacillw, cerewr.

The numbers of antigens detected in the various extracts are summarized in Table 1 and the appearance of the precipitin lines and the changes between the stages are shown in Plate 1. A diagrammatic representation of the thermostable antigens can be seen in Fig. 1. Of these antigens, c, d, e, f, g, k, 1, m, n and o gave a positive result when tested for the presence of polysaccharide.

Enzymes As reported by Baillie & Norris (1963) 2 catalases and 2 esterases were found in extracts of B. cereus studied by starch gel electrophoresis. Both esterases occurred in vegetative cells and were also present in all extracts of the asporogenic variant as was

PLATE 2. Photograph of an acrylamide gel stained to detect the presence of proteins. Samples (left to right) were extracts of cells of the asporogenic variant at stages (a), (b), (c ) , (d) and (e ) .

Antigens of B. cereus 235

the vegetative cell catalase. The heat resistant spore catalase of sporulating cells and spores of the parent strain was never found in extracts of the asporogenic variant. Extracts of protoplasts contained both esterases and both catalases.

Proteins Differences in protein constituents of the various stages were followed by electro- phoresis in polyacrylamide gels. This system was chosen in preference to starch as the transparency of the gel gave a greater sensitivity and the resolution of the protein bands was much improved.

Vegetative and sporulating cells appeared to have similar patterns, 17 bands being detected in stage (i) cells, 18 in stage (ii) cells and 15 in stage (iii) cells. In contrast, mature spores (stage (iv)) possessed only 5 protein components all of which had a high electrophoretic mobility, the slower running proteins of the earlier stages having disappeared.

The asporogenic variant had a protein pattern essentially similar to stage (i) cells, but with fewer components, stage (a) having 12 bands, stage (b) 16, stages (c) and (d) 12 bands and stage (e) showing only 11 bands.

Cell wall preparations contained 6 protein bands, and the protoplasts contained 10, whilst the flagella had only a single protein component.

A diagrammatic summary of the occurrence of the proteins and their heat stability is shown in Fig. 2. A typical stained gel can be seen in Plate 2.

Parent strain Asporogenic variant

- Growth stage

- - - - -

Fig. 2. The proteins in cultures and cell fractions of Bacillus cereu8 as detected CW, cell walls; P, protoplaate; E', flagella; H, heated at 80" by gel electrophoresis.

for 10 min.

Discussion The process by which vegetative cells form spores is complex and it has been studied from many different viewpoints. Differences in molecular components of vegetative

236 Ann Baillie

cells and spores should be reflected by differences in antigenic composition. By following the latter, it should be possible to throw some light on the molecular changes involved, and similarly a study of the antigens of isolated cell fractions should help in tracing the origin of the spore constituents. These can be either vegetative cell components altered in some way so as to become heat resistant, or they may be synthesized de novo during the growth and maturation of the spore.

The antigen changes during growth and sporulation of the parent strain have been described elsewhere (Baillie & Norris, 1964). Seven thermostable antigens were detected in stage (i) cells, 4 of which, c, d, e and f, persisted throughout growth and sporulation and were found in mature spores : these 4 contained polysaccharide. The other 3 thermo-resistant antigens of stage (i) were present in stage (iii) cells but were absent from mature spores. Antigen g has a polysaccharide component, and therefore its thermo-resistance is hardly surprising. On the other hand, antigens a and b failed to stain by the periodic acid Schiff reaction. Both antigens have a fairly high electrophoretic mobility and could well be peptides or polypeptides which are much less sensitive to heat denaturation than proteins. The most striking overall fact emerging from these antigen studies is the marked increase in the heat resistance of the cell constituents as sporulation progresses. Of stage (i) cells, for example, <50% of the antigens resist heating a t 80" for 10 min, while few of the antigens of the spore are thermolabile.

In an endeavour to locate the antigens within the cell, the component cell walls, protoplasts and flagella were isolated. The cell walls contained 6 antigens, all of which were thermostable, and 4 of them gave a positive reaction when tested for the presence of polysaccharide. Three of these antigens were detected both in spore extracts and in extracts of vegetative cells.

Walls of Gram positive organisms appear to be composed mainly of a mucoprotein complex consisting of a peptide and amino sugars (Salton, 1960) and up to 30% amino sugar has been found in the walls of B. cereua (Salton, 1958). Strange & Dark (1956) showed that spore coats of B. cereua contain a variety of amino acids in addition to the characteristic components of the mucoprotein complex of the cell wall, and it is suggested that the main constituent of the coat is a structural protein. The cell wall antigens, c, d and e, which are present in the mature spore may well be the muco- protein complex components of the cell wall which Strange and Dark detected in the spore coat. Ultrasonic treatment of the cells could split the link between these com- ponents and the structural protein, leaving the carbohydrate moiety free to migrate in an electric field.

Protoplasts contained fewer antigens than vegetative cells but appeared to share some antigens with the cell walls (antigens c, d, e and g). There are several possible explanations for this observation, the most obvious of which is the use of a protoplast preparation which is contaminated with cell wall remnants as a result of inadequate washing. Another factor to be taken into consideration is the mode of action of the autolytic enzymes. These, obtained by autolysis of sporulating cells of B. cereus, attack vegetative cells and cell walls releasing hexosamine containing peptides (Strange & Dark, 1957). There would appear to be two distinct lytic systems in such autolyzates. The authors suggest that enzyme V (with a pH optimum of 4.6) is

Antigens of B. cereus 237

concerned with the release of free spores from the sporangia and the erlzyme S (pH optimum 8.0) with the lytic process accompanying spore germination. The exact linkages attacked by these enzymes is unknown, but typical cell wall amino-sugar complexes are released from the walls. It is possible, therefore, that the cell wall antigens present in the protoplasts may be the part of the wall which remains unaffected bythesc enzymes. But a third possibility exists. The antigens c, d and e were detected in vegetative and sporulating cells, mature spores, cell walls and protoplasts. Since these antigens contain polysaccharide and are of fairly high electrophoretic mobility they may be hapten groups which are found a t various sites in the cell. On treatment with ultrasonic vibration such groups could be separated from a larger molecule to which they were attached.

Since flagella are protein and are certainly antigenic, failure to detect any antigens in the purified flagella preparation was unexpected. Immunological studies on flagella have used mainly agglutination or complement fixation tests. The presence of a protein band in flagella preparations subjected to electrophoresis in polyacrylamide indicates that the protein component was able to migrate in an electric field. This being so, failure to detect flagellar antigens could be due to the use of inadequate antisera. Bacillus cereus M8 is not a very actively motile organism and hence the disintegrates used for immunizing rabbits probably contained little if any flagellar material. To overcome this difficulty a rabbit was immunized with a purified flagellar preparation, but in spite of a primary course and booster series of injections a satis- factory response has not yet been obtained. Vennes & Gerhardt (1959) studied the isolated cellular constituents of B. megaterium with antisera against these components by quantitative complement fixation tests, and found that their protoplasts were antigenically distinct from cell walls, but this may be accounted for by the different methods of preparation of the protoplasts. Bacillus megaterium is sensitive to lysozyme and this enzyme was used for the removal of cell walls. Lysozyme action is believed to result in complete dissolution of the walls of €3. megaterium as examined microscopically, chemically and immunochemically (McQuillen, 1960).

The most important observation in the antigen and enzyme studies with the asporo- genic variant is that the constituents peculiar to late sporulating cells and spores of the parent strain were never detected. Thermostable antigens a and b were absent from older cultures of the variant. Neither of these antigens contain a polysaccharide moiety and it is suggested that they might be peptide molecules. If this is the case, such molecules might be degraded during the autolysis of the cells. An asporogenic variant is potentially valuable as a careful comparison with the parent strain might help to elucidate some of the key steps in sporulation.

I should like to express my gratitude to Dr. J. R. Norris for his help and encourage- ment throughout this work. The work was carried out during the tenure of an Agri- cultural Research Council Research Studentship ; and I am grateful to Unilever Ltd. and the Royal Society for equipment grants.

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