Enzymatic hydrolysis of the crystals of Bacillus thuringiensis by the proteases of Pieris brassicae II. Toxicity of the different fractions of the hydrolysate for larvae of Pieris brassicae

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

Enzymatic

322430

Hydrolysis

thuringiensis II.

9,

Toxicity Hydrolysate

(1967)

of

of

of Pieris

of

Fractions

the for

des

Crystals

by the Proteases Different Larvae

Polyosides,

of

Pieris

Bacillus brassicae of

the

brassicae

M. LECADET

MARGUERITE Service

the

Institut

Pasteur,

Paris,

France

AND DANIEL Z.N.R.A.,

Station

de Recherches La Miniere Accepted

MARTOCJRET

de Lutte Biologique par Versailles, France August

15,

et de Biocoenotique,

1966

Dispersion of the crystalline structure of the paasporal thuringiensis, under the action of Pieris brassicae proteases, stances that are directly toxic by injection into the hemocoel the crystal itself is only active pm OS) in doses smaller than ing the fractionation of the hydrolysate, it appears that the by the rupture of peptide bonds, can be carried by the peptides with a molecular weight of the order of 5,000. different fractions to thermal denaturation seems to indicate is necessary for the action of the toxic factor.

INTRODUCTION

In the preceding paper (Lecadet and Dedonder, 1967) we have described the conditions of the enzymatic hydrolysis of Bacillus tliuringiensis crystals by the proteases of the cabbageworm of Europe, Pieris brassicae, and fractionation of the constituents of the hydrolysate. The different stages of this fractionation effected by chromatography on Sephadex G-25 and DEAE-Sephadex A-25 led to the separation of proteic and peptidic fractions of the hydrolysate and to the isolation of peptides with a molecular weight of the order of 5,000. One of the subjects of separating these 322

inclusion of Bacillus liberates soluble subof caterpillars (although 1 @g/g of larva. Followtoxic factor, unmasked proteins as well as by The sensitivity of the that a tertiary structure

different constituents was the investigation of fractions showing toxicity for P. brassicae larvae by injection, since we have already shown ( Martouret, 1960; Lecadet and Martouret, 1962) that the P. brassicae proteases transform the crystal protoxin into a true toxin, active by injection into the hemocoel of the caterpillars. A quantitative study of the toxicity of these hydrolysate fractions forms the subject of the present paper, in which we also establish the stability of the preparations and their sensitivity to denaturation by heat. We may recall, as regards P. brassicae larvae, that the symptoms of intoxication (previously described by Martouret, 1960) include buccal paralysis, involving a cessa-

~ox~c~rn

0F Lys,4~E

tion of feeding during the first hours following ingestion; and digestive troubles followed by general paralysis, leading to death within 48 hours following the intoxication. Reduction of feeding and mortality are used as criteria of the toxicity of the preparations (Lecadet and Martouret, 1962) MATERIALS

AND METHODS

Crystals of Bacillus thuringiensis [Serotype 1 (De Barjac and Bonnefoi, 1962)] were prepared by the technique previously described ( Lecadet, 1965). The preparation of hydrolysates and the fractionation of their constituents are described in the preceding paper (Lecadet and Dedonder, 1967) .

Determination

of Toxicity

The determination of the toxicity of the hydrolysates to P. brassicae was effected by biological assay using caterpillars in the fifth instar; this stage represents the optimum conditions required for microbiological tests (Grison and Sylvestre de Sacy, 1956; Burgerjon, 1957). The toxic activity of the preparations was tested on batches of 10 caterpillars. Five microliters of the preparation were administered to each individual by forced ingestion and by injection into the hemocoel with a microsyringe by a technique based on those of Dutky (1942) and Martignoni ( 1957). The reduction of feeding was assessed as follows, by a technique described by Burgerjon ( 1962). A standard size cabbage leaf (conforming to exact specifications) was offered to each individual of the treated batch of 10 caterpillars ( groups of identical mean weight) in a polystyrene box 4.2 cm in diameter and 1.1 cm deep. A control group of caterpillars dosed with sterile water was similarly

OF Bacillus

32:3

CRYSTALS

treated. The leaf area consumed was evaluated quantitatively by a photoelectric technique based on the principle described by Bulger ( 1935). The residual area of the leaf offered to each individual was interposed between a beam of light and a photoelectric cell, and the amount of light transmitted was a direct index of feeding. The reduction of feeding was expressed quantitatively by comparing the mean of the areas consumed by individuals of the treated group with that of those consumed by the control group, according to the formula: Reduction

of feeding

100 X surface surface

=

100

consumed

consumed

by the treated by the control

group

group

The mortality was evaluated on the same groups of 10 individuals on the 3rd, 5th, and 10th day of the experiment. This simple technique enabled the toxicity of the crystals and of the enzymatic hydrolysate to be determined. It could be used for a quantitative measurement by making a series of determinations of the reduction in the feeding on different groups treated with decreasing amounts of the toxin-containing preparations. The percentage reductions in the feeding were directl) proportional to the administered close, c’nabling the dose producing a 50% reduction ( Dso) to be established. These toxic doses are expressed in micrograms per gram of larva, the mean weight of the larvae being 200 mg. RESULTS

1. Comparative

Toxicity of the Proteinaceous and Peptide Fractions of the IlIydrolysate

In the first series of experiments we considered the action of the two groups of constituents of the hydrolysate: the proteinaceous group, not dialyzable and excluded by Sephadex G-75, the peptide

324

LECADET

AND

MAFITOURET

dialyzable and included in Sephadex G-75. The preparations were adjusted to pH 8.0 and their action was compared with that of an alkaline solution of the crystals. The relevant results are given in Table 1, from which we draw the following conclusions : 1. The proteinaceous and peptide fractions are toxic both by injection and by ingestion. 2. The alkaline solution of the crystals in 0.05 N NaOH is toxic only when given orally. 3. The chyle or the partially purified enzymatic preparation administered to the caterpillars under conditions of concentration in which they were used for the preparation of hydrolysates, show no toxicity. 4. The toxic doses are very low, less than 1 pg/g of larva; with 0.075 pglg we obtained a 25% reduction in the feeding. The principal criterion of activity in the case of P. brassicae is evidently the reduction in the feeding; nevertheless, the mortality is always concomitant. In spite of the uncertainty that generally attends biological assay, we obtained a satisfactory approximation of the toxic dose. It clearly follows from these results that enzymatic hydrolysis liberates substances that are highly toxic by injection and that the toxic action resides in molecules of different sizes.

is still fully active at 80°C but loses the greater part of its activity after heating at 90°C. This fraction is distinctly more resistant to heat than the proteinaceous fraction, whose denaturation is always more rapid.

2. Sensitivity of the Different Thermal Denaturation

Fractions to

4. Comparative Toxicity of Isolated Peptides and the Whole Peptidic Fraction

In the second series of experiments we tested the toxicity of the same preparations after subjecting them to various temperatures : 70”, SO”, and 90°C for periods of 10 minutes. The residual toxicities are listed in Table 2. The proteinaceous fraction is partially inactivated at 70” and 80°C and practically completely inactivated at 90°C. In contrast, the peptide fraction at the same dose as the proteinaceous fraction

One of the reasons that led us to isolate the peptides from the enzymatic hydrolysate was to find the smallest peptide still possessingtoxic activity. We submitted the peptides isolated by chromatography on DEAE-Sephadex to the same toxicity tests. We have established the low toxicity of each of these peptides (Table 3), a toxicity much lower than that of the whole peptide fraction. These results could be due

group,

3. Quantitative Determination of the TOXicity of the Proteinaceous Fraction We have endeavored to specify the toxic dose and to’ determine the Dso, i.e., the dose provoking 50% of the toxic manifestations. For this we have shown quantitatively the reduction in feeding as a function of the administered dose. Figure la shows the toxicity by ingestion of preparations of the crystal and the toxicity of the proteinaceous fraction of the enzymatic hydrolysate which has the same effect as the parasporal inclusion. Figure lb shows the toxicity by injection of the proteinaceous fraction of the hydrolysate (the crystals are inactive by this route), a toxicity in every way compnrable with that produced by ingestion. These graphs enable us to obtain the following values for D5”: 0.03 pg/g of larva for the crystal preparation; 0.04 pg/g of larva for the soluble form given by ingestion; 0.62 pg/g of larva for the soluble form given by injection. In the last case, the D5O is more than 10 times higher, but even this amounts to less than 1 pg/g of larva.

TOXICITY

OF

LYSATE

OF

BaciZlu~s

CRYSTALS

EC= SE= =z=

===

==c c rl

L- cc 3

cc= ===

===

S”

C3

/

/

=

0 3 s

326

LECADET

AND

hlARTOURET

TABLE THERMAL

RESISTANCE

i!

OF THE

TOXIC

Forced Quantity of proteins per g of larvae Nondialyzable proteinaceous fraction excluded by Sephadex G-75 Not heated Heated 10 min at ‘70” C Heated 10 min at 80’ C Heated 10 min at 90” C

0.825

0.825

42.5 0.825

ingestion

Injection

Mortalityc

RC

Mortality”

RC

%‘Ch

3

5

10

q*

3

5

10

99

7 3

10 6

10 8

8”

10

10

10

69

3

4

46

6 4

6 7

12

0

0

5 1

0 3 1

0

1

96

8

10

10

98

99 97

9 5

10 10

10 10

99 97

8 9

10 10 10

57 18

2 0

4

7 6

48 9

/.q 90 66

Dialyzable peptide fraction included in Sephadex G-7.5 Not heated Heated 10 min at 70” C Heated 10 min at 80’ C Heated 10 min at 90’ C

FRACTIONS”

0

/.~g

pg pg

3

9 2 1

10 10

6

10 9

1

2

a Aliquots of the same preparation were heated at 70”, 80”, and 90°C for lo-minute periods and after cooling 5 ~1 portions of the different fractions were given to each individual in lots of 10 caterpillars of mean weight 200 mg. * Toxic activity expressed by the reduction in feeding (RC To) o b served after 44 hours relative to the feeding of controls treated with 5 ~1 of water. c Mortality on the 3rd, 5th, and 10th day of the experiment.

RC%

RC%

50. 10. 30,

l /

,0*52

425

.

. .

I= ww

50

/

10.0 JJg/g d brvae

II’NECTION --

S&Me

.

FORCED toxin

-

-

-

a

FEEDING

Soluble Pafvnpoml

toxin inclusion

b

FIG. 1. Quantitative evaluation of the toxicity of the proteinaceous fraction of the drolysate (soluble toxin) compared with a suspension of untreated crystals of BUC&S Dose administered to the larvae, fig/g, versus reduction in feeding, %.

enzymatic hythuringiensis.

TOXICITY

OF LTSATE

OF Bacillus

TABLE COMPARATIVE

TOXICITY

OF ISOLATED

PEPTIDIC

Amount

(a) Peptides”:

u Peptides

s

FRACTIONS

given

hdg larvae)

---~-. 1 ” 3 k

LNI)

THE WHOLE

Y Reduction ---__ Forced ingestion

Injection 8 LL”

41

17.5

&X

(c) ‘Tutal stored

peptide fraction at -10" C

42.5 0 825

97 06

on DEAE-Sephadex

to the fact that the isolated peptides are not the most toxic ones among the constituents of this fraction. However, we must point out that the various chromatographic methods leading to the isolation of these peptides were carried out at 35”C, thus possibly producing denaturation and leading to a diminution in toxicity. If we compare the toxicity of the whole peptide fraction kept at 4°C during chromatography on Sephadex G-75, and thereafter stored at -lO”C, with that of the same fraction after chromatography and storage at 25”C, we observe in the latter case a considerable diminution in the toxicity, which is of the same order as that of the isolated peptides ( Table 3 ) . From this fact, the diminution of the toxicity by denaturation (already suggested by the action of temperature; Table 2) occurring during isolation of the peptides would seem to indicate the necessity for the presence of tertiary structure to produce the toxic effect. These diverse considerations lead us to think that the isolated peptides, and especially peptides 2 and 3, are just as toxic by ingestion as by injection. Consequently, enzymatic hydrolysis unmasks the toxic motive which can be equally well carried

9-05

FRACTION

of feeding

0 “0 35 II

peptide fraction at ‘25” C

by chromatography

PEPTIUE

1 I-2 10 3

(1~) Total stored

isolated

:12'i

CRYSTALS

I_-~-.

_-

2-i 8

columns.

by the proteins as by the peptides (of molecular weight of the order of 5,000) resulting from proteolysis.

5. Preservation of the Toxic Actioity Hydrolysates

of tlrc

Enzymatic hydrolysates of the crystals of the Berliner strain of B. thuringiensis have been kept since their preparation under the following conditions: in solution in 0.1 M phosphate buffer of pH 8.0 in a frozen condition at -22°C; lyophilized after preparation and precipitation by ammonium sulfate at 50% saturation, and kept at +2” to f4”C. After bringing them to laboratory temperature, these hydrolysates were diluted to various degrees with 0.02 N phosphate buffer of pH 8.0, and were administered to homogeneous batches of 10 P. Drassicue caterpillars under the conditions described above under “Materials and Methods.” The results obtained with freshly prepared hydrolysates and with those stored as described above are compared in Table 4. They show that the toxic properties of the enzymatic hydrolysates of crystals of H. tlmringiensis remain relatively stable after 3 years of storage under the various experimental conditions.

328

LECADET

AND

TABLE PRESERVATION

Nature

of preparation of preservation

OF TOXIC

and mode

(a) Fraction nondialyzable against 0.01 u phosphate buffer of pH Fresh activity Undiluted Diluted l/IO Activity after 3 years at -22’C Diluted l/l0 (b) Solution (a) 50y0 precipitated by (NH&S04 and ppt. redissolved in 0.01 M phosphate buffer of pH 8.0 Immediate activity Undiluted Diluted l/l0 Activity after 3 years at -22°C Diluted l/10 (c)

Lyophilized immediate Activity

after

solution activity 3 years

ACTIVITY

Protein con;;;;;;ion

MARTOURET

4 OF ENZYMATIC

Amount given (pg/g of larvae)

HYDROLYSATEd

Forced Rc s*

Intrahemocoelic injection

ingestion MortalityC 3

5

10

Mortalityc

RC y$‘Ch

3

5

10

-

-

8.0 37 3.7

99

10

-

-

96

10

0.148

1.48

99

7

10

-

82

7

7

8

0.148

3.7

99

7

10

-

85

1

5

7

99

10

-

-

0.48 0.048

14

98

9

10

71

4

6

7

61

6

7

7

1.2

98

810-

0.048

1.2

93

5

0.010

0.250 0.05 0.250 0.05

97 69

7103 8

10

-

-

99

9

10

-

64

2

2

30 -

4 ----

7

9

-

(b)d

at 2-4°C

0.002 0.010 0.002

33

7

6

7 -

7 -

4

A

a Each caterpillar in the lots of 10 is treated with 5 ~1 of the different preparations (see “Materials and Methods”). b Toxic activity expressed by the reduction in feeding (RC 7)o o b served after 24 hours relative to the feeding of controls treated with 5 ~1 of water. c Mortality on the 3rd, 5th, and 10th day of the experiment. * The lyophilized preparations were redissolved in 0.02 M phosphate buffer of pH 8.0 immediately before administration to the larvae.

DISCUSSION

The results of the investigation we have described show clearly that enzymatic hydrolysis of crystals liberates soluble proteins which are directly toxic by injection into the hemocoel of caterpillars, and thus co&m the hypothesis of a crystal protoxin put forward by Angus (1956) and by Martouret ( 1960). The structure of this crystal molecule, whose cohesion is ensured by the formation of disulfide bonds (Young and Fitz-

James, 1959; Lecadet, 1966) between the constituent protein chains seems to be organized in such a manner that a certain number of peptide linkages sensitive to the action of P. bmssicae proteases, and corresponding to their specificity, are directly accessible to the active sites of these enzymes. The resulting enzymatic hydrolysis completely disorganizes the structure of the molecule, thus unmasking the toxic factor carried by the different fractions liberated during the reaction. The total hydrolysate or the constituents

TOXICITY

OF

LY~ATE

of the proteinaceous fraction, in whatever manner administered (by ingestion or injection ) , provoke symptoms identical with those resulting from ingestion of the crystals themselves in similar or slightly smaller doses, doses below 1 pg/g of larva; this places the crystal and its derivatives in the category of highly toxic substances. It is interesting to find that the toxic factor is also carried by the smallest molecules, the peptides, the entire toxicity of which is as great as that of the proteinaceous fractions. However, the peptides isolated by successive chromatographic separations at 35°C show a lower toxicity, resulting probably from inactivation during preparation. It would otherwise seem that a tertiary structure is necessary to support the toxic group since we have shown the temperaturesensitivity of these fractions. This aspect of the enzymatic actiontransformation of the crystal protoxin into a true toxin-brings up the more general problem of activation. The formation of a molecule endowed with biological activity (enzymatic or otherwise) by a proteolytic action starting from an inactive precursor is frequent in biological media. The transformations of trypsinogen ( Kunitz, 1947), chymotrypsinogen ( Northrop et al., 1948 ), and fibrinogen are the best known examples. The elimination of a peptide or the opening up of a cyclic structure are sufficient to bring about the appearance of biological activity by a very specific enzymatic process, e.g., the rupture of the arginyl-leucine linkage in the case of chymotrypsinogen. As regards the toxin of B. thuringiensis the dispersion of the crystal structure by rupture of the peptide linkages effects activation of the precursor by liberating several proteins and peptides carrying the toxic factor. One may, therefore, think that phenomena of this kind are produced in viva. In the course of intoxication of P. brassicae by B. thuringiensis, the ingested crystals

0~

Bacillus

CRYSTALS

329

are hydrolyzed in the digestive tract by the proteases of the chyle. As a result, the toxic symptoms that we have described appear : buccal paralysis manifested by a cessation of feeding, and ending in death of the larvae. The enzymatic liberation of the toxic factor has further been decisively confirmed by the experiments of Yamvrias ( 1962), who showed that the products of hydrolysis of the crystal by the chyle of P. brassicae are highly toxic for larvae of the Mediterranean flour moth, Anagasta kdzniellu, when they are administered either by ingestion or by injection, while the crystals themselves are inactive towards this species. In other words, the manifestations of different toxicities according to the species appear to be linked to the possibility of the occurrence of in vivo enzymatic hydrolysis of the crystals. Species insensitive to the toxin in question alone can be intoxicated by substances derived from it. This phenomenon of activation of the crystals, which we have demonstrated, contributes to the explanation of the pathogenic power of B. thuringiensis for the larvae of certain species of Lepidoptera (P. brassicae in particular) and allows us to extend the efficiency of parasporal inclusions to species that are not sensitive to it. REFERENCES ANGUS, T. A. 1956. Extraction, purification, and properties of Bacillzrs sotto toxin. Can. J. kficrobiol., 2, 416426. DE BARJAC, H., AND BONNEFOI, A. 1962. Essai de classifications biochimique et serologique de 24 souches de Bacillus du type B. thuringiensis. Entomophaga, 7, 5-31. BULCER, J. W. 1935. A photo electric method for measuring small leaf areas. J. Econ. Entomol., 28, 76-81. BURGERJON, A. 1957. L’utilisation des chenilles de Pieris brassicae L. comme insecte test de laboratoire dans un service de contrSle de preparations pathogenes insecticides. Entomophaga, 2, 129-135.

330

LECADET

AND

BURGERJON, A. 1962. Relations entre l’intoxication provoqu&e par B. thuringiensis Berliner et la consommation chez Pieris brussicae L. Ann. Epiphyties, 1, 59-72. DUTKY, S. R. 1942. Method for the preparation of spore-dust mixtures of type A Milky disease of Japanese beetle larvae for field inoculation. U.S. Dept. Agr. Entomol. Plant Quarantine E.T.T., 192, 10 pp. GRISON, P., AND SYLVESTRE DE SACY, R. 1956. L’klevage de Pie& brassicae L. pour les essais de traitement microbiologique. Ann. Epiphyties, 4, 663-676. KUNI-IZ, M. 1947. Cristallization of saltfree chymotrypsinogen and chymotrypsin from solution in dilute ethyl alcohol J. Gen. Physiol., 32, 265-269. LECADET, M. M. 1965. La toxine figurbe de B. thuringiensis. Technique de skparation et composition en acides aminks. Compt. Rend. Acad. Sci., 261, 5693-5696. LECADET, M. MM. 1966. La toxine figurke de B. thuringiensis. Dissolution par action du thioglycolate ou de la cystCine. Compt. Rend. Acud. Sci., 262, 195-198. LECAUET, M. M., AND DEDONDER, R. 1967. Enzymatic hydrolysis of the crystal of Bacillus thuringiensis by the proteases of Pieris brassicae. I. Preparation and fractionation of the lysates. J. Incert. Puthol., 9, 310-321.

MARTOURET

LECADET, M. M., toxine fig&e enzymatique par injection. 24574459. ~~ARTIGNONI,

cenza Hubn. tore nella For&.,

AND MARTOURET, D. lQ62. La de B. thuringiewis. Production de substances solubles toxiques Compt. Rend. Acad. Sci., 254,

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MARTOURET, D. 1960. Etudes p&liminairc,s mode d’action de B. thuringiensis ringiensis Berliner vis-h-vis de Pieris L. Proc. 11th Intern. Congr. Entomol., 2, 849455.

sur le var. thubrassicae I’icnnu,

NORTHROP, J. II., KUNITZ, nl., ANU HEIWO.I-I.. R. 1948. “Cristalline Enzymes,” 2nd ed. Columbia Univ. Press, Kew York. YAII~RIAS, C. 1962. Contribution it l’ttud<, du mode d’action cle B. thuringiensb vis-A-vis de In teigne de la farine. Anagastu kiihrtielka Zell. ( Lepidoptkre ) Entomophaga, 7 ~ 1Ol159. YOUNG,

E.,

AND

FITZ-JAhfES,

cal studies of Bacterial Spore and parasporal B. cereus var. alesti. C!ytol., 6, 483-498.

C. 19%. (%c?nisporr formation-II. protein fornlation in J. Biophys. Biocf~em. 1'.