Mechanism of Action of Bacillus Thuringiensis Var Israel Ens Is Insecticidal S-Endotoxin

8/8/2019 Mechanism of Action of Bacillus Thuringiensis Var Israel Ens Is Insecticidal S-Endotoxin

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Volume 1.54, number 2 FEBS LETTERS April 1983

Mec~anis~ of action of ~aciZZ~s ~~~i~gie~sis ar is$a~~~nsisinse~ticida~ S-endotoxin

W.E. Thomas and D.J. EIlarDepartm ent of Biochemist ry, University of Cambridge, Tennis Court Road, Cambridge, CB2 IQ W , England

Received 14 February 1983Bacillus thuringiensis var israelensisli-endotoxin protein active against mosquitoes was inactivated by priorincubation with lipids extracted from Aedes a~bopicfus cells. Experiments with lipid dispersions andmu~tilamellar liposomes showed that the toxin binds to phosphatidyl choline, sphingomyelin andphosphatidyl ethanolamine provided these lipids contain unsaturated fatty acids. Phosphatidyl serinebinds toxin less efficiently and phosphatidyl inositol, cardiolipin, cerebroside and cholesterol show noaffinity for the toxin. The results suggest an insecticidal mechanism in which interaction of toxin withspecific plasma membrane lipids causes a detergent-like rearrangement of the lipids, leading to disruption

of membrane integrity and eventual cytolysis.Bacillus thuringiensis var israelensis &Endotoxin Phospholipid receptor InsecticideMosquito

1. INTRODUCTIONCertain ~~~~11~s huri~gie~s~s strains synthesize

intracellular protein inclusions (&endotoxins),which are potent insecticides [l] licensed for com-mercial use on a variety of Lepidopteran pests. Thetoxin from a new serotype H-14, designatedBacillus t huringiensis var israelensis is lethal forDipteran larvae [2] including mosquitoes of thegenera Aedes and Culex, which are vectors ofmalaria and filariasis, respectively. An extensiveWHO programme is currently assessing the poten-tial of this serotype for the control of insect vectorsof these and other tropical diseases [3].

Despite their importance, the mechanism of ac-tion of these toxins is not known. Previous reportshave suggested that the primary target of the 6-endotoxin from B. thuringiensis serotypes activeagainst Lepidoptera is the plasma membrane ofgut epithelia and of susceptible cells in vitro[4-121. In [13] it was demonstrated that, in vivo,the gut epithelium also appears to be a primarytarget for the var israelensis d-endotoxin, Recentlyauthors in 1141observed that a soluble preparation

Liposome binding

of israelensis toxin caused rapid cytolysis of insectand mammalian cells in vitro, but had no effect onbacterial protoplasts. This toxin also showedhaemolytic activity against a range of erythrocytes.The possibility that the toxin causes lysis directlyby interaction with a plasma membrane compo-nent was therefore investigated. In this report weidentify certain membrane phospholipids as theprimary target for the toxin and discuss themechanism of cytolysis.

Initial experiments showed that the toxin is inac-tivated by prior incubation with mosquito celllipids, Using purified lipids we find that the toxinbinds readily to multilamellar liposomes contain-ing phosphatidyl choline, SphingomyeIin, orphosphatidyl ethanolamine provided these lipidscontain unsaturated acyl residues. Phosphatidylserine binds toxin relatively poorly, butphosphatidyl inositol, cardiolipin, cerebroside andcholesterol show no affinity for the toxin, Theresults suggest that in susceptible insects the in-teraction of toxin with specific plasma membranelipids causes a detergent-like rearrangement of thelipids leading to disruption of membrane integrity

362published by Elsevier Science Publishers

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Volume 154, number 2 FEBS LETTERS April 1983and eventually cytolysis. in this respect thei%ze/en& ii-endotoxin may resemble othercytolytic toxins such as Streptu~ysjn S 1151 in itsmode of action.

2.3. Analytical

2. MATERIALS AND METHODSThe sources of strains used [14], growth and

sporulation of the microorganisms [16], purifica-tion of the crystal S-endotoxin and the productionof the alkali soluble preparations [14] have beendescribed previously.

Suckling mice (average body weight 5 g) wereobtained from the Pathology Department, Cam-bridge University. Bioassays were carried out as in[ 171. Growth of Aedes a~~op~cfus cells in 25 cm2flasks (Nunc) and in vitro toxicity tests were asdescribed in [ 141.

Neutr~isation of the alkali-soluble crystal S-endotoxin was detected by measuring the potencyof the toxin after incubation with liposomes for2 h at 0, 4, 20, 37 or 44C (toxin-lipid ratio of1: 10 (w/w), except where stated). The super-natants remaining after centrifugation oftoxin-liposome mixtures at 50000 x g for 10 minwere also tested for toxicity. Toxicity of the soluble6-endotoxin was measured in vivo by bioassay ofA. aegypti 3rd instar larvae [17] and by sub-cutaneous inoculation of suckling mice, and invitro by observation of cytopathic effects on A.aibopjctus cells [ 141. Control experiments were runin parallel with alkali soluble S-endotoxin, orliposome preparations alone.

3. RESULTS AND DISCUSSIONPhosphatidyl choline (egg), cardiolipin (beef

heart) and phosphatidyl serine were obtained fromGeneral Biochemicals (Chagrin Falls, OH);cholesterol, dicetylphosphate, sphingomyelin(bovine brain type l), ethanolamine, a-L-dioleoylphosphatidyl choline, cu-L-dipalmitoyl phospha-tidy1 choline, cu-L-soybean phosphatidyl ethanol-amine, ~-L-dipalmitoyl phosphatidyl ethanol-amine, a-L-dimyristoyl phosphatidyl choline,cerebroside (brain) and stearylamine from Sigma,and phosphatidyl inositol from Koch-LightLaboratories.

In preliminary screening experiments to identifyputative toxin receptors on mosquito cells it wasfound that the toxin was effectively neutralised bypreincubation with a IO-fold excess of a sonicateddispersion of phospholipids purified from culturedAedes aibopictus cells.

These results raised the possibility that plasmamembrane phospholipids may be a target for thecytopathic action of the S-endotoxin. Othercytolytic toxins have been reported to interact withphospholipids either in a detergent-like manner, orby activating endogenous membrane phospho-lipases [15,20-271.

2.1. Preparation of liposomesLiposomes were prepared fromchromatographically pure lipids by the following

modified fig] procedure: lipid, cholesterol anddicetylphosphate, or stearylamine, were mixed inmolar ratios of 2: 1.5 : OS, respectively; the totallipid was 35 pmol. The mixture was dried as a thinfilm in vacua in a 100 ml round-bottomed flaskand then resuspended with gentle hand shaking in3 ml of phosphate buffered saline (PBS). Furtherdispersion of the lipids was accomplished by brieftreatment (2-5 min) in a sonic bath.2.2. Ex@action of cell lipids

As table 1 shows, preincubation with the mos-quito phospholipids neutralised both in vitro andin vivo toxicity in comparison to untreated con-trols. In control experiments with &endotoxinpreincubated with PBS alone, 5 pg of 6-endotoxin . ml- of tissue culture medium causedtotal lysis of A. albopjctus cells in the in vitro assay[14]. This 6-endotoxin, preincubated with PBS alsocaused 1OOolo mortality when inoculated sub-cutaneously into suckling mice at a concentrationof 25 pg. g- body weight [14]. Lipid dispersionsprepared from the Gram-positive bacteriumBacillus megaterium KM failed to neutralise thetoxin even at toxin-lipid ratios of 1: 50 (w/w).

Lipids were extracted from A. albopictus cells In contrast to mosquito cells, Bacihsgrown in 25-cm* flasks and B. megaterium KM megaterium KM membranes contain novegetative cells by a modified Folch 1191 phosphatidyl choline, sphingomyelin, cholesterolprocedure. or significant amounts of unsaturated fatty acids363


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Volume 154, number 2 FEBS LETTERS April 1983[27]. This suggested that the specificity of The results (table I; fig.11 show that preincuba-toxin-lipid interaction may be determined by one tion with sonicated dispersions prepared solelyor more membrane components. A series of ex- from phosphatidyl choline is sufficient toperiments was therefore carried out in which nentraiise the toxin in vitro and in vivo. In an at-sonicaced dispersions of purified lipids with vary- tempt to reproduce more closely the lipid structureing composition were tested for their ability to found in the plasma membrane of susceptible cells,neutralise the &endotoxin. multilamellar liposomes were prepared by addition

Table 1Effect of various lipid preparations on toxin action in vivo and in vitro

Liposome compositiona Toxin : lipid Cytopathicbratio (w/w) effect

In vivo toxicitynumber dead/number used

A. ~~~o~~c~u~ ell lipidB. rn~gu~~ri~rnM ceI1 IipidPhosphat~dy~ cholinePhosphatidyl choline :cholesterol :stearylaminePhosphatidyl choline :cholesterol :dicetylphasphateSphingomyelin :cholesterol :stearylamineSphingomyelin :cholesterol: dicetylphosphateSoybean phosphatidyl ethanolamine :cholesterol :

dicetyl phosphatePhosphatidyi serine : cholesterol : dicetyl phosphatePhosphatidyI serine: cholesterol :dicetyl phosphateCaxdiolipin :cholesterol :dicetyl phosphatePhosphatidy~ inositof :cholesterol: dicetyl phosphateCerebroside :cholesterol :dicetyl phosphate

1: io1:501: 101: 10I : 10I: 100:lO0:lO None1: 10 Lysis1:25 None1:25 LysisI:25 Lysis1:50 Lysis

NoneLysisNoneNoneNoneNone

o/9-o/2O/11O/11O/11O/8Oil0

201204/4

a Molar ratios were 2: 1.5 : 0.5. Preparation, dispersion and sonication of all lipid and liposome preparations was carriedout at 30C except in the case of cerebtoside containing Iiposomes which were prepared at 60C and subsequentlyassayed at 20Cb Cytopathology observed after exposure of A. aibopictus cells to a solution of 5 ,ug &endotoxin/ml after it has beenincubated with the appropriate lipid

Subcutaneous inoculation of suckling mice; an equivalent of 25 fig &-endotoxin/g mouse was used

Fig, 1. Phase-contrast light micrographs of A. ~I~~p~cf~s cells treated with: (A) egg phosphat~d~~ choline dispersionsfor 30 min as controi; (Bf 5 pg alkali-soluble isrffehsis toxic protein/ml for 20 min; fC) 5 pg alkali-soIuble ifraeiensistoxic protein/ml preincubated with egg phosphatidy~ choline dispersions 1: 10 (w/w) for 2 h at 37C (bars, 25 am).

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Qohtme 154, number 2 FEBS LETTERS April 1883of chofesterol to ~hos~hatid~~ chofine and in&-sion of either stearylamine or ~~~tyI~hos~hate~ toconfer, respectively, a net positive or negativecharge on the lipid bilayer [ZSJ. Table 1 shows thatliposomes containing phosphatidyl choline andcholesterol neutralised the toxin at least as effec-tively as sonicated phosphatidyl choline disper-sions. Thus the presence of the sterol does not ap-pear to be essential for toxin neutralisation andthis was confirmed by experiments in whichaqueous chofesterof dispersions f29] were fmmdnot to inhibit &x&z action frrot shuwn).

The mzt charge on the fipasomes did not appearto be a determining factor in toxin-lipid interac-tion since no difference was observed (table 1) be-tween phosphatidyl choline liposomes containingeither stearylamine, or dicetylphosphate.

Preliminary experiments to investigate the in-fluence of the phospholipid headgroup are alsoshown in table 1. Sphingomyelin and phosphatidyl~tb~ol~mine were as effective as ~hosphatidy~choline and at higher t~xi~~l~~id ratios,~bospbatidy~ serine showed some ~eutr~~singabiiity, AH other ~hospho~~~~d~ ested proved inef-

fective in vitro and in viva even at high t~x~~~~~~~dratios. Liposomes prepared from cerebroside werealso unable to neutraiise the toxin.

A mechanism for toxin neutralisation based onbinding to susceptible liposomes appeared themost likely explanation of the above results. Thiswas confirmed by the finding that supernatants ob-tained after centrifugation of egg phosphatidylcholine-toxin incubations were devoid of toxin ac-tivity (not shown), This result was obtained whenthe ~n~ubat~o~s were performed at 0, 20, 37 or44%!.

To examine the influence of the fatty acidmoieties af the phos~ho~i~~ds on toxin-lipid in-teraction, iiposomes were prepared from egglecithin and from various synthetic phcrsphatidylcholines and phosphatidyl ethanolarnines (table 2).The importance of the physical state of the lipidbilayer was also investigated by carrying out theliposome preparation, and toxin preincubation atdefined temperatures chosen with reference to thechain melting temperatures of the ~bosp~oii~ids.

Table 2 shows that fiposomes prepared from egg~hosphatidy~ chohne with, or without cholesterol

Table 2Toxin neutralisation: influence of liposome composition and temperature

Liposome compositiona Toxin : lipidratio (w/w)

Incubationtemp. (C)

Cytopathi?effect

Phasphatidyi choline (egg) :cholesterol: dieetyl phosphate(WC)

~~os~~at~dyI chotine (egg) :dicetylt ~~~~~~ate (2WC)Dioleoyl ~hos~hat~dyl choline : ~~o~e~~e~~~dicetyi

phosphate (zoC)

1:10f:IQI:fQ

0, 20, 37 or 44r&20, 37 or 44

20, 37 or 44

NoneNoneNone

Dioleayf phffs~hat~dyI choline :s~e~~i~rn~ne (Z&C) I:10 2037 or 44 NoneLY&Dipalmitoyl phosphatidyl choline :cholesterol :dicetyl phosphate (45C) I:20 20, 37 or 44 Lysis

Dipalmitoyl phosphatidyl choline : stearylamine (45C) I:20 20, 37 or 44 LysisDimyristoyl phosphatidyl choline :cholesterol :dicetyl phosphate (35C) x:50 4, 20 or 37 tysisSoybean phosphatidyl ethanolamine : cholesterof :dicetyl phosphate (20C) I:IO 20 or 37 None~i~~Imitoy1 phosphatidyl ethanolamine : chofesterof :dicetyl phosphate (45C) I:% 20 or 37 Lysis

a Molar ratios were 2: 1.5 :OS. Preparation, dispersion and sonication of each ii~oso~e ~re~~atio~ was carried outat the temperatures in parentheses

b ~~topathoIogy observed after exposure of A. afbogittus ceIIs to a solution of 5 gg C;-endotoxiniml after it has beenincubated with the appropriate phosphoiipid

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Volume 154, number 2 FEBS LETTERS April 1983were equally able to bind toxin at all 4 testtemperatures. Since the chain melting transition ofegg phosphatidyl choline occurs between -7 and- 15C [30], the hydrophobia region of the lipidbiiayer will be in the liquid crystalline, or fluidstate in these liposomes. Dioleoyl phosphatidylcholine has a chain melting temperature of - 22Cand would therefore also be in the fluid state above0C. Liposomes composed of this lipid pluscholesterol were able to bind the toxin at 20, 37 or44C (table 2).Kn contrast to the above results withphosphatidyl choline containing unsaturated fattyacids, table 2 shows that liposomes prepared fromthe synthetic saturated dipalmitoyl (Cl6 ~0) anddimyristoyl fCl4 : 0) phosphatidyl cholines plus orminus cholesterol, were unable to bind and henceneutralise the toxin at any of the test temperaturesat lipid/toxin ratios of 1: 20 and 1: 50 (w/w).

The ability of the unsaturated phosphatidylcholines to bind toxin could indicate a specific re-quirement for unsaturated hydrocarbon chains fortoxin-lipid association, or the need for a fluidbilayer, or both. The experiments with thesaturated phosphat~dyl cholines enable us todistinguish between these possibiI~ties~ however, asfollows: The chain melting temperature ofdimyristoyl phosphatidyl choline is 23C fl5].Thus at 37C liposomes composed of this lipidplus choIestero1 are in the fluid state, whereas at4C the liposomes would be in the more rigid gelstate, Table 2 shows, however, that theseliposomes were unable to bind the toxin at all testtemperatures including 37C. By analogy withsimilar experiments on the Streptalysin S toxin[15], if bilayer fluidity were the sole factor in thehydrophobic region influencing toxin interaction,dimyristoyl phosphatidyl choline liposomes wouldbe expected to bind the toxin at 37C, but not at3OC.Additional evidence that bilayer fluidity per se isnot sufficient for phosphatidyl choline-toxin in-teraction is provided by the experiments withdipalmitoyl phosphatidyl choline in table 2. Purepreparations of this phospholipid show a gel-fluidtransition at 42C [31] and this is likely to bebroadened by the inclusion of cholesterol in theliposomes. Even at incubation temperatures ashigh as 44C however, the dip~lmitoylphosphatidyl choline liposomes were unable to366

bind the toxin.With respect to interaction of the toxin with the

hydrophobic portion of the lipid bilayer, the aboveresults strongly suggest a specific role for un-saturated fatty acids in addition to a need for afluid bilayer. This suggestion is reinforced by theexperiments with phosphatidyl ethanolamine anddioleoyi phosphatidyl choline in table 2.Liposomes containing highly unsaturated (primari-ly Cl8 : 2, linoleic acid) soybean phosphatidylethanolamine bound the toxin very effectively at20 and 37C, in contrast to the saturateddipalmitoyl phosphatidyl ethanolamine liposomeswhich would also be in the fluid state at 37Cbecause of the additional presence of chofesterol.

When cholesterol was omitted from the dioleoylphosphatidyl choline liposomes they no longerbound the toxin at 37 or 44C {table 2). Aspreviously suggested in [32] and in [33], we inter-pret this result to indicate that above 20Cdioleoyl phosphatidyl choline dispersions lackingcholesterol become increasingly disordered andunable to assume the stable bilayer structure re-quired for toxin interaction. Addition ofcholesterol serves to tighten the bilayer and therebybroaden the temperature range over which dioleoylphosphatidyl choline can form a stable bilayerf34]_Consequently the sterol containing liposomescan bind toxin at temperatures as high as 44C.In addition to demonstrating a requirement forunsaturated fatty acids contained within a fluidbilayer the experiments in table 1 and 2 reveal athird specificity determinant located in the polarportion of the phospholipids. Thus, although theexperiments indicate that net surface charge is nota crucial determinant, the results with the differentphosphol~pids suggest that liposomes containingeither the Zwitterioni~ phosphoryl choline groupor phosphatidyl ethanolamine bind the toxin muchmore effectively than liposomes containing theanionic phosphatidyl serine. No toxin binding oc-curs to liposomes containing either strongly acidheadgroups (cardiolipin or phosphatidyl inositol)or cerebraside.The result with cardiolipin (beef heart)liposomes is particularly informative since thisphospholipid contains primarily unsaturated(Cl8 : I, G 18 :2) fatty acids f35] and when combin-ed with cholesterol in liposomes is likely to form alamellar fluid bilayer at 20C. The fact that these


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Volume 154, number 2 FEBS LETTERSliposomes possess a hydrophobic phase containingthe two specificity determinants of unsaturationand fluidity and yet are unable to bind the toxin,emphasises the key role of the phospholipid polargroup in bilayer-toxin interaction.

to investigate the importance of other factors suchas gut conditions and the chemistry of gutepithelial cell surfaces in determining insecticidalpotency and specificity.

In summary, these data suggest thatphosphatidyl choline, sphingomyelin,phosphatidyl ethanolamine and to a lesser extentphosphatidyl serine are the cellular targets of the 6-endotoxin, provided these lipids contain un-saturated acyl residues. The in vitro experimentsalso point to a need for an organised fluid bilayerfor toxin binding.

All the above experiments were carried out usingthe alkali-solubilized toxin described in [14].However, recent work (to be published elsewhere)has shown that the Mr 28000 component of thissoluble preparation is the polypeptide responsiblefor the cytolytic activity of this S-endotoxin.

ACKNOWLEDGEMENTSIn other experiments (not shown) the toxin was

observed to cause a decrease in the turbidity ofliposome suspensions, suggesting that smaller lipidstructures are formed upon interaction with toxin.This suggestion was confirmed by electronmicroscopy of negatively stained toxin-liposomepreparations which showed blebbing of themultilamellar vesicles similar to that observedpreviously with insect cells [14].

This work was supported by the AgriculturalResearch Council. We thank Lesley Futter foreditorial assistance.

REFERENCESPI

In many respects therefore the israelensis 6-endotoxin resembles a number of other cytolytictoxins which bind specifically to membrane lipidcomponents and cause lysis by perturbing andrearranging the lipid bilayer. In some instances[36] membrane cholesterol is the primary target,while others [15,20,21,23-251 resemble theisraelensis toxin in binding specifically tophospholipids. A number of these cytolytic toxinshave been reported to stimulate endogenousphospholipase activity in the membrane of suscep-tible cells [23,37] leading to the suggestion thatcytolysis results indirectly from the damaging ef-fects of the resultant lysolipids and fatty acids. Nosuch stimulation was observed when Aedesalbopictus cells labelled by biosynthetic incorpora-tion of lipid precursors [37] were exposed to theisraelensis toxin (not shown), and we conclude thatlike Streptolysin S and Staphylococcus a toxin[15,23], this toxin acts directly as a protein-surfactant to destroy plasma membrane integrity.

PI131

Lilthy, P. and Ebersold, H.R. (1981) in:Pathogenesis of Invertebrate Microbial Diseases(Davidson, E.W. ed) pp.235-267, Allanheld,Osmun and Co., USA.Goldberg, L.J. and Margalitt, J. (1977) MosquitoNews 37, 355-358.Lacey, L.A., Escaffre, H., Phillippon, B., Seketeli,A. and Guillet, P. (1982) Tropenmed. Parasitol.33, 97-101.Murphy, D.W., Sohi, S.S. and Fast, P.G. (1976)Science 194, 954-956.Fast, P.G., Sohi, S.S. and Murphy, D.W. (1978)Experientia 34, 762-763.Geiser, P. (1979) Diss. ETH Zurich, Switzerland,nr. 6411.Nishiitsutsuji-Uwo, J., Endo, Y. and Himeno, M.(1979) J. Invertebr. Pathol. 34, 267-275.Johnson, D.E. (1981) J. Invertebr. Pathol. 38,94-101.

These results do not immediately suggest an ex-planation for the insensitivity of other insectspecies to the toxin; e.g., Lepidoptera, comparedto Diptera. Although Lepidopteran larvae are in-sensitive when fed the toxin orally [14], they suc-cumb readily to toxin injected into the haemocoel(not shown). Further experiments are in progress

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Nishiitsutsuji-Uwo, J., Endo, Y. and Himeno, M.(1980) Appl. Ent. Zool. 15, 133-139.Angus, T.A. (1970) Proc. IV Int. Colloq. InsectPathol., pp.183-189.Ebersold, H.R., Liithy, P., Geiser, P. andEttlinger, L. (1978) Experientia 34, 1672.Endo, Y. and Nishiitsutsuji-Uwo, J. (1980) J.Invertebr. Pathol. 36, 90-103.De Barjac, H. (1978) CR Acad. Sci., Paris Serie D286, 1629-1632.Thomas, W.E. and Ellar, D.J. (1983) J. Cell Sci.60, 181-197.Duncan, J.L. and Buckingham, L. (1981) Biochim.Biophys. Acta 648, 6-12.

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f29] Freer, J.H., Arbuthnott, J.P. and Billcliffe, B.J.(1973) J Gen. Microbial. 75, 321-332.[30] Chapman, D. (1973) in: Form and Function ofPhospholipids (Ansell, G.B. et al. eds) pp.127-130,Elsevier Biomedical, Amsterdam, New York.

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Mechanism of Action of Bacillus Thuringiensis Var Israel Ens Is Insecticidal S-Endotoxin

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Transcript of Mechanism of Action of Bacillus Thuringiensis Var Israel Ens Is Insecticidal S-Endotoxin