Partial purification and characterization of an escherichia coli toxic factor that induces morphological cell alterations

Partial

Purification and Characterization

of

an

Escherichia coli

Toxic

Factor

That Induces

Morphological Cell Alterations

A.CAPRIOLI,l* V. FALBO,' L.G. RODA,2 F. M.RUGGERI,' ANDC. ZONA3

Laboratorio di Ultrastrutture, Istituto Superiore diSanita,' andCattedra di Tecnica Fisiologica, Universita degli Studi di Roma,3 Rome; and Laboratorio di Farmacologia, Universita degli Studi diAncona, Ancona,2

Italy

Received 20 July 1982/Accepted14December 1982

Afactor produced by several strains ofEscherichia coli isolated from

enteritis-affected children has been shown to produce both a necrotizing effect onrabbit

skin and striking morphological alterations onCHO, Vero, and HeLa cells. The same strains were foundto have hemolytic activity on sheep erythrocytes. The toxic, cell-altering factor was demonstratedto bedifferent from both heat-labile and heat-stable enterotoxins and from Vero toxin.The main effect induced bythe

isolated factoroncultured cells wasthe formation of large multinucleated cells.

The partial purification achieved suggests that the same factor (most likely a

protein with amolecular weight of 70,000 to80,000) is responsible for toxicand

cell-altering activities, whereas a different molecular species is responsible for

hemolytic activity.

A number of Escherichia coli strains are known to produce heat-labile enterotoxin (LT) and heat-stable enterotoxin, whose properties

and role in diarrheal disease have been widely investigated (1-4, 9, 11, 18, 19). A cytotoxin active onVerocells has also been described by Konowalchuk et al. (13, 14), and its role in humaninfection byenteropathogenicE.coli has

subsequently been discussed (17). In addition,

several E. coli strains involved in sepsis or

urinarytractinfectionshavebeenreportedtobe hemolysin producers (12).

Inthis paper we report thefindingof a new E.

coli factor, designated CNF, obtained from strains of clinical relevance and showing an

activity which is cytotoxic for HeLa cells and

necrotizingonrabbit skin. Wealsodescribethe

mainbiological properties andchemical

charac-teristics of CNF.

MATERIALSANDMETHODS

Bacterial strains. Atotal of 213 E.ccli strainstested fortoxicactivitywereisolated from stoolspecimensof 110children (age, 2 years andunder) hospitalized for acutediarrheal disease. Strainswereidentifiedbythe

API20Etechnique (LaBalme, LesGrottes, France).

The followingE. coli strains were used as controls: E9638 (LT') and E1106/0 (positive for Vero toxin), kindlysupplied byB. Rowe(PublicHealthLaboratory Service,London,UnitedKingdom);andISS132

(posi-tivefor heat-stable enterotoxin)and K-12 J62carring

thehemolysinplasmid p212, fromourlaboratory

col-lection. E. coli K-12 J53 (pro met) was used as

negative controlin allexperiments.

Culture conditions. Cultures were maintained on

nutrientagarslantsat25°C. Erlenmeyer flasks(250ml)

containing20 mlofTrypticasesoybroth(BBL Micro-biology Systems, Cockeysville, Md.)wereinoculated

with 104 bacterial cells from exponentially growing

cultures ofcontrol and teststrains. After incubationin a rotary shaker at 37°C for 18 h, the cultures were

centrifugedat 10,000 x gfor30min at 4°C, and the supernatants werefilteredthrough 0.45-,umMF mem-branes(Millipore Corp., Bedford,Mass.). Ifrequired, mitomycin C was added (0.5 ,ug/ml) 3 h after the

inoculationofthe cultures. To obtain higheramounts

ofLT(2),cultures weresonicatedinan icebathat an amplitude of14 p.mpeaktopeak (three strokes,20 s

each) in an MSE apparatus (MSE Scientific Instru-ments, Crawley, United Kingdom). The cell extracts

thusobtainedwerecentrifugedat20,000xg at4°C for

20 min, and supernatants were filtered as described above.

Cell culture assays. Chinese hamsterovary(CHO), Vero, and HeLacells, obtained from the American Type Culture Collection (Rockville, Md.), were

pas-sagedby trypsinization and grown as monolayers at 36°C in Eagle minimal essential medium (GIBCO Laboratories, Grand Island, N.Y.) with Earle salts. The medium was supplemented with 5% fetal calf serumand 1%nonessentialaminoacids(GIBCO). LT assays were carried out in microtiter plates (Falcon Plastics, Oxnard, Calif.)as describedby Guerrant et

al. (10). Bacterial extracts were tested in Vero and HeLa cultures by the same procedure. To obtain

monolayers in eight-chamber tissue culture slides

(Lab-Tek 4808; Lab-Tek Products, Naperville,

Ill.)

eachchamberwasseeded with about104cellsin 0.36 ml of minimal essential mediumcontaining 1% fetal

calfserum and then was inoculated with 0.04 ml of

bacterialsample.Afterincubationat36°C, monolayers

were fixed in methanol and stained with May Grun-wald-Giemsa(E.Merck, Darmstaadt, Federal

CELL-ALTERING E. COLI TOXIC FACTOR -.I 4. * .4 . * -.4 -

'I

a., 4S.,

IF

;,.o" .9 a.', "1! Sb .--Wl

*v-

_ 14~

I

*w

A..l

1AF I; I}'." * I.&M .1 .. i'll lld x. : !i t

.It

4, __s,

-w

.' ; .AM a <¾./ ;.t C9Jr 4it.

FIG. 1. ToxineffectonmonolayersofCHO(A), Vero (B). and HeLa(C)cells. From lefttoright,cells treated with: K-12J53 cell extract,18 h; ISS51 cell extract, 18 h: ISS51 cellextract, 3days.

lic ofGermany). To quantify toxin effects in HeLa

cultures,20microscopic fields(x400)foreach sample

wereexamined;viable cells (bytrypanblue exclusion) and cells containing one, two, or more nuclei were

counted. Data were expressed as cells per square

millimeter.Adenylate cyclase stimulationin CHO cell cultureswasevaluatedasdescribed by Guerrantetal.

(10); cAMP was determined by the protein binding

i_ . mkkI ..S6 . -.

C

4 VOL. 39, 1983 1301 -:.

toi-041"

4w

.---& il. .I

Or,

A

l.

t IV' 1; I le Is .pl

0

TABLE 1. Biologicaleffectsof cell extracts obtained from CNF-producing E. :oli strains"

Test results

Strain Infantmouse Ileal loop Skin cAMP increase Cell culture" Hemolysin

(intestinewt/ (mloffluid/

nb

(meanlevel/

_odu

ction

bodywt) cmofgut) test control level) CHO Vero HeLa production

Control' 0.061 0.15 - 0.90 - - -K-12J53 0.060 0.20 - 0.48 - - -ISS132 0.140 0.40 - 1.08 - - -E9638 0.058 1.43 PF 6.08 E E -E1106/0 0.061 1.11 - 0.82 - K -K-12J62 0.064 0.16 - 0.66 - - - + ISS2 0.064 0.19 N 0.71 M M M + K + ISS4 0.060 0.22 N 0.48 M M M + K + ISS18 0.058 0.25 N 0.39 M M M + K + ISS51 0.065 0.22 N 0.65 M M M + K + ISS92 0.070 0.19 N 0.54 M M M + K + ISS94 0.061 0.28 N 0.91 M M M + K + ISS107 0.057 0.19 N 0.86 M M M + K +

Experimentswere performedasdescribed in the text. h - No effect;PF, vascular

response;

N.

necrosis.

-.

No

effect;

K,

killing;

M. multinucleation; E,

typical

morphologicalalterationsbyLT

(elongation

inCHO

cells,enlargementin Vero cells).

dUninoculatedTrypticase soy broth.

method with a reagent kit from the Radiochemical Centre(Amersham Corp.).

Serial dilutionsof eachsampleweretestedonHeLa cellculturestofollowevery stepofpurification. Data wereexpressedastoxic units permilliliter, determined fromthereciprocal ofthe lastdilution showing

detect-able morphological alteration.

Hemolysis test. Twodifferentprocedures were used.

(i)E. coli strains were tested on 5% sheep blood agar

plates, and afterovernightincubation (37°C), hemoly-sis halos were measured. (ii) Hemolytic activity on

fractionated samples was assayed by the following

procedure. Samples(100 ,ul each)of 5% washed sheep

erythrocytesandofthetestsample(adjustedto afinal concentration of0.9% NaCI)weremixedinglass test tubes (3 by 30 mm), incubated (37°C, 4 h), and

centrifuged. Hemoglobin release in the supernatant was evaluatedby absorbance at 540 nm.

Animal assays. To reveal LT, the ileal loop assay and skin test in rabbitswere carriedoutas described by Evansand co-workers (7, 8). The sucklingmouse

testfor heat-stable enterotoxin was performed bythe methodofDeanetal.(5). Necrotic reaction in rabbit skin wasevaluatedbyinspecting the ulceration inthe injection site.

Fractionationprocedure.High-pressuresteric exclu-sionchromatographywasperformedonacolumn (7.5

by 500 mm) of TSK G4000 SW (Toyo Soda Co., supplied byVarianAssociates, PaloAlto, Calif.)on a

Perkin Elmer series2/1 chromatographequippedwith

an LC 75 variable wavelength detector. The steric exclusion column was calibrated with the following proteins as standards: bovine serum albumin

(mono-mer and dimer), egg albumin, chymotrypsinogen, myoglobin (from horsehearts). Astandardcurve was

fitted withaleast-squares computerprogram.

Detection methods. Proteincontentwasfollowedby absorbance at 280 nm and by a microbiuret method (16). Amino acidanalysesofhydrolyzed samples(24 h in constant boiling 6 N HCI at 110°C underreduced

pressure) wereperformedon aCarlo Erbamodel3A29 amino acidanalyzer (CarloErbaStrumentazione, Cor-sico, Italy)equippedwith ninhydrin detection.

Electrophoresis. Electrophoresis was performed by

the method of Laemmli (15) on an LKB Uniphor

apparatus (LKBInstruments, Bromma, Sweden). RESULTS

Sonicated cell extracts of isolated E. coli strains were tested for LT in CHO cultures. Four of these proved to be LT producers,

whereas seven others (ISS2, 4, 18, 51, 92, 94, 107) induced unexpected morphological cell al-terations consisting of remarkable cell

enlarge-ment and the presenceof morethanonenucleus in each cell (Fig. 1). Similar alterations were

1000 / 800-E

,

E600- 400-,----'W ---200' 'N 20 40 60 80 100 time( hours)

FIG. 2. Survival of HeLa cells with time. Cells treated with ISS51 cell extract (---), K-12 J53 cell

extract (- - -), and medium control ( ). Experi-ment wasperformedasdescribed in the text.

CELL-ALTERING E. COLI TOXIC FACTOR 1303

-60-

' U 40- i' 20-10 20 30 40 50 60 tim*(hours)

FIG. 3. Relative frequencies of HeLa cells with

one(---),two(- - -), andmore( )nuclei,by

timeofexposurewithISS51 cellextract.Percentages ofmononucleated cells in controlmonolayerstreated

withK-12 J53cellextract wereconstantlyabout97%.

observedwhencultures of the samestrainswere treated withmitomycin C,thus inducingrelease

oftheinner cellcontents.Theoccurrenceof cell

lysing was confirmed by a 50% decrease in

turbidity, asdetermined by Klettreadings, and

bya drop of >90%in viability. Conversely, no

effectwasdetected whenCHOmonolayerswere

inoculated with the supernatants of untreated cultures ofthe toxic strains. All 7toxic strains werefoundtobepositive when tested for

hemo-lyticactivity,whereasonly 20hemolytic strains werefound among theremaining 206.

To characterizethe seven toxic strains, their cell extractswerecompared with extracts from

control strains in a set of biological assays

(Table 1). Cellextracts did notelicit fluid accu-mulation in rabbit ileal loops (the same result was obtained by inoculating about

1010

living

bacteriaperloop),nordidtheystimulate adenyl-ate cyclase in CHO cultures. In addition, anti-cholerahyperimmune serumfailed to neutralize thecytotoxic activity. These results as awhole suggest the presence of a specific factor other than LT. Furthermore, all seven E. coli strains werenegative in the infant mouse assay for heat-stableenterotoxin.

Cell extracts werealso able to produce mor-phological alterations in Vero and HeLa cell cultureswithin18 h, and the formation of giant multinucleated cells was observed after a few days (Fig. 1). HeLa cells died within 60 h of incubation unless the medium was changed dai-ly. CHO and Vero cells appeared to be more

resistant,their viability being unaffected over 5

days.Medium substitution 6hafterinoculation didnotinfluence the outcomeof morphological

changesin any of the cell lines tested.

Multinucleation wasobservedatlowcell

den-sities(lessthan 5,000cellspercm2)aswellasin

confluent monolayers.

Because theE. coli strains testedwere more

toxic forHeLacells, this cell line was used for the quantitative evaluation of the alterations induced by the cytotoxic factor. Monolayers

were treated withbacterialextracts andstained

at different times as described above. Determi-nations were made of cell viability (Fig. 2) and therelativefrequenciesof cellswith one, two,or more nuclei (Fig. 3).

Intradermalinjection of cellextractsinrabbits caused anindurationareawithanecroticcenter up to a dilution of1:20 (Fig. 4). Subcutaneous inoculationin the abdomensofguineapigs

elicit-ed a large necrotic reaction; slightly more than 50%oftheanimals diedwithin 48 h andshowed

adiffusehemorrhagic patternduring autopsy.

Heating at75°C for 15 min or at 60°C for1 h

destroyedthetoxicactivity ofthesample,which wasfound tobestableatpHvaluesranging from

6 to 10.5. Since the above-mentioned observa-tions led us to suppose that toxic

activity

was

carried by proteinaceous material, further

ex-periments were carried out to obtain evidence

on thispoint.

Samples of bacterial culture (20 ml) were

centrifuged (20minat

4°C, 1,500

x g),

suspend-ed in 5 ml of25 mM

Tris-hydrochloride

buffer

(pH7.2), spun downagain, suspendedin 1.5 ml

FIG. 4. Local reactions in rabbit 40 h after intra-dermalinjection(0.1 ml)of cell extracts obtained from

ISS51 (a), ISS4(b), K-12J53(c), ISS18(d), and ISS2

(e).

VOL. 39, 1983

:.m r.7t

A'

A

C 1.5 -°0 .0 10 C'r-Q5 c-c)0 'Nj I.: 8 10 12 14 16 18 20 22 24 26 Elutionvolume (ml)

FIG. 5. Chromatogram fromTSK G4000SWcolumn(7.5 by 500 mm). The column, equilibrated in 25 mM

Tris-hydrochloride(pH7.2)and loaded with 175,uloffilteredsupernatant(45,000xg),waseluted at 1 ml min '.

Thesolidline represents absorbance at 280 nm; hatched bars representtoxicactivity onHeLa cells; dark bars represent hemolytic activity measured as absorbance at 540 nm; the number of asterisks is proportional to

necrotic activity in theskin testassay. TU,Toxic units.

of Trisbuffer, and sonicated as described above.

Sonicated cellswere finally centrifuged (20 min at40C, 45,000 x g),and the supernatant,filtered througha45-urmMillipore MFfilter, was imme-diately applied toaTSK G4000 SW column (7.5 by 500 mm). The column, equilibrated in the same buffer, was eluted at 1 ml/min, and the effluent was monitored at 280nm.

The chromatogram obtained from the G4000 column is shown inFig.5together with the toxic

activity determined by HeLa cell and skin test assays. Furthermore, the hemolytic activity of theTSK G4000fractionswasalsodeterminedas described above. Figure6shows thesame chro-matogram as Fig. 5 together with the carbohy-drate and protein contents of each fraction. From the results shown, it is evident that toxic activity is eluted by the G4000 column from15 to 17 ml, approximately 75% of the total toxic

activity being localized at 16 ml, whereas

ac-cordingtothe biuret assay,only 22% ofthetotal protein is localized in fraction 16. On the con-trary, hemolytic activity (Fig. 5, dark bars) is eluted at 19 ml. Figure 7 shows the electropho-retic pattern of the fractions from the TSK column.

From Fig. 6, it is also clear that the total

carbohydrate content of the original sample is

quite low, andno morethan 25

p.g

ofequivalent

hexose isassociated with eachfraction, whereas theprotein contentis considerable. Indeed, the maximum protein concentration is associated

with thesecondmain peak in Fig. 5 and 6. Toxic

activity is also associated with the same peak.

This datum makes it very likely that the toxic

activity is due to material of a proteinaceous

nature. To confirm this hypothesis, amino acid analyses offractions 14 through 18 were run as

described above. It isclearfromthese analyses

that amino acids are associated with all of the

V) 50l 1'5 105 404-400 30 20 10 -300 ct-200 & -100 8 10 12 14 16 18 20 22 24 26 Elution volume (ml)

FIG. 6. Chromatogram from the TSK G4000 col-umn. Chromatographic conditions were the same as

for theexperimentsshown inFig.5.Symbols:

absorbance at 280 nm; - -- - protein as deter-minedbybiuret

assay:

carbohydrate(as hex-ose) in micrograms per milliliter determined by the method of Duboisetal.(6).

CELL-ALTERING E. COLI TOXIC FACTOR( 1 2 3 4 5 6 7 8 9 MWx104 6.65 4.50 1.78 1.44 1.24 -.35

-FIG. 7. Polyacrylamide gelelectrophoresis inthe presenceofsodiumdodecyl sulfate, performedon0.6-ml fractionsfrom the TSKG4000SWcolumn. Fractionscorrespondingtoeffluentat15to17 ml (wells1 through 4) arecytotoxicfor HeLacells: fractionscorrespondingto19 ml (wells 7 and8)arehemolytic. Inactive fractions. correspondingto18and 20ml,are in wells5. 6. and9.

assayed fractions, and 4.5 nM amino acid is present in fraction 16, confirming again the proteinaceous nature of the material to which

toxic activity is due.

The TSK G4000 column was calibrated as described above. Theresultingcalibrationcurve regression coefficient was 0.98, and in the cali-bration curve (data not shown) the elution vol-ume of the main toxic fraction (fraction 16)

corresponds to the elution volume ofaglobular

protein withamolecularweight of

approximate-ly 76,000.

DISCUSSION

The most relevantbiological properties of this E. coli toxin, termed CNF, can be summarized as follows. (i) It appears to be cytotoxic for HeLacells. (ii) Itsaction on CHO and Vero cells distinguishes it from Verotoxin and LT. (iii) It causes the formation of large multinucleated cells when tested in cell cultures; this effect is moreremarkablein HeLa andCHO cells than in Verocells. Vero and CHO cells remained viable

just as long as untreated controls did, whereas HeLacultures died within60 hafter inoculation. (iv)Thereis good evidence that the toxinis ofa proteinaceous nature.

The outcome of morphological alterations is notinfluencedby thedensity ofthetoxin-treated

monolayers; sucharesultmakes the occurrence ofacellfusion processunlikely, thussupporting

the hypothesis ofan inhibition of cell division. In vivo experiments show that CNF causes not only a local necrotic reaction but also sys-temic damage consisting of widespread hemor-rhage. No target organ was evidenced, and the observed alterations appear rather to be the result ofadiffuse vascular involvement.

The data also tend strongly to support the hypothesisthat toxicactivity is associated with material ofa proteinaceous nature, and specifi-cally with a protein with a molecular weight of about 70,000to80,000. Furthermore, hemolytic activity is consistently separated from toxic ac-tivity and is related to material with a much lower apparent molecular weight. The low fre-quency ofhemolyticstrains among E. coli

isolat-VOL. 39. 1983 1305 move ",14c. - "O..

40

* AqF. 4w.

4w

40,

ed from stool specimens (12), confirmed in the present study, makes it extremely difficult to hypothesize a casual association of the two activities in seven out of seven toxic strains examined. However, our findings very clearly show that the two activities are indeed due to different molecular species.

Inconclusion, our findings suggest that CNF plays the role of virulence factor. Although bacterial extracts and living bacteria themselves werenegative in the rabbit ileal loop test, it must be stressed that CNF-producing strains were all

derived from infants who had severe diarrhea and whose stool specimens proved negative for all otherbacterial or viral enteric pathogens.

Furtherinvestigations are in progress to char-acterize thebiochemical properties of CNF and its mode of action in vivo and in vitro.

ACKNOWLEDGMENTS

We are verygratefulto ValeriaDe Marco (University of Rome) for the aminoacidanalysesand to Ida Luzzi(Istituto Superiore diSanita, Rome) forherhelpinrabbit surgery.

This workwaspartially supportedby the Progretto Finaliz-zatoChimica FineeSecondaria oftheItalian Nation Council forScientific Research.

LITERATURECITED

1. Alderete, J.F., and D. C. Robertson. 1978. Purification and chemical characterization ofthe heat-stable entero-toxinproduced byporcinestrain ofenterotoxigenic Esch-erichia c oli. Infect. Immun. 19:1021-1030.

2. Clements,J.D., and R. A. Finkelstein. 1979. Isolation and characterization ofhomogeneous heat-labile enterotoxins withhighspecific activity from Escherichia colicultures. Infect. Immun. 24:760-769.

3. Clements, J. D., R.J. Yancey, and R. A. Finkelstein. 1980.Properties ofhomogeneous heat-labile enterotoxin from Escherichiaco/i.Infect.Immun. 29:91-97. 4. Dallas, W. S., and S. Falkow. 1980. Aminoacidsequence

homology between cholera toxin and Escherichia co/i

heat-labiletoxin. Nature(London)288:499-501. 5. Dean, A.G.,Y.Ching,R.G.Williams,andL. B. Harden.

1972. TestforEscherichia colienterotoxin usinginfant mice: application in a study of diarrhoea inchildren in Honolulu. J. Infect. Dis. 125:407-411.

6. Dubois, M., K. A.Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith.1956. Colorimetric methodforthe determi-nation of sugar and related substances. Anal. Chem. 28:350-356.

7. Evans, D. G., D. J. Evans, Jr., and N. F. Pierce. 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli.Infect.Immun.7:873-880.

8. Evans, D. J., Jr., D. G. Evans, and S. L. Gorbach. 1973. Production of vascularpermeabilityfactor by enterotox-igenic Escherichia coliisolatedfrom man. Infect.Immun. 8:725-730.

9. Gill, D.M., J. D.Clements,D. C. Robertson, and R. A. Finkelstein. 1981. Subunit number and arrangement in Escherichia coliheat-labile enterotoxin. Infect. Immun. 33:677-682.

10. Guerrant, R. L., L. L. Brunton, T. C. Schnaitman, L.I. Rebhun, and A. G. Gilman. 1974.Cyclicadenosine mono-phosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Esc/herichia coli. Infect.Immun. 10:320-327.

11. Guerrant, R. L., J. M. Hughes, B. Chang, D. C. Robert. son, and F. Murad. 1980.Activationof intestinal guanyl-atecyclase byheat-stable enterotoxin of Escherichiaco/i: studies of tissuespecificity, potentialreceptors and inter-mediate.J. Infect. Dis. 142:220-228.

12. Hughes, C., D. Muller, J. Hacher,and W. Goebel.1982. Genetics andpathogenicrole of Escherichia c oli haemoly-sin. Toxicon20:247-252.

13. Konowalchuk, J., N. Dickie,S.Stavric, and J. I. Speirs. 1978.Properties ofanEscherichia colicytotoxin.Infect. Immun. 20:575-577.

14. Konowalchuk, J.,J.I. Speirs,andS. Stavric. 1977. Vero response to a cytotoxinof Escherichia (oli. Infect. Im-mun. 18:775-779.

15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature(London)227:680-685.

16. Munkres, K. D., and F. M. Richards. 1965. The purifica-tionandproperties ofneurospora malatedehydrogenase. Arch. Biochem.Biophys. 109:466-479.

17. Scotland, S. M., N. P. Day, G. A.Willshaw,and B. Rowe. 1980.CytotoxicenteropathogenicEscherichia coli. Lan-ceti:90.

18. Smith,H. W., and C. L.Gyles. 1971. The effect of cell free fluids prepared fromculturesofhumanand animal enteropathogenic strains of Escherichia coli on ligated intestinal segments ofrabbitsandpigs. J. Med.Microbiol. 3:403-409.

19. W.H.O. ScientificWorking Group.1980.Escherichiacoli diarrhoea.Bull.W.H.O.58:23-36.