Rev. Inst. Med. trop. São Paulo22(4) :184-191, julho-agosto, 1980

UDC 593.t61 .13

IN VITRO EXOANTIGEN FROM HERPETOMONAS SAMUELPESSOAI WHICHPROTECTS MICE AGAINST TRYPANOSOMA CBUZI INFECTION

Maria do Carmo Moreira de SOUZA f, K. MIZITTA (1) and H' IKEMOIIO (1)

SUMMARY

This paper describes the extraction, purification and characterization of Her-petomonas samuelpessoai exoantigen. The antigen was obtained from a complexmedium after 48 hours incubation at 28'C and characterized as a pÌotein with a

molecular weight of 58,800, having pH 4.2 as isoelectric point, without proteolyticactivity and an Erro* value of 39,900. Mice immunized by this exoantigen showa considerable protection against T. cruzi challenge.

INDEX KEY WORDS: Herpetomonas samuelpessoai; exoantigen; Trypanosomacrutzi; immunity to T. cruzi.

RUNNING HEAD: Exoantigen from Herpetomonas samuelpessoai

INTRODUCTION

SOUZA & ROITMAN 17 reported a partialprotection against Trypanosoma cruzi infectionin mice inoculated with viable culture forms ofHerpetomonas samuelpessoai, earlier namedLeptomonas pessoai 12. However the disruptedforms of this microorganism were not able toelicit resistance against T. cruzi 1r. This factwor¡ld suggest that a soluble exoantigen pro-duced by the living cells such as those detec-ted in the surface coat of T. brucei and in othertrypanosomatidsT,e'2z may participate in the de-

velopment of resistance in immunized animals.

Our present findings are related to the "invitro" exoantigen production by Herpetomonassamuelpessoai and its isolation and characteri-za;tion. This exoantigen was also assessed inprotection experiments against T. cruzi chal-lenge in mice.

MATERIAL AND METHODS

Protozoa: Herpetomonas samuelpessoai, iso-lated from Zelus leucogrannmus in the Institutode Patologia Tropical (Universidade Federal de

Goiás) 8; Trypanosoma cruzi (CL strain) pro'vided by Drs. I. Roitman and N. Alvarenga(Universidade de Brasília) and Trypanosomahastatus hasúatus from the Instituto de Pato-

logia Tropical (Universidade Federal de

Goiás) r.

. Culture media - LIT (liver infusion tryp'tose) was prepared as described by YAEGER3

with the following composition (g/1): NaCI

4.0g; KCI 4.0g; NaTHPO+ 8.0g; tryptose 5'09;

liver infusion 5.0g; glucose 2.Oe; LO% bovine

seÌum and this mixture was heated at 68'C for60 minutes with shaking. Hemoglobin (2%)'

crystallized penicillin (50 U,hl) and strepto-

rnycin (100 pg/r¡rl) were added and the pH was

adjusted with 2.0N HCI to 7.2. This mediumwas sterilized by filtration in a "Seitz" filterwith an EKS membrane with positive pressure.

Complex medium - It was prepared withthe following composition (g/l): tryptose 5.0g;yeast extract 7.0g; glucose 15.09; hemin 20.0m9;

folic acid 2.0mg. This medium was adjustedto pH 7.0 with NaOH and autoclaved for 20

minutes at 120"C.

(T) Fatecida em 19?9. Instituto de Patotogia Tropic¿l, Universidade Federal de Goiás - 74.000 - Goiânta' Goiás -Brasil

(1) Departamento ate Biologia Celular, Universid.aale ate B¡asflia - ?0.910 - Brasflia - DF - Brasil

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SOUZA, M. do C. M. de; MIZUTA, I<. & IKEMOTO, II. - In viúro exoantigen from Herpetomonss samuelpessosi whichprotects mice against Trypanosom¡ cruzi infect:on. Rev. Insú. Med, trop, São Paulo 22:184-191, 1980'

Maintainance of organisms - H. samr.lel'pessoai and T. hastatus hastatus Ïvere grownin LIT medium at 28 C for 2 and 15 days res-pectively. After growth, they were kept in thecold room. Every 20 days they were transferr-ed to a new medium. Trypanosroma cruzi (CL

strain) wa,s maintained in a albino mice (18-209)

by ïreekly transfers.Immune sera preparation - The T. hasta.

tus hastatus antigen was prepared as follows:the microorganisms rrvere grown in LIT medium at 2B'C for 48 hours; the cells were thencollected by centrifugation at 15009 and wa-shed 3 times with 0.15M NaCl. The cell con'centration was estimated by counting in a Neu-bauer's chamber and diluted to I x 10 8 cells./ml in distilled water. This suspension wasfrozen and thawed 5 times during 5 days andthe cell rupture completed by cycles of 30 se-

conds sonication followed by 30 seconds inter-val during 5 minutes in a Broson Sonifier CelIDisrupter. The cell fragments and intact cellswere separated by centrifugation. The proteinconcentration in the supernatant was adjustedto 3mglml and sterilized by filtration in Milli-pore membrane (0.45p).

The antigenic emulsion was prepared bymixing 1 ml antigen plus I ml dead flagellatesuspension (2 x 106 cells in formalin) and 2 mlFreund's adjuvant.

The immune serum was prepared as describ-ed þy COOMBS & GELLó. The Ner¡r¡ Zeallandrabbits (3 kg body weight) received, in the firstapplication, an intramuscular injection of 1 mlantigenic emulsion in each gluteal region. Fif-teen days later, an equal volume was infectedin each scapular rnuscle. On the 2lst døy, LÙVo

concentration of the initial antigenic emulsionwas injected intraperitoneally; afúer which, 0.5

ml of Lorc diluted antigenic solution was inject-ed every day intravenously for 5 days. Sevendays after the last injection, the rabbits werebled and their sera collected and pooled.

Purification of exoantigen from.the cultivat'ed media - After 4B hours incubation of H. sa-

muelpessoai in the complex medium with shak-ing at room temperature, each culture waschecked by microscop5l for possible contamina-tion with other microorganisms. The cells wereseparated by centrifugation and the supernatantwas fractionated by protein precipitation withsolid ammonium sulfate. Two fractions (0-50%

and50-90% saturation) were collected and, afterexhaustive dialysis, they were lyophilized.

The material precipitated at 50-90% ammo-nium sulfate saturation, which gave a positivereaction in the immunodiffusion test, was frac-tionated in a Sephadex G-?5 column (2.6 x 40

cm) equilibrated in 0.05M phosphate buffercontaining 0.2M KCI and0.O2Vo sodium azide atpH ?.0. For each chromatography 300 mg oflyophilized material was applied; the flow wascalibrated to 25 ml,/hour and 3 ml fraction werecollected after recording the absorbance at2BOnm.

The poot of fractions, which gave a positi-ve reaction in the immunodiffusion test, afterdialysis and lyophilization, was gel filteredagain in a Sephadex C*150 column equilibratedwith the same buffer system and the flow cali-brated to 15m1,/hour. The appropriate fractionwas dialyzed, tyophilized and stored at -18"C'

Polyacrylamide gel electrophoresis - Thesimplified method described by CLARKE s wasused with 7 .\Vo gel concentration. The polyme-rization in 0.6 x 8.0cm tubes was carried outwith TEMED, riboftavin and U.V. light. Thebuffer in both electrode chambers was 5mMTris-Glycine, pH 8.1 and the run ïvas made at2m&"/tube at 4.'C temperature. The protein con:centration applied was 100 pB þer tube. After 20

minutes run, the gels were stained with Amido-black and destained wit]n 7.0Vo acetic acid. Thepossible glycoprotein character of the exoanti-gen lvas analysed by staining the gel with AlcianBlue according to ïtrARDI & MICHOS te.

Isoelectric focusing - Two tubes (0.6 x 10

cm) containing ïVo polyacrylamide gel and2.0% Ampholine (3-10 pH range) were used' Theupper chamber (cathodic) was filled with 0'05Mphosphoric acid and the lower (anodic) with0.05M triethanolamine. Samples of 400 pg proteindissolved in LÙVo sucrose and 20/o Ampholine so-

lution were applied to the top of the gel andoverlaid by L0% sucrose and ZVo Ampholine so-

lution. The focusing vvas carried out at cons-tant voltage for 4 hours, by setting the currentto 2 mAltuþe in each hour. After running, onegel was fixed in trichloroacetic acid and stain-ed- with Coomassie Brillant Blue as describedb[' CHRAMBACH + and the other gel was slicedin 1cm segments to determine the pH gradientby elution in double distilled water for 2 hoursand by measuring the pH in a digital potentio-meter.

Evaluation of m¡rlecular weighú by hydrody'namic parameters - The molecular weight de

185

SOUZA, M. do C. M. de; MIZürA, K. & IKEMOTO,protects mice against TrJrl¡anosoms cruzi infection.

H. - fn viúro exoantigen from Herpsúomonas samuelpessoai whichRev. Inst. Med, trop, São Paulo 22:184-191, 1980.

termination ïvas made by gel filtration in Se-phadex Cr150 column (2.6 x 40cm) equilibratedwith 0.05M phosphate buffer containing 0.2MKCl,0.O2% azide, pII 7.0 at room temperature.The flow wa,s calibrated to lSmvhour and thevoid volume was determined with Blue Dextran.Ttre column calibration was made with thefollowing standard proteins: bovine serum al-bumin, egg albumin, alpha{hymotrypsin randmyoglobin.

Protease acúivity - To test possible proteo-lytic activity of exoantigen, the caseinolytic me-thod of KUNITZ 10 was used. A series of .13 tubescontaining different amounts of purified exoan-tigen (2 to 200 pg/tube) in 0.1M phosphatebufer pH 7.0 was incubated for 20 minuteswittt LYo heat denaturated casein. After thistÍme, 3 ml of íVo trichloroacetic acid lvas addedand filtered after standing 30 minutes, at roomtemperature. The proteolytic activity lyas corre-lated with 280nm absorbance of filtered solution.

Absorpúion spectrum - The purified andlyophilized exoantigen was dried on PrO, in adesiccator for 12 days. The absorbance of a so-lution containing 0.972 mg/ml in 0.05M phos-phate, 0,2M KCl, pH 7.0 buffer was read in aZEISS PMQ II spectrophotometer.

Agar immunodiffusion meúhod - ft wasperformed as described by OUCHTERLONY 11,

witJil O.LYî agar concentration. Anti-serum wasapplied in a central well and homologous or he-terologous antigens in the peripheral wells.Samples of complex medium freshly preparedand without inoculation of protozoa were alsotested. Diffusion was carried out at room tem-perature for 72 hours, followed by washing inO.g%,'NaCl soluti-on for 24 hours. After drying,the gels were stained with acid fuchsin.

Biological proúecúion tesús - Two groups of10 albino male mice (15-20S) were used, one ascontrol and the other as the test group. The so-lution containing different concentrations of thepurified exoantigen (without Freund's adjuvant)was injected subcutaneously in mice of the testgroup at seven darys intervals during threeweeks. They received 0.1 ml volume containing16mg in the firsi, 50mg in the second and 20mgin the last injection. Mice of the control groupreceived physiological solution at the same in-tervals. Seven days after the last injection, themice of the two groups were challenged intrape-ritoneally with 50,000 bloodstream forms of T.crazi (CL strain). Trypanosoma count was made

186

by the method described by BRENER, z and themortality evaluation was carried out for 3

months.

RESULTS

After 4B hours incubation of H. samuelpes-soai in the complex medium using a controlledenvironment incubator shaker, the yield wasabout 1.5 to 2.0g (wet weight) cells in 500 ml.This medium free from micrgorganism aftercentrifugation at 10,0009 for 40 minutes wassubmitted to salt fractionation. In order to testthe best salt concentration, it was precipitatedin a pilot experiment, with solid. ar,nmoniumsulfate in 0-60Yo, and 6O-90Vo fractions. After'exhaustive dialysis and lyophilizatipns, bothfractions sho¡ved a positive reacúion. in the im-munodiffusion test but in the polyacrylamidegel electrophoresis the active fraction was pre-dominant in the material precipitated with thehigher salt concentration. We decided then touse 50-90% saturation to get the exoantigencrude extract.

Figure 1 shows the electrophoresis pat-tern of: 1) the 50-90% precipitate from themedium without microorgariism growth; 2) thecrude exoantigen extract obtained by 50-900/o(NII4)rSO4 saturation and 3) the'somatic extractobtained by the same procedure as above fromthe supernatant of the sonicated trtr. samuelpes.soai cells. The exoantigen yvhich corresponQs tothe stronger band has a mobility similar toone of the components from the somatic crudeextract,

Figure 2 shqws the result of immunodiffu-sion tests with T. hastatus hasúatus immune se-

rum. This T. cruzi - like micfoorganism whosecharacteristics are being studied in our labo-ratory shows many similarities to the behaviorof T. cruzi, but it is not pathogenic to labora-tory animals. the immune sera obtained withT. cruzi and T. hasúatus hasúatus showed thesame pattern of precipitation bands when react-ed with H. samuelpessqai exoantigen. :

Gel filtration of crude extract in Sephad.exG-75 separated the exoantigen fraction frombrownish inactive material. The immunological-þ active fraction (the first peak material) wasagain gel filtered in Sephadex G-150; its elutionpattern and electrophoretic behavior are shownin Fig. 3 and 4. The exoantigen was present inthe last peak (see Fig.3). The biological tests

SOUZA, M. do C. M. de; MIZUTA, K. & IKEMOTO, H. - In vitro exoantigen from Herpeúomonas samueltrEssoai whichprotects mice against TrJ4tanosome cruzi infection. Rev. fnst. Med. trop. São Paulo 22:184-191, 1980.

PH

./'

r0 cm

Fig. 6 - Determination of the isoelectric point of H. samuel.pessoa¡ exoantigen by polyacrylamide gel electrofocusing. The

experimental conditions are described in METHOÐS.

same electrophoretic mobility as the antigen de-tected in LIT medium without inoculation ofH. samuelpessoai was isolated. by the same pÌo-cedures and inoculated in mice to analyze theprotection effect against T. cruzi (CL strain)challe.nge. All animals died within 30 days afterinjeètion. In view of these results we conclu-ded that the protection is not retated to anyfraction of the medium.

One of the components from the somaticcrude extract of H. samuelpessoai has anelectrophoretic mobility similar to the strongerband corresponding to exoantigen (Fig- 1). Pro.;bably because of the very low proportion ofthis protein in the somatic extract, the crossreàction rvas not detected in the immunodiffu-sion test. To clarify. this point it is necessaryto get a sufficient quantity of the purified so-matic fractions, which will be done in the fu-bure.

o

I2.0

Fig. ? - Estimation oi.the molecular weight of H. samuelpessoalexoantigen. The Sephadex G-150 column was equilibrated wiih 0.05M phosphate buffer containing 0.2 M KCI ând 0.02,1¡ sodium âzide,pH ?.0. Flow mte:15 mllhr; (-o-) exoantigen; (-O-) refe-

. rence prolelns,

WEïT220'21 ¡lsssriþsd a soluble exoantigenin the serum of rats infected with T. bruceiwhich protected animals against homologousinfection. TARRANT et al. 18 obtained T. cruziexoantigens in viúro which were better thansomatic antigen when tested by complement fi-xation activity. Recently GOTTLIEB e detecteda polysaccharide originating from T. cruzi inplasma of mice acutely infected with this para-site. SEED ls separated, by elution, a precipi-

tinogen and a protector factor from T. rhó.densiense. The H. samuelpessoai exoantigenmolecule characterized in the present study isaþparently not a glycoprotein. This conclusionswas obtained only by Alcian Blue staining tech-nique. Others methods will be used to confirmthis result since TARRANT 18 found the carbo- ,

hydrate moiety in the T. cruzi exoantigen in aratio 1:1 5 to 1:2. 0 between carbohydrate andprotein.

189

SOUZA, M, do C. M, d€; MIZUTA, K. & IKEMqrO, H. - In v¡úro exoantigen from Herpetomonas smuelpessoal Írhichpxotêcts mice against TrJæa¡rosonr.a cruzi infection. Rcv. Inst. Illed. fuop. São P¿ulo 22:184-191, 1980.

Exoantigen from other genera have alsobeen descriþed. SCHNUFù 13 demonstrated an-tigenic components released by Leishmania tro-pica and Leishrnania donovani which can beused for a differential diagnosis of leishma-niosis by intradermic test. Recently, SCHNUR 14

extended these observations to other generasuch as Leptomonas, Crithidias and He4leto-monas.

An experiment to characterize the exoanti-gen is being carried out in our laboratory. Itconsisús of the incubation of H. samuelpessoaiin the medium containing radioactive aminoacids. There is evidence of the radioactive ele-ment being incorporated into the exoantigenmolecule. These results, if confirmed, will de-finitively show that the exoantigen is biosyn-thesized by cells.

It is a commonly recongnized fact thatsome exoproteins have proteolytic activity;this property, however, was not detected in theH. samuelpessoai antigen. The apparently highvalue of molecular weight confirmed the factthat the dialysis water of exoantigen solutionwas negative in the immunodiffusion test. Thelow ratio (1.21) of absorption 278 nm:255 nmindicates that the molecule is poor in aromaticchromophore residues. On the other hand,the maximum at 278 nm means that this pro-tein has more tyrosine than tryptophan residues.

The literature is pratically virgin witJ¡respect to size and conformation of antigenicmacromolecules. Considering that many bio-logical events occur at the level of molecularinteractions, more attention should be dedicatedto these studies since they colrld contribute tothe elucidation of Chagas' disease immune me-chanisms.

RESUMO

Exoantígeno de Herpetomonas sarnuelpessoaique protege camundongos contra infecção de

Trypanosoma cruzi

O presente trabalho descreve a extração,purificação e caracterização de um exoantígenode Herpetomonas samuelpessoaÍ obtido do meiocomplexo após 48 horas de crescimento a zBC.Trata-se de uma proteína de peso molecular58.800 daltons, ponto isoelétrico em pH 4,2, sematividade proteolítica e o valor de Erro* =39.900. Os camundongos imunizados com este

190

exoantígeno mostraram uma considerável pro-teção contra uma dose maciça de I'rypanoso-ma, crvzi injetada intraperÍtonealmente.

ACI(NOWLEDGEMENl'S

The Authors gratefully acknowledge thesuppofi of Ðrs. Z. Bteîer, lvr. rvt. Ventura, \ü.Barþosa anct I. ltortman. Tha"nxs are que to Dr.Lrnda Styer Cald.as lor lingurstrc advrce andto the Conselho Nacronal o.e uesenvolvrmentoOren[Ífico e 'l'ecnologico (Ct\Pq) tor grants.

ITEI'ERENCES

1. BARBOSA, ÌV.; MARTINS, S. P. ó¿ Ol,Ivrjilì,a, r¿. L.

- i\oùd pteuìru¡at soor€ lrypmosû¡uó !4r. n¡staruslsola(to qe f4y¡lostomus I¡steûs oa wve¡îa ¡ ercat -.u¡'., Brasil. ll.ev. Pat, Irop. 4: 36?-3?6, 19'rJ.

2. BRENER, i¿. .- Therapeutic activl[y il]d crr¡eflon oIcue On üuce ffiperlmen[¿lly üUec[eu wru¡ì rryp6¡¡uËo¡n4cruzi. Rev. Inst. lYled. trop. São tsauro 4: ódy-¡rùú, luttz.

3. CAMARGO, E. P. - Crescimento e (¡Ilerenciação emrtypanosoms cruzi. A€v. Ilst. 1l¡d(¡. ¡rop, Sho Pauloti: 93-100, 1964.

4. CHRAMBACH, A.; REISFED, &. A.; WYCKO¡F, M. &ZACAnRI, J. - A procedure lor rapid ar¡d sensitives[ai¡ing of protein fractioned þy polyacryìamide gel elec-

trophoresis. An¿I. iBiochem. i U: ibu-154, 19tt?.

5. CLARKE, J. J. - Simplified disc (po¡yac¡y,arnrde)

gel electrophoresis. Am. N. Y. Acad, Sci. I¿r: 428-43tt,

r964.

6. COOMBS, R. R,. A. & GFr.r., P. G. rf. - Ð.iagnosticmethods in sorology a,nd irnrnmopachology. In: clinicalaspects of immunology. l"t eq. GELL, P. u. ¡I, &COOMBS, &. R. A. Ordord, New York, 1963.

?. DE SIQUEIRA, A. F.; FEn'RIOLLI, F. & CARVAIÍIEI-RO, J. - Um antlgeno solúvel no soro de ratos iniec-tados com T. crur¡. Nota Prévia, R€v. Inst. Med. trop.São Paulo E: 148-149, 1966,

s. GALV.ã,O, A. B.; OLMIRA, R. t.; CARVALHO, A.L. M. & VEIGA, P. G. - Leptononas Bessoei sp.n.(TrJpanosom¿tidae, Kinetoplastida, Protozoa). Rev.Goiânla Med. 16: 229"236, 1970.

9. C,OTTLIEB, M. - Å caxbohydrate-containing ar¡tigenfrom TrJ4ranosome cruzi and its detection in the cir-culation of infected mice. J. Imunol. Ll.Sr 465-470,

7977"

10. NORTHROP, J. H.; KuNITZ, M. & HER'R'IOT, R. M.

- In: CrJ¡stalune Enzj¡mes. New York, Columbia uni-versiry Press, 1948.

11. OUCHTERT,oNY, O. - Diffusion in gel methods forimmunological analysis. Prog. Allergy 53 l-?9, 1958.

SOUZA, M. do C. M. de; MIZUTA, K. & IKEMOIO, H. - fn vltro exoar¡tigen from Herpetomonâ,s sarnuelpesso¡i whicl¡protects mice agairist Trypanosom cruzi infect¡on, A€v. Insú, Med. troB. São P¡ulo 22:184-191, 1980r

13.

ROITMAN, I.; BRENER, Z.; ROITMAN, C. & KITA-JIMA, E. W. - Demonstration that Leptomonas trl€s.soel. Galvão, Olivelra, Carvalho & Veiga, 1970 is âHeq)etomonas. J. Protozool. 23: 291-293, 1976.

SCHNI'R, L. F. - Leishmanial excreted factors asskin test reagents in the diagnosis of Leishmariia,sis.Proc. Brlú. Soc. Par¿sit. ?3: 9.10. 19?6.

SCHNI'R, L. F. - Insect flagellate excreted factorsfrom leptomonad crithidial an leptomonâd strains. J.Protozool, (Suppl.) 24t 5A-6A, 7977.

SEED, J. R. - The characterization of antigens iso.lated from TrJ4ranosom¿ rhodesiense, J. Protozool. l0:380-389, 1963.

SOUZA, M. C. M.; REIS, A. P.; DIAS, \ry. S. &BRENER, Z. - Mechanism of acquired immunity in-duced by "Leptomonas pessoai" âga,inst Trypanosomecruzi in mice. J. Proúozool, Zl.. 579-584. 79'14.

SOUZ.A, M. C. M. & ROITMAN, I. - Protective effect,of lÆpúomonas pessoai against the infection of miceþy TrJæ¿nosoma cruzi. Bev. Mictobiol. 4: 187-189, 1971.

18. T.ARRANT, C. J.; FIFE, E. H. & ANDERSON, R,. I.- Serological characteristics and general chemical na-ture of the in vitro elroantigens of T. ctui. J, Pilasi.tol. 5l: 277-285, 1965.

19. WAADI, .4. H. & MICHOS, c. A. - Alcian blue stai-ning of glycoptoteins in acrylârnide disc electrophoresis..Anal. Blochem. 49: 601-609, 7972.

20. WEITZ, B. - .A soluble protective antigen of Try-BânosomÐ brucei. Nature 185: ?88-789, 1960.

21. WEITZ, B. - Irnmuity to Protozoe. lsted. GARNHAM,P. C. C.; PIERCE, A. E. & REIS. trold, L. BIa-ckwell, 1963.

22. ZUCKERMAN, A. & RISTIC, M. - Blood parasiteantigens and antibodies. In: Infecúions Blood Diseaseof Man and Animals, David Weinrnan & M. Ristic. 1:?9-122. 1968.

Recebido parâ publica4ã,o em 2L/3/1979,

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