Trypanosoma Cruzi Biological Characterization of 19 Clones Derived From

8/2/2019 Trypanosoma Cruzi Biological Characterization of 19 Clones Derived From

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COSTA E SILVA FiLHO-SURFACE CHARGE OF TRlTRICHOMONAS FOETUS 5559. Diamond, L. S. 1957. The establishment of various trichomon-ads of animals and man in axenic cultures. 1. P aras i t o f . ,43: 488-490.10. Eylar, E. H., Madoff, M. A., Brody, 0. V. & Oncley, J. L.1962. The contribution of sialic acid to the surface charge of the eryth-rocyte. J . Biol . Chem. , 231: 1992-2000.1 1 . Furghgott, R. F. & Ponder, E. 1941. Electrophoretic studies

on human red blood cells. J. G en . P h ys io l. , 24: 447-457.12. Gasic, G. J., Berwick, L. & Sorrentino, M. 1%8. Positive andnegative colloidal iron as cell surface electron stains. L ab . In ves t . , 18:13. Grinnel, F., Tobleman, M. Q . & Hackenbrock, C. R. 1975. Thedistribution and mobility of anionic sites on he surfaces of baby ham-ster kidney cells. J . Cell Biol. , 66: 470479. 1963. Theelectrophoretic behaviour of some trypanosomes. Biochem. J . , 89: 123-127.15. Honigberg, B. M. 1978. Trichomonads of veterinary impor-

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14. Hollingshead, S . , Pethica, B. A. & Ryley, J. F.

tance, in Kreier, J. P., ed., Parasit ic Protozoa , Academic Press, NewYork, : 163-273.16. James, A. M. 1979. Molecular aspects of biological surfaces.C h em . S oc . R ev . , 8: 389418.17. King, C. A. & Preston, T. M. 1977. Studies of anionic sites onthe cell surface of the amoeba Naegleria gruberi using cationized fer-ritin. J. Cell Sci . , 28: 133-149.18. Meherishi, J. N. 1972. Molecular aspects of the mammaliancell surface. Prog. B iophys . Mol . B iol . , 25: 1-70.19. Nicolson, G . L. 1973. Anionic sites of human erythrocytemembranes. I. Effects of trypsin, phospholipase C, and pH on thetopography of bound positively charged colloidal particles. J . Cell Biol.,57: 373-387.20. Pereira, M . E. A., Loures, M . A., Villalta, F. & Andrade, A. F.B. 1980. Lectin receptors as markers for Trypanosoma cruzi. De-velopmental stages and a study of the interaction of wheat germ agglu-tinin with sialic acid residues on epimastigote cells. J . Exp. M e d . , 152:1375- 1392.

21. Phillips, H. J. & Terrybery, J. E.22. Seaman, G. V . P. & Heard, D. H.

1957. Counting activelymetabolizing tissue cultured cells. E xp . C ell R es . , 13: 341-347.

1960. The surface of thewashed human erythrocyte as a polyanion. J . G en . P h ys io l. , 44: 251-268.23, Seaman, G . V . P. & Uhlenbruck, G. 1963. The surface struc-ture of erythrocytes from some animal sources. Arch. Biochem. Bio-

24. Tenforde, T. 1970. Microelectrophoretic studies on he surfacechemistry of erythrocytes, in Lawrence, J. H. & Gofman, J. W., eds.,Advances in Biological and Medical Physics, Academic Press, NewYork, 13: 43-105.25. Trissl, D., Martinez-Palomo, A,, Arguello, C., De la Torre, M.& De la Hoz, R. 1977. Surface properties related to concanavalinA-induced agglutination. A comparative study of several Entamoebastrains. J. Exp. M ed. , 145: 652-665.26. Wallach, D. F. H. 1979. Plasma membrane of eukaryotic cells:some general principles, in The Membrane Pathobiology of TropicalDiseases , Schwabe & Co. AG., Basel, Switzerland, pp. 1-34.27. Warton, A. & Honigberg, B. M . 1979. Structure of trichomo-nads as revealed by scanning electron microscopy. J . Protozool . , 26:56-62.28. Weiss, L . 1969. The cell periphery. In t . R ev . C y to l . , 26: 63-105.29. Weiss, L. & Subjeck, J. R. 1974. The densities of colloidal ironhydroxide particles bound to microvilli and the spaces between them:studies on glutaraldehyde-fixed Ehrlich ascites tumour cells. J . Cell

30. Weiss, L . , Zeigel, R., Jung, 0.S. & Bross, I. D. J. 1972. Bind-ing of positively charged particles to glutaraldehyde-fixed human eryth-rocytes. Exp. Cell Res. , 70: 57-64.31. Wessells, N. K . , Nuttall, R. P., Wrenn, J. & Johnson, S. 1976.Differential abeling of the cell surface of single ciliary ganglion neuronsin v i t ro. P roc . N a t l . A cad . S c i . U . S . A . , 73: 41004104.Received 3 II 82; accepted 5 IV 82

ph ys . , 100: 493-502.

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J . Protozool., 2%4), 1982, pp. 555-560@ 1982 by the Society of Protozoologists

Trypanosoma cruzi: Biological Characterization of 19 Clones Derived fromTwo Chronic Chagasic Patients. I. Growth Kinetics in Liquid MediumJUAN C. ENGEL,*,** JAMES A . DVORAK,*J ELSA L. SEGURA,** and MARK ST . J. CRANE*.*

*Laboratory of Parasitic Disease s, National Institute of Allergy and Infectious Diseases,National Institutes of Hea lth, Bethesda , Maryland 20205 and**lnstituto Nacional de Diagnostico e Investigacidn d e la Enfermedad de Ch aga s,D r. Mario Fatala Chaben, Buenos Aires, ArgentinaABSTRACT. Nineteen clones of Trypanosoma cruzi were obtained as single-cell isolates from Triuroma infestans. Ten of the cloneswere isolates from a patient with chronic Chagas disease; nine clones were isolates from a dog infected with T . cruzi strain CA-Iisolated originally from a chronic chagasic patient. The growth kinetics and peak modal Coulter volume of these clones were charac-terized. Significant inter- and intra-group differences between growth rates and peak modal volumes were found. These data indicatethat subpopulations and, consequently, genetic heterogeneity of T. cruzi exist in chronic chagasic patients. Al l of the clones infectedvertebrate cells in vitro.

HE causa tive agent of Chagas disease, T ry panosom a cruzi ,T has been isolated from various infected mammals and in-vertebrate vectors . Such isolates are composed of a variableThis work was supported in part by a Research Training Grant(JCE) from the UNDP/World BanWWHO Special Program for Re-search and Training in Tropical Diseases. The authors express appre-ciation to Ms. D. Hartman for technical assistance and to Dr. A. Mar-teleur and Dr . 0.Ledesma, Centro de Enfermedad de Chagasy PatologiaRegional de Santiago del Estero, Argentina for providing the infectedinsects used to isolate some of the clones of T. cruzi used in this study.Present address: Merck, Sharp, and Dohme Research Laboratories,P.O. Box 2000, Rahway, New Jersey 07065.

3 To whom all correspondence should be sent.

and nonquantified number of organisms. Laboratory stocks ofthe par asites, classified a s strains, ar e maintained using variousprotocols (22). Differences have been shown to exis t betweenstrains of T . cruzi using diverse chara cteristics, such as: tissuetropism, morphology of blood stream trypom astigotes, parasit-emia (1, 4, 14), zymo deme pa ttern (15), and profile of specificrestriction endonuclease products (18).However, several characteristics such as the infectivity ofculture forms, rate of differentiation to metacyclic trypomas-tigotes, and zymo dem e patter n, may change on maintenance ofthe strain in the laboratory (2, 6, 12 , 20). To explain thesechang es, it has been postulated th at, in natu re, T . cruzi is com -posed of a genetically heterogeneous population of organisms


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T C R U Z I CLONESFig. 1. Graphical representation of the doubling time (in hours) obtained from growth rates in L IT medium at 26C for 19 cloned stocks ofTrypanosoma cruzi and the Tulahuen strain. Legend:a = Tulahuen strain;0 59-73) = CA-I clones; A 75-91) = Miranda clones. C lones withsignificantly different doubling times with respect to the mean group doubling time are represented as 0 and A .

( 1 1 ) . The conditions used to m aintain stock s of T . cruzi in thelaboratory may impose selection pressure on subgroup s withinthe population, resulting in a modification of the n ative geneticprofile.Stocks derived from single-cell isolates of T . cruzi are ge-netically homogeneous. Such cloned stocks exhibit stable iso-enzym e patterns and growth kinetics (8), and a re excellent m a-terial for studies of intraspecific variation. In this report, wedescribe and compare the kinetics of growth in liquid mediumand infectivity in cell culture of 19 clon es of T . cruzi obtainedfrom two chronic chagasic patients.MATERIALS AND METHO DS

Parasite isolation and cultivation. Third instar Triatomainfestans were fed on a 26-year-old asymptomatic chronic cha-gasic patient (Miranda) from Santiago del Este ro, A rgentina, ora dog infected with the CA-I strain, originally isolated from apatient with chronic Cha gas' disease from Sa n Luis, A rgentina,and maintained for 4 y ears in laboratory animals prior to cloning(10).Seventeen isolates from the chagasic patient (Miranda) and14 isolates from T . cruzi strain CA-I were obtained by a micro-manipulation technique (23) from insects infected 30-45 dayspreviously. The isolates were inoculated into sterile vials (NUN Ccryotubes, 5 mi, A . H. Thomas Co., Philadelphia, Pennsylva-nia) containing an enriched biphasic medium composed of ablood nutrient agar base (1.0 ml) and a liver-infusion tryptose(LIT) (3) liquid medium overlay (0.2-0.4 m l), supplemented with

20 p d m l hemin, 1% (v/v) heat-inactivated fetal calf serum, 100Ufml penicillin, and 100 pg/ml streptomycin (subsequently re-ferred to as LIT medium). The cultures were incubated at 26Cand monitored for growth at regular intervals for one year. Nineof the CA-I isolates, clones CA-I/59, 64, 5, 67, 6S 73 , and 10of the Miranda isolates , clones Mirandd75-78, 80, 81, 82, 84,88, 91, showed growth.Th e parasites were transferred and exp anded in LIT mediumand subcultured every 14 day s at a dilution of 1/100 to 1/200.Stocks obtained between passages 2 to 9 were stabilized bycryopreservation.Growth kinetics in liquid medium . The g rowth kinetics of th eindividual clones were studied with stocks from passages 5 to18. Parasites in the exponential phase of growth were inocu-lated into plastic tissu e culture flasks (25 cm' surface area) atan initial concentration of 5 x lo5 or lo6 parasites/ml (10 mlLIT medium) and incubated at 26C. Two replicate cultureswere inoculated fo r each experim ent, and a minimum of threeindependent experiments per clone was performed. The Tula-huen strain of T . cruzi (19),maintained in liquid medium cul turefor more than 20 years in this laboratory, was used as a com-parative standard.Parasite growth and modal Coulter volume were monitoredevery two days from day 0 up to stationary phase, using aCoulter Counter M odel B o r ZB1 quipped with 70 pm aperturean d a Coulter Channalizer and X-Y Recorder. An external stan-dard of 3.4 pm diameter latex spheres was used to calibrate theChannalizer.


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EN GEL ET AL.-CLONES OF TRYPAN O S O M A CRUZI FROM CHRONIC PATIENTS 557I I I I I I I I I I

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T . C R U Z I CLONESFig. 2. Graphical representation of the modal Coulter volume (in pn3) t peak growth of epimastigotes of T . cruzi grown in LIT m edium at26C. Legend: fx = Tulahuen strain; 0 (59-73) = CA-I clones; A 75-91) = Miranda clon es. Clones w ith significantly different modal volumeswith respect to the mean group modal volume are represented as 0 and A.

Grow th kinetics da ta collected during exponential growth overa minimum of 10 days w ere analyzed with a Tektronix Plot 50computer using software developed specifically for this pur-pose. Students t test was used for statistical analyses of thedata. Differences b etween da ta sets at the 95% or greater con-fidence level were judged as statistically significant.Infection of v ertebrate cells. The infectivity of clones of T.cruzi was tested in cell culture. Bov ine embry o skeletal musclecells (BESM) (9) were cul tured in 75 cm2 surface area cul tureflasks with 10 ml RPMI 1640 medium (17) supplemented with5% (v/v) heat-inactivated fetal calf serum, 100 Uimi penicillin,and 100 pgiml streptomycin at 37C in a 5% COz, 95% air at-mosphere.The initial infection of BESM cells was established by in-oculating the cultures with approximately lo8 organisms har-vested at s tat ionary phase from LIT cul ture. Twenty-four hourslater, the BES M cultures were w ashed twice w ith fresh mediumto remove parasites in the supernatant fluid and were reincu-bated. The presen ce of tissue culture-derived trypomastigoteswas then determ ined by phase con trast light microscopy of theflasks during the next several days. Serial passages were sub-sequently established in BESM cultures with trypomastigotes.Trypomastigotes from passages 1 to 3 were cryopreserved.

RE SU L T SOf the original 31 single-cell isolates collected, 10 (59%) ofthe Miranda isolates and 9 (64%) of the CA-I strain isolatesgrew to a cell density sufficient for expan sion within one year.

Outgrowth was observed as early as 45 days and as late as 200day s after cloning. The time required for outgr owth was inde-pendent of the origin of the isolate and th e grow th rate of theorganisms.Inter-experimen tal variability in both the growth rate and peakmodal volume of the clones occurred. However, the degree ofvariability was negligible, ranging from 0.2 t o 6% for growthrate and from 1.6 to 6.7% for peak modal volume.Significant intra-group differences in the growth rates andpeak m odal volumes w ere found in both the M iranda and CA-Igroups. The se differences were analyzed by com paring the datafor individual clones to the arithmetic mean of the group fromwhich they cam e. Th e individual doubling times and peak mod-al volumes calculated from the exponential pha se of growth areshown graphically in Figs. 1 , 2. The doubling times of CA-I/59(45.1 * 2.7 h) and CA-I/70 (42.1 * 1 h) ar e significantly higherand that of CA-W1 (28.3 f 3 h) is significantly lower than theCA-I group mean doubling time (34.1 * 1.9 h). Th e doublingtime of Mirandd83 (33.1 * 1 . 1 h) is significantly lower th an th eMiranda gro up doubling time (51.6 ? 2.4 h). The peak modalvolumes of CA-1/69 (21.9 * 0.4 pm3) and CA-I/70 (18 * 0.6pm3) are significantly smaller than the CA-I group mean peakmodal volume (25.2 * 1 . 1 pm3 ). Th e peak modal volume ofMirandd83 (24.1 0.4 pm3) is significantly smaller than theMiranda group mean peak modal volume (31 * 1.6 pm3).Significant inter-group differences in the growth rates andpeak modal volumes were found among the Miranda group, theCA-I group, and the Tulahuen strain. The inter-group differ-


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558 J . PROTOZOOL., VOL. 29, NO . 4 , NOVEMBER 1982

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Fig. 3. Graphical representation of the growth rates of three populations of T. cruzi epimastigotes in exponential phase of growth in LITmedium at 26C. Legend: $t= mean growth rate of Tulahuen strain (25 f 0.7 h) ; 0 = mean growth rate of CA-I clones (34.1 2 1.9 h) ; A =mean growth rate of Miranda clones (51.6 f .4 h) .

ences in the growth rates among the three populat ions areshown graphically in Fig. 3. These differences, analyzed bycomparing the arithmetic mean doubling time of the Mirandaclones (51.6 -C 2.4 h), CA-I clones (34.1 f 1.9 h), and theTulahu en strain (25 * 0.7 h), showed that these three popula-tions were statistically unrelated w ith res pect to this charac -teristic. The peak modal volume of the Miranda group (31 *1.6 pm3) is significantly higher than that of the CA-I group(25.2 2 1.1 pm3). Howe ver, the ari thmetic mean peak modalvolume of the Tulahuen strain (27.8 2 0.8 pm3) is not signifi-cantly different from the Miranda and CA -I clone groups. D ueto the dispersion in peak modal volume within the M iranda andCA-I groups, there is no clear separat ion between them; anoverlapping distribution represen ted by clones CA-I/59 (27.6 ?1.5 pm 3), CA-1/64 (28.6 ? 0. 8 pm3),CA-1/72 (28.3 f .3 pm3 ),Mirandd88 (29 r 0.6 pm3), and M irandd91 (27.5 f 0.7 pm3)occurs. The growth rates and peak modal volumes of thoseclones that were significantly different from their respectiveintra-group means were analyzed further by testing them indi-vidually against the arithmetic mean of the other group or th eTulahuen strain. The doubling time of CA-1/59 was statisticallysimilar to that of the M iranda group. Th e doubling time of CA -1/70 was significantly different from the me an d oubling time ofboth the CA-I and the Miranda groups, presenting an inter-mediate position between the two groups of clones. The dou-bling time of CA-1/70 was intermediate b etwee n the CA-I groupand th e Tulahuen strain but significantly different from the Tu-

lahuen strain. Th e doubling time of Mir andd 83 was statisticallysimilar to the CA -I group. T he peak m odal volume of Mirandd83was statistically similar to the CA-I group. The peak modalvolumes of CA-1/70 and CA-1/69 were significantly smaller thanthe M iranda, CA-I, and the Tulahuen strain mean peak modalvolumes. N o correlation was found between the kinetics ofgrow th and the pea k modal Co ulter volume of the 19 clonedstocks of T. cruzi .All 19 clones of T. cruzi readily infected BE SM cells, a com-plete intracellular cycle occurred, and serial passages of try-pomastigotes in BESM cultures were established. The lengthof time betwee n the infection of BE SM cells and the appearan ceof trypomastigotes in the medium was ca. 6-7 days for the CA-I c lones with the e xception of CA-1/59 and C A-I/70, which ap-peared a t ca. 9-10 day s. Trypomastigotes appea red in the me-dium ca . 9-11 da ys after infection of BESM cells with theMiranda clones.

DISCUSSIONFrom the first description of T. cruzi in 1909 (5) until thepresent tim e, hundred s of strains of T. cruzi have been isolatedand characterized from various hosts over a wide geographicarea (24). Differences in characteristics between strains haveconfirmed the heterogeneity of this species. Changes in straincharacteristics that have occurred during routine laboratorymaintenance (13) imply that intrastrain heterogeneity also ex-ists. The biological heterogeneity of T. cruzi demonstrated in


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ENGEL ET AL.-CLONES OF TRYPANOSOMA CRUZI FROM CHRONIC PATIENTS 559this study with single-cell-isolate clones confirms the conceptof intrastrain heterogeneity; both of the strains isolated fromthe chagasic patients studied harbored more than one parasitepopulation.If it is assumed that the characteristics reported here arestab le, the biological diversity of the C A-I and M iranda parasitepopulations could ha ve occ urred in one of two different ways.The patients could either have b een infected onc e with a singleheterogeneous population of parasites or multiply infected witha variety of genetically homogeneous po pulations of parasites.It has been demonstrated that strains of T. cruzi can retainsom e biological heterogeneity during many ye ars of routine lab-oratory m aintenance. For exam ple, the Y strain of T. cruzi wasoriginally isolated in 1953 (21) and maintained under diverselaboratory conditions for 28 years prior to cloning. However,recently isolated clones of the Y strain still exhibit two-folddifferences in their growth rates (7). Selection pressures im-posed by the conditions used to maintain uncloned parasitesprobably influence the relative proportions of each subpopu-lation in a strain. The CA-I strain used in these studies wasmaintained by serial passage in laboratory anim als for four yearsprior to cloning. The proportion of each subpopulation origi-nally present in the chronic patient may have changed duringthis period. H owe ver, th e dispersion in the grow th rates of nineCA-I clones proved that the CA-I strain is still a mixture ofparasites with at least four statistically different subpopula-tions. Indirect evidence exists in the original description of theCA-I strain that a selection-induced chang e in the parasite pop-ulation may have occurred during routine laboratory mainte-nance (10). The authors reported that when the CA-I s t rain wastransferred at passage three from mice to biphasic medium, thedoubling time of the parasit e population du ring the first passagein vitro was 48 h and subsequently decreased to 30 h. Thischange in population doubling time may have occurred as aresult of selection of subpopulations with higher growth rates.The finding that the doubling times of seven of th e nine clonesisolated ranged arou nd 31 h and only two clones had doublingtime over 41 h supports this premise. A similar change couldhave occurred in the CA-I strain during the four years it wasmaintained in mice prior to cloning.Biological and biochemical characteristics of clones of T. cru z ihave been shown to be stable (8). The study reported hereconfirms and extends these observa tions. Both the growth ratesand modal volumes of 19 clones were stable during the four-month period require d for these studie s. In addition, two clones(CA-1/64 and Miran dd83) repeatedly analyz ed during an eight-month period showed no significant variation in their growthrates or modal volumes. When these two clones were trans-ferred to BESM cell culture, passaged serially as trypomasti-gotes, and subsequently re-established in LIT medium, thegrow th rates and modal volumes of the parasites were identicalto the original LIT-grown stocks. Thus, these characteristicswere not influenced by th e culture system used to maintain theparasites.Th e volume of a eukary otic cell varies a s the cell progressesthrough its growth cy cle (16). In a freq uen cy distribution of cellvolum es, the modal volume is a stable charac teristic for a pop-ulation of cells in exponen tial growth. T he kinetics of cell growthof T. cru z i in culture can be described using both the numberof cells present and a frequency distribution of their volumes.Significant differences in modal volumes were found among thecloned stocks of the Miranda and CA-I isolates. The lack ofcorrelation betw een growth rate an d modal volum e implies thatthese characteris t ics are independent . How ever, these data fur-ther support the concept that distinct subpopulations of para-sites exist within a single strain. Con sequen tly, modal volume

is another ch aracteristic that can be used to classify differencesin cloned stocks from a population of T. c r u z i .Trypanosoma cru z i infects mammalian cells and multiplesintracellularly as part of its life cycle. Following inoculationinto BESM cell cul tures , all 19 clones of T. cruzi completed anintracellular cycle, an d trypomastigotes we re liberated into themedium. These data imply that the procedure of single-cell-isolate cloning of T. cruzi does not affect the infectivity of theresulting clones.The demonstration that heterogeneity in populations of T.cru z i exists, and is probably of usual occurrence in chronicpatients, may help to explain the diverse clinical signs and man-ifestations of Chagas disease. The utilization of clones of T.cru z i would thus offer numerous advan tages to stud y the inter-relationship between the host and biologically different sub-populations of the parasites.

L IT E RA T U RE CIT E DI . Andrade, S. G . 1976. Tentative for grouping different Trypano-

soma cruzi strains in some types. R ev . Inst . M e d . T r o p. Siio Paulo,18: 140-141.2. Bice, D. E. & Zeledon, R. 1970. Comparison of infectivity ofstrains of Trypanosoma cruzi (Chagas, 1909). J . Parasi tol . , 56: 663-670.3 . Bone, G . J . & Steinert, M. 1956. Isotope s incorporated in thenucleic acids of Trypanosoma mega. Na ture, 178: 308-309.4. Brener, Z . 1969. The behavior of slender and stout forms ofTrypanosoma cruzi in the blood stream of normal and immune mice.A n n . Trop. Me d. Parasi tol . , 63: 215-220.5 . Chagas, C . 1909. No va tripanosomiase humana. Estudos sobrea morfologia e o ciclo evolutivo do Schizotrypanum cruzi, n. gen. , n.sp. , agente etiologic0 de nova entidade morbida do hom em. M em . In s t .Oswaldo Cruz, 1: 15S218.6. Chiari, E . 1974. Growth and differentiation of Trypanosomacruzi culture forms kept in laboratory for different periods of time. R e v .Ins t . M ed. T rop . S6o Paulo, 16: 81-87. 1982. Trypanosoma cruzi:Spontaneous transformation by a Y strain variant in liquid medium.E x p . Parasi tol . , 54: 87-92.8. Dvorak, J. A. , Hartman, D . L. & Miles, M . A. 1980. Trypanoso-ma cruzi: correlation of growth kinetics to zymodeme type in clonesderived from various sources. J . P ro tozoo l ., 27: 472-174.9. Dvorak, J . A . & Hyde , T. P. 1973. Trypanosoma cruzi: inter-action with vertebrate cells in vi tro. I. Individual interactions at thecellular and subcellular levels. Exp. Parasi tol . , 34: 268-283.10. Gonziilez-Cappa,S. M ., Chide, P., del Prado, G . E ., Katzin, A.M ., de Martini, G. W ., de Isola, E . D. , Orrego, L. A. & Segura, E. L .1980. Aislamiento de una cepa de Trypanosoma cruzi de un pacientecon miocardiopatia chagasica cronica y su caracterizacion biologica.Medicina, 40: 63-68.

1 1 . Lambrecht, F. L. 1965. Biological variations in trypanosomesand their relation to the epidemiology of Chagas disease. R e v . I ns t .Med. Trop. Sao Paulo, 7: 346-352.12. Lana, M . de, Chiari, C. A ., Chiari, E ., Romanha, A. J. & Morel,C. 1980. CaracterizaGLo de formas de culturas d o Trypanosoma cruzi,amostras Berenice isoladas da paciente em diferentes periodos. Pes-quisa Basica em DoenGa de Chagas. VII Reuniiio Anual. Caxambu,Brasil.

13 . Luban, N . A. & Dvorak, J. A. 1974. Trypanosoma cruzi: in-teraction with vertebrate cells in vitro. 111. Selection for biological char-acteristics following intracellular passage. E x p . Parasi tol . , 36: 143-149.14. Melo, R. C. & Brener, Z . 1980. T issue tropism of differentTrypanosoma cruzi strains. J . Parasi tol . , 64: 4 7 5 4 8 2 .15 . Miles, M. A ., Souza, A ., Povoa, M ., Shaw, J . J. , Lainson, R.& Toye, P. J. 1978. Isoenzym ic heterogeneity of Trypanosoma cruziin the first autochthonous patients with C hagas disease in AmazonianBrazil. N atu re , 27 2 819-821.1971. The Biology of the Cel l Cycle. Cam-bridge University Press, London.

7. Crane, M. St. J. & Dvorak, J. A.

16. Mitchison, J. M.


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560 J . PROTOZOOL., VOL. 29 , NO . 4, NOVEMBER 198217. Moore, E., Gerner, R. & Franklin, A. 1967. Culture of normalhuman leukocytes. J . Am . M e d . As s o c. , 199:519-524.18. Morel, C., Chiari, E., Camargo, E. P., Mattei, D. M., Romanha,A. J. & Simpson, L. 1980. Strains and clones of Trypanosoma cruzican be characterized by patterns of restriction endonuclease productsof kinetoplast DNA minicircles. Pr o c. Na t f . Ac a d . Sc i . U . S . A . , 77:68 10-6814.19. Pizzi, T. 1957. lnmunologia de la Enfermedad de Chagas. Uni-versidad de Chile, Santiago.20. Romanha, A. J., da Silva Pereira, A. A., Chiari, E. & Kilgour,V. 1979. Isoenzyme patterns of cultured Trypanosoma cruzi: changesafter prolonged subculture. Com p. Biochem. Physiol . , 62: 139-142.21. Silva, L. H. P. & Nussenzweig, V. 1953. Sobre uma cepa deTrypanosoma cruzi altamente virulenta para o camundongo branco.Folia Clin. Biol. , 2 0 191-207.

22. Tayler, A. E. R. & Baker, J. R. 1%8. The Cultivation of Par-asites In Vitro. Blackwell Scientific Publications, Oxford and Edin-burgh.23. Van Meirvenne, N . , Janssens, P. G . & Magnus, E. 1975. An-tigenic variation in syringe passaged populations of Trypanosoma (Try-panozoonf brucei. I. Rationalization of the experimental approach. Ann.SO C .Belge . Med. T rop. , 55: 1-23.24 . Zeledon, R. 1974. Epidemiology, modes of transmission andreservoir host of Chagas disease, in Trypanosomiasis and Leishman-iasis with Special Reference to Chagas Disease, Ciba FoundationSymposium 20, Elsevier Excerpta Medica, North Holland, pp. 51-77.

Received 20 t82; accepted I III 82

J . Prorozool., 244) , 1 1 2 , pp . 560-5650 982 by the Society of ProtozoologistsBiochemical and Structural Analyses of Microtubules in thePellicular Membrane of Leishmania tropica

CLEMENT BORDIER,* R. MICHAEL GARAVITO,** and BARBARA A RMBRUSTER***Institut de Biochimie, Universite de L ausanne, CH-1066 Epalinges, Switzerland and**Biozentrum der Universitat Basel, CH-4056 Base l, Switzerland

ABSTRACT. The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein iscomposed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the a and psubunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S . aureus V8 protease, andthe peptides obtained analysed by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the p subunitsof Leishmania and pig tubulin have very similar primary structures, while the a subunits have evolved divergently. These experimentsdemonstrate that the major polypeptides found in the pellicular membrane of L . tropica are a and p subunits of tubulin. Immuno-electron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania.Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negativestaining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity correspondingto the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12. A distance of 47 nm separates each microtubule fromthe next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identicalin the pellicular membrane of L . t ropica and in mammalian brain.

ARLY morphological studies on protozoa of the familyE Trypanosomatidae showed microtubule-like structuresunderlying the cytoplasmic membrane of the cells. These mi-crotubules are aligned parallel to each oth er and form a regularspiral network abou t the w hole cell. Thei r classification as mi-crotubules was based primarily on the observation that theybore morphological similarities to mammalian brain microtu-bules (3 , 4, 18, 23).More recently the proteins of the purified pellicular mem-branes (PM) of trypanosomes and Leishmania have been ana-lysed by sodium dodecyl sulfate polyacrylamide gel electro-phoresis and a major protein was found at 53,000 d , a positioncorresponding to the molecular weight of mammalian tubulin(8-10). Since the subpellicular microtubules remained associ-ated with the membranes during their purification, it was sug-gested that these microtubules were built from the major pro-tein, which was assumed to be tubulin (8-10). Furthermore,

We thank R. Etges for a generous supply of parasites and pellicularmembrane fractions. We are also indebted to R. Meyer, R. Franklin,J. Louis, and E. Moedder for purified immunoglobulins and serum. Thisinvestigation received financial support from the UNDP/WorldBanWWHO Special Programme for Research and Training in TropicalDiseases and from grants FN 3.489.79 and FN 3.069.81 to J. Mauel andE. Kellenberger, in whose laboratories the experiments were per-formed.

two major developmentally regulated proteins of L . mexicanawere found to co-migrate approximately with the subunits ofChlamydomonas axonemal tubulin in two-dimensional gel elec-trophoresis, and the lower of these bands cross-reacted im-munologically with the /3 subunit of Chlamydornonas axonemes(11).In this investigation, we have analysed the major protein ofthe pellicular membranes of the human parasite L. fropica. Wefound that this protein is formed by two different subunits andis located in the microtubule array of the membrane. They wereidentitied as Leishmania tubulin subunits by imm unological andbiochemical comparison with the subunits of mammalian braintubulin. Finally, the supramolecular organization of the micro-tubules and their association with the membrane was studiedby electron microscopy and optical diffraction.MATERIALS AN D METHODS

Pellicular me mb ran es. Leishmania tropica major (hereaftershortened to L. t ropica) was grown at 25C in 50% HEPES-buffered Dulbeccos medium and 50% medium 199 supple-mented with 5% heat-inactivated fetal calf serum (GIBCO) and4% type 0 human erythrocyte lysate. Promastigotes were har-vested at a cell density of ca. 4 x 107/ml,washed twice in Trisbuffered saline (25 mM Tris-HC1, 160 mM NaC l, 1 mM E DTA ,pH 7.8) at 4C. The preparation of the pellicular membraneswas carried out according to Dwyer (8), except that the equi-

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