Characterization of Trypanosoma cruzi from Argentina by electrophoretic zymograms

Acta Tropica, 50(1992)125 133

125

© 1991 Elsevier Science Publishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00174

Characterization of Trypanosoma cruzi from Argentina by electrophoretic zymograms Enrique E. Montamat, 1 Gloria De Luca d'Oro, 1 Beatriz Perret ~ and Cristina Rivas 2 1Chtedra de Quimica Biol6gica, Facultad de Ciencias Mkdicas, Universidad Nacional de COrdoba, Argentina; 21nstituto Nacional de Diagnbstico e lnvestigaci6n de la Enfermedad de Chagas, Buenos Aires, Argentina

(Received 28 January 1991; accepted 20 June 1991) Polyacrylamide gel electrophoretic patterns for six enzymes in 73 isolates and 38 clones of Trypanosoma eruzi from different areas of Argentina were classified into 12 zymodemes. The isolates were obtained from human patients with acute, chronic or congenital Chagas' disease, vector insects, domestic and sylvatic animals. Two out of 8 isolates cloned were shown to be heterogeneous. Zymodemes 1 and 12 exhibit widespread geographic distribution; isolates belonging to both zymodemes account for 55% of the total analyzed. The other zymodemes are not widely geographically dispersed. Although there is a clear predominance of zymodeme 1 among asymptomatic patients, the data do not show a clear relationship between particular zymodemes and the clinical picture. The results suggest that the sylvatic and domestic transmission cycles overlap. This remarkable heterogeneity of T. cruzi in Argentina supports the possible multiclonal origin of these parasite populations. Key words: Trypanosoma cruzi: Argentina; Electrophoretic zymograms

Introduction T r y p a n o s o m a cruzi is the p r o t o z o o n responsible for C h a g a s ' disease, an e n d e m i c infection affecting millions o f h u m a n p a t i e n t s in C e n t r a l a n d S o u t h A m e r i c a . T h e acute stage o f the disease m a y p r e s e n t a variety o f s y m p t o m s o r m a y evolve, w i t h o u t clinical evidence, to the c h r o n i c p h a s e several years later. The c h r o n i c p h a s e m a y exhibit a variety o f clinical pictures (Prata, 1985). In s o m e cases, lesions are localized in the heart; in others, in the digestive t r a c t and, in m a n y instances, the infection takes a 'silent' course w i t h o u t a p p a r e n t clinical m a n i f e s t a t i o n s . This s y m p t o m a t o l o g i cal diversity poses the q u e s t i o n o f w h e t h e r it is related to genetic h e t e r o g e n e i t y o f the p a r a s i t e p o p u l a t i o n s . The fact t h a t different clinical pictures p r e d o m i n a t e in different areas (for e x a m p l e , c a r d i o p a t h i e s are d o m i n a n t in A r g e n t i n a , while digestive tract i n v o l v e m e n t is relatively m o r e frequent in Brazil), c o u l d be due to the prevalence o f genetically distinct T. cruzi p o p u l a t i o n s in different areas.

Correspondence address." Dr. Enrique E. Montamat, C~ttedra de Quimica Biol6gica, Facultad de Ciencias M6dicas, Universidad Nacional de C6rdoba, C. C. 35 Suc. 16 5016 C6rdoba, Argentina [FAX 54-51695101].

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The electrophoretic zymogram has been the most widely utilized method to demonstrate variability among parasite populations (Miles, 1983; Tibayrenc and Ayala, 1988). Previous studies in different areas of Latin America where the disease is endemic have shown the existence of protein polymorphism in T. cruzi (Miles, 1983; Tibayrenc and Ayala, 1988; Brener, 1985). In Argentina, the first zymogram analyses were conducted in this laboratory by studying 18 clones derived from two isolates from chronic patients (Montamat et al., 1987). The findings indicated the existence of genic variability and led to a survey of a large number of isolates from acute and chronic chagasic patients, vector insects and domestic and sylvatic animals collected over an extensive area of Argentina. Our results are presented in this paper.

Material and Methods

Isolates 73 isolates from different areas of Argentina were analyzed: 42 from human patients with acute, chronic or congenital Chagas' disease, 14 from Triatoma infestans (the insect vector), 1 from a domestic dog, 1 from a guinea pig, and 15 from sylvatic animals (13 weasels, 1 skunk and 1 rodent of the genus Octodontomys). Eight of the human isolates were cloned, resulting in 38 clones. Tables 1 and 2 list the isolates, clinical symptoms and sites of origin. The map in Fig. 1 shows the provinces where isolates were collected. Brazilian stocks of T. cruzi Silvio-X10/1, Esmeraldo/1 and CAN-III/1 belonging to Brazil zymodemes 1, 2 and 3 respectively (Miles et al., 1980), were provided by the Instituto de Diagn6stico e Investigaci6n de la Enfermedad de Chagas (Buenos Aries). Human and other mammalian isolates were obtained by xenodiagnosis. Parasites from the intestinal tract of infected triatomids were transferred to culture by one of the following methods: (a) triatomid feces were inoculated in pre-weaning mice. After 7 days, those mice showing parasitemia were aseptically bled and the blood added to a biphasic culture medium (Montamat et al., 1987); (b) the whole intestine from infected triatomids was homogenized in brain-heart-tryptose infusion culture medium (BHT) (Boveris et al., 1978) and the parasites isolated following the technique described by Engel et al. (1982), and added to agar-blood culture tubes. Once grown in culture, parasites were transferred to BHT medium plus 10% heat inactivated bovine fetal serum. After 10 days, parasites were harvested by centrifugation for 15 min at 3000 × g and the pellet was washed twice with 0.25 M sucrose 5 mM KC1 solution. The parasites were suspended in bidistilled water and lysed by repeated freezing-thawing. The final preparation was centrifuged at 20 000 × g for 45 min at 4 ° C. The supernatent was divided into 20 ~tl aliquots and stored frozen in liquid nitrogen until used. Single, cloned parasites were derived from isolates as described by Engel et al. (1982).

Electrophoresis Extracts were subjected to polyacrylamide gel electrophoresis using the micromethod of Ogita and Markert (1979); an acrylamide concentration of 7 to 9% was used for

127

I

.I

\/

~g

Fig. 1. Argentina. Provinces or Federal States where samples were collected. 1, Jujuy; 2, Salta; 3, Chaco; 4, Santiago del Estero; 5, Santa Fe; 6, Corrientes; 7, C6rdoba; 8, San Juan; 9, San Luis; 10, Mendoza; 11, Buenos Aries; 12, La Pampa.

the running gel and of 4% for the stacking gel. After electrophoresis, the gels were stained for aspartate amino transferase (AAT, EC 2.6.16.1) (De Lorenzo and Ruddle, 1970), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), malic enzymeNADP (ME, EC 1.1.1.40), phosphoglucomutase (PGM, EC 2.7.5.1), alcohol dehydrogenase (ADH, EC 1.1.1.2), lactate dehydrogenase (LDH, EC 1.1.1.27) and glucose-phosphate isomerase (GPI, EC 5.3.1.9), as in Montamat et al. (1987). GPI presents two zones of activity. The region with faster mobility is revealed by staining for a much longer time than is needed to reveal the slower zone. Staining reagents are mixed with agar and poured on to the polyacrylamide gel. When the agar slab

128

is removed after long-exposure staining, most of the dye precipitate from the slow band region is transferred to the agar and only a diffuse zone of weak staining remains in the polyacrylamide, as seen in Fig. 3. This is the reason why each region is shown in a different slab (Fig. 3).

Results

All the variants of electrophoretic patterns obtained for the 73 isolates and 38 clones of T. cruzi from Argentina for each of the six enzymes analyzed are schematically represented in Fig. 2. Photographs of the patterns are shown in Fig. 3. The PGM and GPI patterns for the three Brazilian stocks were different from those of the Argentinian isolates (Montamat et al., 1987). LDH was monomorphic. For GPI, G6PD and ME, two groups of bands were observed. The band pattern of each zone varies independently of the other, suggesting that they are controlled by different genes. In the three enzymes, variants with different mobility for each of the groups were detected, except for the fastest migrating band of ME, which was monomorphic. GPI, G6PD and PGM exhibited greater variation in band mobility than did the other enzymes. According to the patterns +

+ ME

ASAT

ADH

I I

l mum

iIIIIlI I I

/

A

B

A

C

A

B

G6PD /

/

/

/

I

B

I

/

I

I

m

B

C

/

I /

A

C

PGM

D

E

A

F

B

/

/

C

D

E

GPI uIuIII

i i i

/ /

m

l

i i i i

m m

i A

B

C

D

iliiiilii

i i i

m

m /

l

/ E

F

G

H

Fig. 2. Schematic representation of gel electrophoretic patterns of enzymes from extracts of T. cruzi. ME, malic enzyme; ADH, alcohol dehydrogenase; ASAT, aspartate aminotransferase; G6PD, glucose-6-phosphate dehydrogenase; PGM, phosphoglucomutase; GPI, glucose-phosphate isomerase. The arrow indicates the origin. Letters designate the different patterns for each enzyme. Zymograms characteristic of each zymodeme are indicated in Table 3.

129 ASAT

ME

G6PD

ADH

GPI.1

PGM

GPI.2

Fig. 3. Polyacrylamide gel electrophoretic patterns of enzymes from extracts of T. cruzi. Abbreviations as for Fig. 2.

observed for AAT, G6PD, ME, PGM, GPI and ADH, all the isolates and clones studied could be classified into 12 different electrophoretic zymodemes (Table 1). The isolates showing the same combination of zymograms are considered to belong to one zymodeme (Z) (Fig. 2, Table 3). Parasites for T. infestans collected in different areas of Salta province belong to zymodemes 5 to 8. These zymodemes only differ in the GPI zymogram. Zymodeme 11 (Z11) differs from Z5, Z6, Z7 and Z8 in GPI and PGM patterns. Isolates from sylvatic hosts, [1 from an Octodontomys, 1 from a skunk (Conepatus chinga) and 13 from weasels (Didelphis albiventris)], were obtained mainly in Santiago del Estero province, and belong to Z3, Z9 or Z10 (Table 1). Eight of the isolates were cloned (Table 2) and of these two (SE14 and SE6) were found to be heterogeneous, presenting patterns of either Z1 or Z2. The original SE14 and SE6 isolates when analysed periodically during 3 years of maintenance in culture, always showed zymograms corresponding to Z1, never to Z2, or to the combination of Z 1 + Z2.

Discussion

The electrophoretic patterns of six enzymes permit the classification of 73 isolates of T. cruzi collected in different areas of Argentina into 12 Zymodemes. Zymodemes 1 and 12, comprising isolates from acute and chronic chagasic patients, show a widespread geographic distribution. 40 of the 73 isolates (55%) belong to those zymodemes. Two other isolates obtained at widely separated sites (one from a

130 TABLE 1 Zymodeme patterns, host, geographic origin and clinical picture of the 73 T. cruzi stocks studied No. stocks

Zymodeme

Host

Province

Clinical

7 l l 1 5 5 1 1 1 1 1 13 1 1 1 2 1 1 1 2 1 l 1 1 2

1 12 1 1 l 12 1 12 1 12 12 10 3 9 9 1 1 1 12 2 12 1 1 1 4

Adult human Adult human Adult human Child Child Child Adult human Adult human Adult human Adult human Adult human Weasel Skunk Domestic dog Octodontomys Adult human Adult human Child Child Adult human Adult human Child Child Child Adult human

Asymptomatic Unknown Unknown Congenital Acute Acute Asymptomatic Asymptomatic Unknown Unknown Chr. Ch. card a --

1 5 2 1 2 3

4 5 6 7 8 1

1 1 1 2

1 11 12 1

C6rdoba C6rdoba C6rdoba C6rdoba Sgo. del Estero Sgo. del Estero Sgo. del Estero Sgo. del Estero Sgo. del Estero Sgo. del Estero Sgo. del Estero Sgo. del Estero Sgo. del Estero Sgo. del Estero Juj uy San Juan Santa Fe Buenos Aires Buenos Aires San Luis San Luis La Pampa Salta Salta Salta Salta Salta Salta Salta Salta Salta Salta Corrientes Mendoza Chaco

T. T. T. T. T. T.

infestans infestans infestans infestans infestans infestans

Guinea Pig Adult human Child Child

--Asymptomatic Unknown Congenital Acute Chr. Ch. card? Asymptomatic Acute Chr. Ch. card" Unknown Unknown ------Chr. Ch. card. a Acute Acute

aChronic Chagas with cardiopathy.

d o m e s t i c d o g , S a n t i a g o del E s t e r o p r o v i n c e a n d the o t h e r f r o m an O c t o d o n t o m y s 9 p a t t e r n . T h i s w i d e s e p a r a t i o n is n o t o b s e r v e d for t h e o t h e r z y m o d e m e s . Z y m o d e m e s 4 to 8 f r o m c h r o n i c p a t i e n t s a n d v e c t o r insects w e r e o b s e v e d o n l y in S a l t a p r o v i n c e . I s o l a t e s f r o m s y l v a t i c a n i m a l s , f r o m S a n t i a g o del E s t e r o p r o v i n c e , b e l o n g to Z3 a n d Z10. Z2 has b e e n f o u n d o n l y in i s o l a t e s f r o m San L u i s p r o v i n c e a n d Z11 in C o r r i e n t e s p r o v i n c e . A p p a r e n t l y , the p a r a s i t e s in A r g e n t i n a s h o w a h i g h e r d e g r e e o f g e n e t i c v a r i a t i o n t h a n t h o s e f r o m Brazil, w h i c h , a c c o r d i n g to M i l e s et al. (1980), c a n be g r o u p e d in f e w e r z y m o d e m e s . N o n e o f o u r z y m o d e m e s is i d e n t i f i e d w i t h a n y o f t h o s e d e s c r i b e d by M i l e s et al. ( M o n t a m a t et al., 1987).

sp., J u j u y p r o v i n c e ) s h o w e d t h e z y m o d e m e

131 TABLE 2 Zymodeme pattern, geographic origin and host, of the 38 clones studied Stock

Clone no.

SL1

59 64 65 67 69 70 71 72 73 K78

SE6

Zymodeme

Origin

Host

Clinical

2 2 2 2 2 2 2 2 2 2

San Luis

Adult human

Chr. Ch. card a

1 2

1 2

Sgo. del Estero

Child

Acute

SE8

1 2 3

1 1 1

Sgo. del Estero

Child

Acute

SE9

1 2 3 4 5 6 7 8 9

12 12 12 12 12 12 12 12 12

Sgo. del Estero

Child

Acute

SE14

75 76 77 78 80 81 83 84 88 91

1 2 1 2 1 1 2 1 1 l

Sgo. del Estero

Adult human

Asymptomatic

SJl

l

1

San Juan

Adult human

Asymptomatic

BAI

l 2

1 I

Buenos Aires

Child

Congenital

CH I

1

I

Chaco

Child

Acute

"Chronic Chagas with cardiopathy. P a r a s i t e s f r o m p a t i e n t s w i t h a c u t e a n d c h r o n i c d i s e a s e b e l o n g p r e d o m i n a n t l y to Z 2 a n d Z12, b u t t h e r e is n o d e f i n e d c h a r a c t e r i s t i c p a t t e r n f o r a c u t e o r c h r o n i c cases. I s o l a t e SE6, f r o m a n a c u t e p a t i e n t , w a s l a t e r c l o n e d a n d s h o w e d h e t e r o g e n e i t y , since p a r a s i t e s b e l o n g i n g to Z1 a n d Z 2 c o u l d be d i s t i n g u i s h e d in t h e s a m e isolate. It is i n t e r e s t i n g to n o t e t h a t m o s t i s o l a t e s f r o m a s y m p t o m a t i c p a t i e n t s b e l o n g to Z1. A l l e x c e p t o n e o f t h e i s o l a t e s f r o m s y l v a t i c a n i m a l s w e r e o b t a i n e d in S a n t i a g o del

132 TABLE 3 Electrophoretic zymograms corresponding to each one of the 12 zymodemes presented by Argentinian stocks. Letters correspond to the type of pattern as indicated in Fig. 2.

Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Zll Z12

EM

ASAT

ADH

PGM

G6PD

GPI

B C B A A A A A B A A C

C C A B C C C C -A C A

B A B B B B B B B A B A

C A B E D D D D C A C A

A B C D A A A A E F A F

A B A C D E F G A F H B

Estero province. Zymodemes Z3 and ZI0, found in isolates from the weasels and the skunk, have not been found in isolates from humans yet, suggesting that, at least in Santiago del Estero, the sylvatic and domestic transmission cycles may not overlap. There is one exception, however, where overlapping may exist; an isolate from an Octodontomys sp. in Jujuy showed the same patterns (Z9) as one from a domestic dog from Santiago del Estero. Parasites from Triatoma infestans belong to five different zymodemes (Z4 to Z8). Two isolates from chronic patients in Salta exhibited the same patterns (Z4) as an isolate from a domiciliary T. infestans collected at a site far from the locality where the patients lived. Analysis by means of DNA probes of the same isolates permitted a classification of parasites into similar groups to those obtained with zymograms (Macina et al., 1987). The observation of patterns for only one zymodeme (Z1) in isolates where cloning demonstrated the existence of Z1 and Z2 parasites, could be explained by differential adaptation to the culture conditions. Z1 development would be favored in the medium used. So far, we have characterized 73 isolates and 38 clones. The results demonstrate that at least 12 zymodemes exist in Argentina. This diversity supports the possible multiclonal origin of T. cruzi populations present in this country. The correlation between T. cruzi genetic structure and the clinical picture the parasite produces is still an intriguing question. Our observations on the predominance of zymodeme ZI in isolates from asymptomatic patients, and of Z2 and ZI2 in those from acute and chronic cases, encourage further studies in a larger number of isolates from Chagasic patients in order to substantiate this matter.

Acknowledgements This investigation received financial suport from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and from the Consejo de Investigaciones Cientificas y Tecnol6gicas de la Provincia de C6rdoba

133 (CONICOR). manuscript.

W e t h a n k D r . A n t o n i o B l a n c o for a d v i c e a n d critical r e v i s i o n o f the

References Brener, Z. (1985) General review on T. cruzi classification and taxonomy. Rev. Soc. Bras. Med. Trop. 18, 1-8. Boveris, A., Docampo, R., Turrens, J.F. and Stoppani A.O.M. (1978) Effect of Beta-lapachona on superoxide anion and hydrogen peroxide production in Trypanosoma cruzi. Biochem. J. 175, 431-439. De Lorenzo, R.J. and Ruddle, F.H. (1970) Glutamate oxaloacetate transaminase (GOT) in Mus musculus: linkage, polymorphism and phenotypes of the Got-2 and Got-I loci. Biochem. Genet. 4, 259-273. Engel, J.C., Dvorak, T.A., Segura, E.L. and Crane, M.St.J. (1982) T. cruzi: Biological characterization of 19 clones derived from two chronic chagasic patients. I. Growth kinetics in liquid medium. J. Protozool. 29, 555-560. Macina, R., Arauzo, S., Reyes, M., Sanchez, M.A., Basombrio, M., Montamat, E.E., Solari, A. and Frasch, A.C. (1987) T. cruzi isolates from Argentina and Chile grouped with the aid of DNA probes. Mol. Biochem. Parasitol. 25, 45-53. Miles, M.A., Lanham, S.M., de Souza, A.A. and Povoa, M. (1980) Further enzymic characters of T. cruzi and their evaluation for strain identification. Trans. R. Soe. Trop. Med. Hyg. 74, 221-237. Miles, M. (1983) The epidemiology of South American trypanosomiasis: biochemical and immunological approaches and their relevance to control. Trans. R. Soc. Trop. Med. Hyg. 77, 5 23. Montamat, E.E., Arauzo, S., Cazzulo, J.J. and Subias, E. (1987) Characterization by electrophoretic zymograms of 19 T. cruzi clones derived from two chronic chagasic patients. Comp. Biochem. Physiol. 87B, 417-422. Ogita, Z. and Markert, C.L. (1979) A miniaturized system for electrophoresis on polyacrylamide gels. Analyt. Biochem, 99, 233 241. Prata, A. (1985) Significance of T. eruzi differentiation and selection, relationship with clinical and epidemiological varieties. Rev. Soc. Bras. Med. Trop. 18 (suppl.), 9-16. Tibayrenc, M. and Ayala, F.J. (1988) Isozyme variability in T. cruzi the agent of Chagas' disease: Genetical, taxonomical and epidemiological significance. Evolution 42, 277-292.