Isoenzyme patterns of cultured Trypanosoma cruzi: Changes after prolonged subculture

Comp. Biochem. Physiol.. Vol 62B, pp. 139 to 142 © Peryamon Press Lid 1979. Printed in Great Britain

0305-0491/79/0215-0139S02.00/0

ISOENZYME PATTERNS OF CULTURED TRYPANOSOMA CRUZI: CHANGES AFTER P R O L O N G E D SUBCULTURE* ~LVARO JOSl~ ROMANHA,IA. A. DA SILVA PEREIRA,2 E. CHIARI3 and V. KILGOUR4 1Centro de Pesquisas "Rene Rachou" (FIOCRUZ), 2Departamento de Bioquimica-Imunologia, 3Departamento de Parasitologia, Instituto de Ci6ncias Biol6gicas, Universidade Federal de Minas Gerais, 30000 Belo Horizonte, MG., Brasil and 4Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Kepple St, London WCIE 7HT, England (Received 9 May 1978)

Abstract--1. The isoenzyme patterns of four soluble enzymes in seven stocks of T. cruzi were determined by electrophoresis. According to their patterns they could be classified into four sets. 2. The isoenzyme patterns of two stocks were influenced by the number of subcultures. 3. Five stocks from man are distinct from those derived from a silvatic reservoir. 4. Since the isoenzyme patterns of a stock isolated from a patient with acute disease were similar to those of a silvatic reservoir, its recent introduction into the domiciliary cycle is postulated.

INTRODUCTION

Biochemical techniques are now providing objective markers to distinguish species and subspecies of Protozoa (Newton, 1976). For instance, Peters et al. (1977) employed the D N A buoyant density and a serum factor in taxonomic studies of Leishmania; the analysis of the digest of T. cruzi kinetoplast D N A by restriction enzymes is also a tool for strain characterization at the genotype level (Mattei et al., 1977). Determination of the electrophoretic patterns of isoenzymes has been widely used as a taxonomic method for several groups of Protozoa, such as Paramecium (Tait, 1970), Plasmodium (Carter, 1970), Trypanosoma (Kilgour et al., 1973, 1975; Bagster & Parr, 1973; Toy6, 1974; Godfrey & Kilgour, 1976; Miles et al., 1977) and Leishmania (Kilgour et al., 1974; Gardener et al., 1974; Peters et al., 1977). In this paper we report the electrophoretic behaviour of four soluble enzymes derived from seven stocks of T. cruzi from varied sources. Of these, five stocks originated in m a n - - f o u r of recent origin from asymptomatic, chronic and acute forms of the disease, while the fifth was an old laboratory stock from an acute form; one stock originated in a bug and one from a silvatic animal. MATERIAL AND METHODS The T. cruzi cultures were obtained by hemoculture from infected animals or man and maintained in LIT (liver infusion-triptose) monophasic liquid medium (Camargo, 1964) by serial passage. Trypanosoma cruzi stocks 1. From man. Gerson (Ge) and Jesa (Je), isolated in 1976, from patients with the chronic cardiac form of the disease; Gilmar (Gi), 1973, from a lethally acute form; Neuza (Ne), in 1976, from an asymptomatic patient; Y, isolated by Silva & Nussenzweig (1953), from an acute form of the disease. 2. From a naturally infected bug. CL isolated in Rio Grande do Sul, Brazil, from a Triatoma infestans (Brener & Chiari, 1963).

3. From a naturally infected animal. Gamba (Ga), from Didelphis marsupialis (opossum) from the hinterland of S~o Paulo, Brazil, isolated in 1976. All stocks were maintained in mice by weekly i.p. blood passages. At different periods of time, the blood of heavily infected mice was inoculated in LIT medium, and the flagellates serially transferred to fresh medium approximately every 10 days until the experiments were completed. The Y and CL cultures were maintained in the laboratory over a period of years, but lines were stabilized at intervals: Y, at 3 months; Yim, 3yr; YPF (usually known as PF), 23yr (17 in agar blood, plus 6yr in LIT): CL17, 8 months and CLsg, 2 yr. Enzyme extracts preparation Cultures of trypanosomes were harvested and washed 3 times with KRT (Krebs, Ringer and Tris) buffer pH 7.3 after centrifugation at 1000 g, 4°C for 10 min. The enzymatic extract was prepared according to Kilgour & Godfrey (1973) and stored as beads in liquid nitrogen (Godfrey & Kilgour, 1976). Starch-gel electrophoresis Horizontal thin-layer starch-gel electrophoresis was used essentially as described by Wraxall & Culliford (1968). L-aspartate: 2-oxoglutarate aminotransferase (ASAT) (E.C. 2.6.1.1) and L-alanine: 2-oxoglutarate aminotransferase (ALAT) (E.C. 2.6.1.2) were stained by Karmen's (1955) and Chen & Giblett's (1971) method, modified by Kilgour & Godfrey (1973). For both enzymes, the electrode buffer was 150mM Tris-citrate, pH 9.0 and diluted x 10 in water for the gel buffer: electrophoresis was carried out for approx 1.5 hr at 30 V/cm. Phosphoglucomutase (PGM) (E.C. 2.7.5.1) and glucosephosphate isomerase (GPI) (E.C. 5.3.1.9) were visualized via the tetrazolium salt. For PGM, the electrode buffer was 150mM Tris-maleate, pH 7.4, which was x 10 diluted in water for the gel. For GPI, 200 and 15 mM phosphate buffer pH 7.4 were used as electrode and gel buffers, respectively. For both PGM and GPI, electrophoresis was carried out for 2.5 hr at 20 V/cm. RESULTS AND DISCUSSION

The strains examined for ALAT, ASAT, GPI and 139

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Fig. 1. Diagrammatic representation of enzyme patterns in T. <'rum P G M may be classified into four different sets, according to their isoenzyme patterns (Fig. 1). The sets are not mutually exclusive and there are many common bands among them. The first set includes five stocks (Ge, Je, Ne, YPF and YIMT) with the same patterns for all four enzymes. The second comprises a single strain (Y) that is distinct from the others. Both the third and fourth sets have a subset, differing only in one enzyme, either ASAT or ALAT. A typical experiment is shown in Fig. 2. The stocks Ge, Je and Ne of set I were isolated from patients with Chagas' disease, the other two, YPI and Y~M-,, resulted from successive subcultures of the Y strain originally obtained from man. The frequently passaged YPF and Y.MT strains differ from the original Y strain (set II): the slow anodic GPI bands has disappeared, the ALAT became more anionic (or its molecular size reduced), while in P G M the fast anionic band has become retarded and diminished in intensity although the intensity of the slow band has increased. In the CL stock, set III, an increase in the number of subcultures was concomitant with the appearance of a fast anodic ASAT band in the subset. It would seem that serial subcultures are conducive to a partial change of multiple bands in the electrophoretic patterns of some enzymes, for Godfrey (1975) also reported similar phenomena in the GPI and ASAT patterns of Sonia strain of T. cruzi after numerous subcultures. He also reported that two Y stocks of T. cruzi from different laboratories differ markedly in their enzyme patterns. The change in isoenzyme

patterns occurred after serial subcultures (Fig. 3). It is possible that the Y stocks reported by Godfrey had also been submitted to different numbers of serial passages in the culture media. While selection through laboratory manipulation from a heterogeneous population, or some form of genetic exchange could account for the T. cruzi changes, it could not account for the gradual weakening and disappearance of an ALAT band over many passages from a T. hrucei that had been cloned (Kilgour, 1976). Stability was not in doubt since one ALAT band remained constant throughout, and from field experiments Kilgour & Godfrey (19771 found that T. vivax sets direct from naturally infected cattle remained stable for over a year. To date the phenomenon has only been seen in isoenzymes having multiple bands: if the significance of the many bands (which is not understood) were related to the alternative directions a metabolic pathway may take, the suppression of one or more bands may be related to the lengthy dissociation from the alternative host concomitant with frequent sub-passaging. However, no explanation is clear and the events need further investigation, particularly as Chiari (1974) noticed modification of the differentiation rate in vitro of T. cruzi Y and YPv with frequent serial passages. It is interesting to observe the similarity between the stocks isolated from a lethal, acute case (Gi) and from a silvatic reservoir (Ga), both classified in set IV, The epidemiological data of Gi show that the infection was acquired after contact with wild reservoirs and vectors of 7". cruzi. The patient who lived

Isoenzyme patterns of cultured Trypanosoma cruzi

Fig. 2. Thin-layer starch-gel electrophoresis of T. cruzi phosphoglucomutase (PGM). Anode to the top. (left to right): CL17, CL89, Ga, Ge, Gi, Je, Ne, YPF, Y and YIMT. For strains abbreviations and conditions see Material and Methods.

Fig. 3. T. cruzi electrophoresis. (A) Alanine aminotransferase (ALAT) and (B) glucosephosphate isomerase (GPI). Strains positions in A and B (left to right): YPF, Y and YIMT.See Fig. 2.

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in an u r b a n area spent, during a fishing excursion, one single night in a primitive but heavily infested with triatomine-bugs and located in a silvatic uninhabited area where h u m a n transmission was very unlikely to occur. Three out of four enzymes examined display identical patterns for b o t h stocks, which, however, can be distinguished by A L A T isoenzymes. O u r results agree with those of Miles et al. (1977) which describe two distinct strain groups of T. cruzi, one characteristic of h u m a n infections and domiciliated animals, and the other t h o u g h t to be characteristic of silvatic animals but now found in m a n (Miles et al., 1978). O u r set I corresponds to their first group, and our set IV is similar to Miles's second group. A more extensive investigation will be necessary to correlate the isoenzyme patterns of T. cruzi strains with biological characteristics, especially their pathogenicity to man, and to examine the nature of the changes that may occur on prolonged culture. Acknowledgements--Our thanks are due to Dr J. C. P. Dias, who has supplied us with human infected blood: Dr I. Roitman for assistance in this study, Dr G. Gazzinelli for helpful discussion: and Dr Z. Brener and Dr D. G. Godfrey for critically reading the manuscript. This work was supported by "Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico'" (CNPqt and FINEP, The Ministry of Overseas Development of HM Government and the World Health Organization.

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