Genetic Diversity and Genetic Exchange in Trypanosoma cruzi: Dual Drug-resistant "Progeny" from Episomal Transformants

Genetic Diversity and Genetic Exchange in Trypanosoma cruzi: Dual Drug-resistant "Progeny" from Episomal Transformants

Suppl. I: 189-193

JR Stothard*, IA Frame, MA Miles⁺

Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Key words: Trypanosoma cruzi - genetic diversity - genetic exchange - transfection

Trypanosoma cruzi occurs widely in many mammal and triatomine bug species (Hemiptera: Reduviidae) in the New World, although there are few human infections in the United States or the Amazon basin. It has been estimated that there are around 16 to 20 million people infected and 100 million exposed to infection. There are five main domestic triatomine vector species: Triatoma infestans (southern cone countries and Peru), Rhodnius prolixus (northern South America and Central America), Panstrongylus megistus (eastern and central Brazil), Triatoma brasiliensis (northeastern Brazil) and Triatoma dimidiata (Central America).

The diverse clinical outcome of human T. cruzi infection and biological differences between T. cruzi strains led to the hypothesis that T. cruzi was a heterogeneous species. T. cruzi strains were reported to differ antigenically, in virulence and histotropism in experimental animals, in infectivity to triatomine bug species and in susceptibility to drugs. The diversity of T. cruzi was first conclusively confirmed by phenotypic characterization of isolates using isoenzyme electrophoresis. There were shown to be at least three main T. cruzi strain groups or principal zymodemes (Z1, Z2, Z3 ) and many distinct strains within these three major groups. The diversity within Z2 was particularly marked. T. cruzi Z2 was originally described from central and eastern Brazil in domestic transmission cycles where heart disease and megasyndromes were reported to be common. T. cruzi Z1 was predominantly sylvatic where Z2 occured but was found in both sylvatic and domestic transmission cycles north of the Amazon basin. T. cruzi Z3 has so far been primarily associated with Panstrongylus geniculatus and burrowing animals, such as the armadillo, and has rarely been isolated from humans. T. cruzi Z1 appeared to be associated with opossums, especially the genus Didelphis. Host associations for Z2 were less obvious. It was suggested that the original mammalian host for Z2 could be guinea pigs in the sylvatic cycle in Bolivia and that Z2 may have spread with T. infestans to the six southern cone countries of South America (Argentina, Bolivia, Brazil, Chile, Paraguay, Uruguay) and to southern Peru. T. cruzi transmission cycles were described as either (a) non-overlapping or discontinuous, where there are separate domestic and sylvatic transmission cycles in a single locality (e.g. Bahia, Brazil), (b) as overlapping or continuous, where the same vector and similar T. cruzi strains occur in adjacent domestic and sylvatic cycles (e.g. parts of Venezuela) or (c) enzootic, where there is abundant sylvatic transmission, but domestic transmission has not yet become established (e.g. the Amazon basin and the USA) (Miles 1998).

Isoenzyme characters could be used in numerical taxonomy to estimate the similarities or genetic distances between T. cruzi strains. A series of strains was selected, many of which are biological clones, so that experimental work could represent naturally occurring epidemiologies (Table).

Although there is circumstantial evidence, there is still no formal proof that infection with a particular T. cruzi strain determines a poor clinical prognosis. The high prevalence of T. cruzi Z2 in human infections in central and eastern Brazil, where megasyndromes are common and the contrasting high prevalence of T. cruzi Z1 in human infections in the north of South America, where megaoesophagus and megacolon are said to be rare, suggested that Z2 was more likely to cause chronic Chagas disease. Luquetti et al. (1986) isolated only Z2 from symptomatic chronic Chagas disease, but there was no proof that Z1 was not present earlier in the infections. Z1 and Z2 have each been isolated from symptomatic acute phase human infections and each could relapse after unsuccessful treatment. There are no conclusive studies of the relationship between human genotype and disease prognosis (Miles 1998).

It was soon demonstrated that biological clones of Z1, Z2 and Z3 differed radically in other characters, such as in kinetoplast DNA minicircle fragment patterns (schizodemes), in DNA content, in virulence to experimental animals, in extracellular and intracellular growth rates in vitro, in response to experimental chemotherapy, in antigenic profiles, in oxidative metabolism and in elemental composition of iron, zinc and potassium (Nozaki & Dvorak 1993, Miles 1998). Zymodeme groupings and schizodeme groupings of T. cruzi strains and the surface abundance of a particular antigen broadly correlated.

In further extensive studies of isoenzyme phenotypes of T. cruzi, Tibayrenc and colleagues subdivided the Z1, Z2 and Z3 groups into many individual zymodemes. They also demonstrated a strong correlation between the strain clusters established by isoenzyme phenotypes and by genotyping using random amplified polymorphic DNA (RAPD) profiles (Tibayrenc et al. 1993). Two further genotyping methods have been introduced for T. cruzi isolates, one based on analysis of polymorphisms in polymerase chain reaction (PCR) amplified ribosomal RNA genes and a second based on polymorphisms in mini-exon genes. These two methods have indicated the presence of at least two major T. cruzi genetic lineages, named genetic lineage 1 _ which appears to correspond with Z2, and genetic lineage 2 _ which appears to correspond with Z1 (Stothard et al. 1998, Fernandes et al. 1999). Microsatellite analysis and functional analysis of gene promoters have also partitioned T. cruzi into two major groups (Nunes et al. 1997, Oliveira et al. 1998).

Recent studies of sylvatic T. cruzi transmission cycles in Rio de Janeiro have confirmed the hitherto proposed link between genetic lineage 2 (presumed Z1) and Didelphis. It has also revealed a previously unsuspected association in Brazil between genetic lineage 1 (presumed Z2) and a primate host, the golden-lion tamarin, Leontopithecus rosalia (Fernandes et al. 1999). Genetic lineage 1 (again presumed Z2) has also been reported from lion-tailed macaques (Macaca silenus), from a ring-tailed lemur (Lemur catta) and from racoons (Procyon lotor) in the USA (Pung et al. 1998). In both these recent studies the principal zymodemes are presumed from correlations established with reference T. cruzi strains and it appears that isoenzyme phenotypes were not determined for all the new T. cruzi isolates involved. The finding of T. cruzi genetic lineage 1 (presumed Z2) in primates is of particular interest as pre-adaption to primates may well have facilitated the establishment of T. cruzi Z2 in the human host and domestic transmission cycles. Furthermore, marmosets, especially Callothrix species, are common pets and this could have introduced T. cruzi Z2 into dwellings from sylvatic cycles. Based on a single isolate, the suspected triatomine vector of genetic lineage 1 (presumed Z2) in Rio de Janeiro is Triatoma vitticeps.

Potential for antigenic variation within biological clones of T. cruzi is not the primary focus of this brief article. Nevertheless, attention is drawn here to the presence of complex gene families in T. cruzi, which may have a role in evasion of the host immune response. The large family of putative mucin genes consists of hundreds of copies per haploid genome. The copies share a signal peptide on the N-terminus and a presumed glycosyl-phosphatidylinositol anchoring sequence on the C-terminus, with hypervariable central regions (Di Noia et al. 1998).

GENETIC EXCHANGE
CROSSING EXPERIMENTS WITH EPISOMAL TRANSFORMANTS
CONCLUSION
REFERENCES

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