Home Economics American Trypanosomiasis Chagas Disease, Second Edition: One Hundred Years of Research
Experimental attempts to produce recombinants between TcI and TcII, then referred to as Z1 and Z2, were first undertaken in the 1970s (M. Miles, unpublished data). The experimental approach was either simply to passage together mixed populations of the two lineages in blood agar cultures or sequentially from mouse to triatomine to mouse and back into culture, then re-characterizing the population mixtures. No recombinants were detected and either a mixture of TcI and TcII or TcII alone were recovered from these experiments. In retrospect it was ambitious to attempt an experimental cross between these genetically divergent lineages. Furthermore, there were no genetic markers available for the selection of recombinants, which would have been undetectable if they had been present as minor populations in the output of such experiments.
Experimental genetics for T. cruzi became feasible with the development of recombinant DNA technology that allowed genetic transformation of parasite populations to confer resistance to different drugs. Trypanosomes could now be marked to carry a wide range of reporter genes including fluorescent markers of
various colors: it is possible to direct such markers to intracellular organelles of special interest or to tag genes of interest that are expressed during different life cycle stages.
In 1996, putative parental homozygous and recombinant heterozygous genotypes of TcI were described on the basis of phosphoglucomutase (PGM) isoenzyme phenotypes in isolates from a single locality in the Amazon basin of Brazil, potentially compatible with active intra-lineage recombination.16 Accordingly, two putative parental strains from this locality were transformed with episomal recombinant DNA plasmids bearing genes conferring resistance to specific antibiotics, either hygromycin B or G418. Experimental crosses were attempted in vitro in mammalian cell cultures, and in vivo in mice and triatomine bugs by coinfection with both transgenic parental strains.17 Parasite populations subsequently derived from the co-infections were subjected to selection with both drugs simultaneously. Six double drug-resistant T. cruzi recombinant hybrid clones were obtained from mixed infections in the mammalian cell cultures and were shown to contain both drug resistance marker genes indicating that they were the products of genetic exchange between the co-infecting strains. The six clones were characterized by MLEE, karyotypes, microsatellites, and sequencing of some housekeeping genes. This analysis demonstrated that parental genetic markers had not been inherited in typical Mendelian ratios. Rather than inheriting one allele per locus from each of the parent strains, as expected in typical meiotic F1 heterozygous progeny, the hybrid clones contained all alleles from both parents at most loci. However, at a small minority of loci, some parental alleles were not present. Each of the six hybrid clones had one of the parental kDNA maxicircle genotypes but not both. It was concluded that fusion of the diploid parental strains had occurred to produce a tetraploid hybrid, with limited subsequent genome erosion and an unclear level of concomitant genetic recombination between parental sequences.
In terms of virulence, pathogenesis, and epidemiological relevance it was of considerable interest to see how these hybrid T. cruzi clones behaved in experimental infections. The hybrid clones proved to be at least as a virulent as the parental strains in immunocompromised mice.18 They produced abundant pseudocysts in heart and skeletal muscle, with some detectable infection of smooth muscle of the alimentary tract. This showed that these in vitro generated hybrids were capable of all the morphogenic transitions required to complete a full life cycle, and were able to survive in a mammalian host. Whether the hybrid clones display increased virulence or “hybrid vigor” in comparison with their parents, or would compete with the parents in co-infections, is a topic that requires further study.
Flow cytometric analysis of DNA content provided further insight into the genomic composition and ploidy of the experimental hybrid clones and the process of genome erosion. This approach was originally applied to T. cruzi by James Dvorak in the 1980s and revealed substantial variation in the DNA content of natural isolates.19 The DNA content analysis demonstrated that all six hybrids had, on average, 69% more DNA that the parental strains. This was compatible with an aneuploid chromosome complement intermediate between 3n and 4n, and so the hybrids were considered to be subtetraploid.18 There was no dramatic decline in DNA content when the
clones were recovered from infected mice or in response to stressful growth conditions, such as heat shock, indicating that their genomes were relatively stable. The DNA content analysis thus further supported the hypothesis that the hybrids underwent limited genome erosion from a tetraploid fusion product.
The ploidy level, however, is not absolutely stable. Following prolonged passage in axenic cultures a gradual, progressive decline in DNA content has been observed (M. Lewis, unpublished data), with a pattern that is not compatible with any true meiotic reductive division that would result in rapid, ordered reduction of ploidy. Further comparisons are ongoing of the parental and experimental hybrid karyotypes and genotypes, particularly using heterozygous parental allelic markers, to understand the mechanism of genome erosion in T. cruzi. Meanwhile, it is clear these in vitro-generated intra-lineage TcI hybrids are not the result of the typical eukaryotic programme of genetic exchange, because neither the parents nor the hybrids underwent a meiotic reductive division. The process of fusion of diploids followed by genome erosion is reminiscent of the parasexual reproductive cycle of the pathogenic yeast Candida albicans, which is characterized by cellular and nuclear fusion of diploid cells producing tetraploid intermediates, followed by random, concerted chromosome losses, giving rise to recombinant progeny with an approximately diploid chromosome complement.20 Whether the diploid fusion— genome erosion model of genetic exchange applies to wild populations of T. cruzi or whether sexual reproduction involving normal meiosis could occur under different conditions or between different strains remains an open question.
Performing experimental crosses in T. brucei using transgenic strains expressing different fluorescent proteins cytoplasmically or tagged to specific proteins has proven to be a powerful tool for studying recombination under laboratory conditions since hybrid organisms co-expressing multiple markers due to inheritance can be identified microscopically. This has helped to pinpoint the developmental stage of the parasite that is involved in genetic exchange.21,22 The same approach may prove fruitful for experimental crosses in T. cruzi. Red and green fluorescent strains of T. cruzi as well as the closely related species T. rangeli have been described, and the potential for exploiting these reporters to track co-infections in vitro and in mice and triatomine bugs in vivo has now been demonstrated.23,24 As yet no hybrid parasites have been recovered from such experiments, although relatively few conditions or strains appear to have been tested.
In T. cruzi the experimental hybrid clones described above were derived from mammalian cell cultures but it was not proven that hybridization was an intracellular event. In T. brucei and Leishmania sp. recombination occurs in their invertebrate vectors—the tsetse fly and sand fly, respectively.25,26 In the T. cruzi cross the mammalian cells were infected with a mixture of metacyclic trypomastigotes and epi- mastigotes from stationary phase cultures. It is therefore possible that hybridization occurred between epimastigotes prior to invasion and establishment of intracellular forms. Alternatively hybridization may have taken place between trypomastigotes emerging from the mammalian cells during the prolonged infection, which could have encompassed up to four rounds of invasion and intracellular multiplication. This implies that hybridization between extracellular forms that would be found in natural infections of triatomine bug vectors should not be ruled out. This possibility requires further investigation, ideally with transgenic T. cruzi strains carrying both drug resistance and fluorescent markers to allow visualization of interactions between coinfecting strains. This will require careful experimental design because there are around 140 known species of triatomines, and the 6 known lineages of T. cruzi each encompassing considerable genetic diversity. Triatomine species appear to differ in their susceptibilities to infection with different T. cruzi lineages and so the behavior of T. cruzi strains in one combination of triatomine species and T. cruzi lineage will not necessarily be typical.
The experimental demonstration of hybridization was a milestone in research on T. cruzi. It proved that T. cruzi has an extant capacity for genetic exchange. It also revealed an unusual, nonmeiotic mechanism involving fusion of diploids followed by genome erosion, for which the precise details remain to be understood. This mechanism may be operating among natural populations of T. cruzi, but the occurrence of other genetic mechanisms among natural populations cannot be excluded.
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