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Taxonomy 15/10/2022

Morphological taxonomy

Morphological methods

Wanderley de Souza and Juliana Vidal

Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro

Email: wsouza@biof.ufrj.brTrypanosoma cruzi belongs to order Kinetoplastida, familyTrypanosomatidae and sub-genus Schizotrypanum. In this family there are flagellated organisms with 1 or 2 flagella originating from a single opening, known as flagellar pocket, and normally have a flagellum with an additional structure called paraflagellar structure (except in those trypanosomatides that have an endosymbiont bacterium). In addition, specimens from this family have a structure known as kinetoplast, a region that concentrates mitochondrial DNA. The kinetoplast position with relation to the nucleus dictates the development form the protozoan presents (Figure 1). Figure 1 is a diagram showing the development forms that trypanosomatides can present. Also note the swimming direction adopted by each form. It should be emphasized that these protozoa have a single mitochondrion that ramificates throughout the cellular body. The Trypanosomatidae family consists mostly of monoxenic protozoa, with a single invertebrate host, and heteroxenic protozoa, which alternate their life cycle between two hosts. Some of the genuses of this family are: BodonídeosParatrypanosoma, Trypanosoma, Blechomonas, Sergela, Wallacemonas, Hepertomonas, Phytomonas, Blastocrithidia, Leptomonas, Lotmaria, Leishmania, Crithidia, Angomonas, Strigomonas and Kentomonas. Information obtained from the article titled: New Approaches to Systematics of Trypanosomatidae: Criteria for Taxonomic (Re)description. Votýpka et al., 2015. (Figure 2).

Figure 1: Schematic diagram of the development forms found in the trypanosomatide family. Note the position of the kinetoplast regarding the nucleus and the swimming direction each form adopts.
Figure 2: Phylogenetic tree of the Trypanosomatidae family. Note the three subfamilies (in blue letters) which have been acknowledged so far: Blechomonadinae, Leishmaniinae and Strigomonadinae. Bodonida species are written in green. In yellow are the monoxenic trypanosomatides and in red, the heteroxenic trypanosomatides.

Genus Trypanosoma is one of the most important within the Trypanosomatidae family, as it includes a series of species that cause relevant human diseases, such as Trypanosoma cruzi, the agent of Chagas disease (or American trypanosomiasis), Trypanosoma rhodesiense and Trypanosoma gambiense, agents of sleeping sickness, and animal diseases, such as Trypanosoma bruceiTrypanosoma equiperdum and Trypanosoma equinum. The Trypanosoma genus was divided in two groups, according to the parasite’s behavior in its hosts. The first, called Stercoraria, includes trypanosomes that develop in the vector’s digestive tube, progressing towards the intestinal portion and releasing infecting forms through the feces. This group includes T. cruzi and T. lewisi. The second, called Salivaria, includes trypanosomes that develop in the digestive tube at first, and later cross through the digestive epithelium to reach the salivary glands, where we can find the infecting forms that are mechanically inoculated. In this group we can find T. bruceiT. congolense and T. rangeli. The great complexity of the biological behavior of trypanosomatides of genus Trypanosoma has led to the creation of a few sub-genuses. Sub-genus Schizotrypanum includes specimens that go through an intracellular development form, amastigote forms that live and multiply within host cells. This group also includes T. cruzi and other trypanosomes found mainly in bats of the Old World and the New, such as T. dionisiiT. vespertilionisT. myoti, T. erneyi, T. herthameyeri. All protozoa mentioned above have a development cycle in axenic cultures and represent cells that are morphologically similar to those of T. cruzi.

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Biochemistry and Molecular

Biochemichal and Molecular Methods

Bianca Zingales

Chemistry Institute, University of São Paulo

Email: bszodnas@iq.usp.br

T. cruzi is represented by a group of populations that circulate in mammal hosts and in vector insects. These populations, also called isolates or strains, have largely heterogeneous biological behaviors, such as different degrees of virulence for experimental animals and humans, variations in sensitivity to drugs, and tissue tropism. The explanation for this phenotypical diversity resides in the fact that T. cruzi is a diploid organism that multiplies predominantly through binary division. The genome of each isolate therefore evolves independently. It is interesting to note that Chagas disease also has various clinical presentations (indeterminate, cardiac and digestive forms). Thus, a huge challenge for the scientific community has been identifying genetic markers of the isolates in order to gather then in discreet groups, with the goal of characterizing them from the epidemiological standpoint and in terms of pathogeny.

Electrophoretic profiles of isoenzymes

The first genetic studies of T. cruzi populations were made by Michael Miles’s group, beginning in the late 1970’s. Analyzing the polymorphism of the electrophoretic profiles of six enzymes, the isolates were gathered in three main groups, called zymodemes. Zymodemes Z1 and Z3 grouped predominantly strains of the wild cycle (opossums and triatomines), while zymodeme Z2 included strains of the domestic cycle (humans and domestic mammals). These conclusions were very promising, as zymodemes could be associated to different patterns of epidemiological transmission. However, the analysis of a higher number of isoenzymes showed a considerable increase in the number of zymodemes, which jumped to 43. Therefore, the analysis of isoenzyme patterns strengthened the concept of genetic diversity among strains of T. cruzi, although it suggested the possibility of groups.

Polymorphic genotypical markers

Scientists began to search for other genetic markers in the parasite’s genome. Different techniques were used to generate polymorphic genotypical markers: (i) polymorphism of the size of DNA digestion products (RFLP); (ii) random amplification of polymorphic DNA via PCR (RAPD) and (iii) DNA fingerprints.

Historically, these markers were initially researched in mitochondrial genome, represented by a complex network of thousands of circular DNA molecules called kinetoplasts (Figure 1A). In T. cruzi, mitochondrial DNA (kDNA) represents from 20 to 25% of the overall DNA in the cell, and consists of two types of molecules, maxicircles and minicircles. Maxicircles (20,000 pb; 50 copies per cell) codify for proteins of the electron transport chain and mitochondrial ribosome RNA. Minicircles (1,400 pb; 10,000 to 20,000 copies per cell) codify for small RNAs (guide RNA) that participate in the editing process of maxicircle transcripts. The minicircle contains four regions of preserved sequence separated by four regions of variable sequence (Figure 1B). The latter present high mutation rates, providing diversity among the minicircles of the isolates. Morel and collaborators explored this characteristic and established the pattern of RFLP of the minicircles of various strains of T. cruzi (Figure 1C). The patterns obtained (called schizodemes) confirmed their high heterogeneity.The characterization of strains was also explored through the technique of nuclear DNA fingerprints (Figure 1D); RAPD and micro satellite analysis. In all cases, genetic heterogeneicity between the strains was observed. It should be pointed out that RAPD data can be used to build dendograms, and that micro satellites can determine whether an isolate of T. cruzi is a monoclonal or multiclonal population. The characteristics of the maxicircle dictated a highly sensitive and specific PCR parasitological assay.

Figure 1: (A) Electron micrography of epimastigotes, K= kinetoplast; (B) Minicircle structure; (C) Digestion profiles of the minicircle of T. cruzi isolates with restriction enzymes and gel electrophoresis analysis. (D) Nuclear DNA fingerprints.

T. cruzi is divided in two main groups

In contrast to the great genetic diversity of the strains, highlighted by the techniques mentioned above, the analysis of sequences with a smaller evolution rate: ribosome RNA genes – classic evolution markers – and mini-exon genes, used for the taxonomy of trypanosomatides, showed a clear dimorphism between the isolates (Figure 2), resulting in their division in two groups. The analysis of 50-60 loci through RAPD showed that the two groups correspond to two great phylogenetic lineages. It was observed that the two lineages diverged a long time ago (between 40 and 10 million years ago). The two lineages were confirmed using other methodological approaches, and were named, by consensus, T. cruzi I and T. cruzi II, in 1999.

Figure 2: Molecular typing of T. cruzi isolates PCR assays for the rRNA gene 24Sα and for the intergenic spacer of the mini-exon gene make it possible to divide isolates in two groups. PCR for 24Sα originates a product of 110 pb for TcI and 125 pb for TcII. PCR for mini-exon originates a product of 350 pb for TcI and 300 pb for TcII. Initiators: red arrows (Souto et al. 1996).

Later, based on the analysis of other genetic markers, a subdivision was proposed of the T. cruzi II group into five sub-groups, called Iia – Iie. The evidence of the presence of hybrid isolates originated from genetic exchanges between parental strains was also observed. It is interesting to note that clone CL Brener, the reference organism for the T. cruzi genome project, is a hybrid isolate.The epidemiological distribution of groups T. cruzi I and T. cruzi II was investigated. The conclusion was that strains of group T. cruzi I were prevalent in the wild cycle and strains of group T. cruzi II were predominant in the domestic transmission cycle of the parasite. In countries of the Southern Cone, isolates of T. cruzi II are the main responsible for the clinical manifestations of Chagas disease (Figure 3).

Figure 3: Epidemiological cycles of T. cruziT. cruzi I parasites are prevalent in wild reservoirs, while T. cruzi II parasites are responsible for Chagas disease in the domestic cycle. The connection between the two cycles is provided by vectors that host T. cruzi II and invade domiciles. (Zingales et al. 1998).

Two points should be highlighted: (i) in countries of northern South America and in Central America and Mexico, Chagas disease can be caused by strains of the T. cruzi I group; and (ii) in countries of the Southern Cone, isolates of the T. cruzi II group induce the different clinical presentations of Chagas disease. Therefore, it seems clear that the pathogenesis of Chagas disease is the result of the interrelation between the genetic (and therefore biological) characteristics of parasite isolates and the immunogenetic characteristics of the human host. These genetic markers are currently being actively researched, as they have a predictive value for the evolution of the disease. Consequently, they will be useful for the adoption of therapeutic procedures within the Health Service.

Conclusions

The genetic diversity between T. cruzi strains is widely documented. This characteristic raises the question whether T. cruzi is a single species or an artificial taxon that includes more than one species. In this sense, genus Leishmania, which has genetic diversity similar to that of T. cruzi, has a high number of species. The scientific community gathered isolates of T. cruzi into two main groups that have particular epidemiological characteristics. Taxonomists can be divided in two categories: those who explore the diversity between the populations and those who prefer to divide these populations in blocks. Although not all taxonomists have these extreme views, many lean towards one position or the other depending on the objective they have in mind. In our more pragmatic view, classificating strains in discreet groups makes it possible to better explore the characteristics related to prognostic and treatment of Chagas disease.

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