Ricardo P. Souto

DNA MARKERS DEFINE TWO MAJOR PHYLOGENETIC LINEAGES OF TRYPANOSOMA CRUZI

Parasitic protozoa within the taxon Trypanosoma cruzi are considered to be derived from multiple clonal lineages, and show broad genetic diversity as a result of propagation with little or no genetic exchange. We have analyzed a wide sample of T. cruzi isolates from vertebrate and invertebrate hosts by PCR amplification of a ribosomal RNA gene sequence, a mini-exon gene sequence and random amplified polymorphic DNA (RAPD). Amplification of the distinct rDNA and mini-exon gene sequences indicated a dimorphism within both of the tandemly-repeated genes: 125 or 110 bp products for rDNA and 300 or 350 bp products for the mini-exon. Within individual isolates, one of three associations was observed: the 125 bp rDNA product with the 300 bp mini-exon product (defined as group 1), the 110 bp rDNA product with the 350 bp mini-exon product (defined as group 2) and the presence of both rDNA amplification products with the mini-exon group 1 product (group 1/2). The RAPD analysis showed variability between individual isolates, however, tree analysis clearly indicated the presence of two major branches. Interestingly, the rDNA/mini-exon group 2 isolates correlated precisely with one branch of the RAPD-derived tree; group 1 and group 1/2 isolates correlated with the other branch. Our studies show a clear division of T. cruzi into two major lineages presenting a high phylogenetic divergence. Hypotheses are discussed to explain the origin of the two lineages as well as isolates that are hybrid for group 1 and 2 rDNA markers.

The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity

The agent of Chagas disease, Trypanosoma cruzi, is divided into two highly divergent genetic subgroups, lineages 1 and 2, which include all typed strains isolated from humans, insect vectors, and sylvatic mammals. The evolutionary origin of these two T. cruzi lineages and the clinical importance of their identification, have been the subject of intense debate. Here, using molecular phylogenetic analysis, we found that the distance between the two T. cruzi lineages is equivalent to the distance between genera Leishmania and Endotrypanum. Also, we confirmed that T. rangeli is more closely related to T. cruzi than to T. brucei using the rDNA sequence from a human strain of T. rangeli. Phylogenetic trees based on small subunit rDNA sequences further suggest that the two T. cruzi lineages diverged between 88 and 37 million years (Myr) ago. We hypothesize that lineage 2 is indigenous to South America while lineage 1 has been introduced to South America recently, along with North American placental mammals, after the connection of the Americas in the Pliocene (5 Myr ago) or with caviomorph rodents and primates in the Oligocene (38 Myr ago). This would explain the preferential association of T. cruzi lineage 2 with marsupials and of lineage 1 with human disease. These two T. cruzi lineages are likely to be distinct species, or at least subspecies, because of their different ecological and epidemiological traits and estimated long period of independent evolution.

Molecular epidemiology of American trypanosomiasis in Brazil based on dimorphisms of rRNA and mini-exon gene sequences

American trypanosomiasis is transmitted in nature via a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, or via a domestic cycle where the parasite comes into contact with humans through domiciliated triatomines. The pool of T. cruzi isolates consists of sub-populations presenting a broad genetic diversity. In contrast to the heterogeneity suggested by isoenzyme analysis, PCR amplification of sequences from the 24S alpha rRNA gene and from the non-transcribed spacer of the mini-exon gene indicated dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 157 T. cruzi isolates obtained from humans, triatomines and sylvatic mammalian reservoirs from 12 Brazilian states were analysed by the 24S alpha RNA and mini-exon typing approaches. The stocks were classified into the two proposed lineages and according to the domestic or sylvatic cycle of the parasite. Data presented provide evidence for a strong association of T. cruzi lineage 1 with the domestic cycle, while in the sylvatic cycle both lineages circulate equally. Molecular typing of human parasite isolates from three well-characterised endemic regions of Chagas disease (Minas Gerais, Paraiba and Piaui) and from Amazonas State, where T. cruzi is enzootic, suggests that in some endemic areas in Brazil there is a preferential linkage between both cycles mediated by lineage-1 stocks.

Sensitive detection and strain classification of Trypanosoma cruzi by amplification of a ribosomal RNA sequence

A sequence of about 100 bp of the 24S alpha ribosomal RNA was investigated for sensitive detection of Trypanosoma cruzi. It was shown that the target sequence is specific for this parasite and no cross-reactivity was observed with different species of pathogenic Leishmania, two strains of Trypanosoma rangeli or human RNA. Amplification of the sequence was obtained by reverse transcription coupled to polymerase chain reaction. Following this procedure the equivalent to 0.1% of the nucleic acid content of a single parasite cell could be detected either by ethidium staining or blot hybridization. The distribution of the target sequence in sixteen strains of T. cruzi was investigated. Positive amplification was obtained for all samples employing the same oligonucleotides as primers. However, amplified fragments of 125 bp were obtained in eight strains, while fragments of 110 bp were detected in the remaining eight isolates. No amplification of both classes of fragments has been detected in any of the strains examined. Dimorphism in the target region was confirmed by hybridization to specific internal probes and sequencing, allowing the division of T. cruzi strains in two groups. It is proposed that sensitive parasite detection could be achieved by rRNA amplification followed by hybridization to two probes derived from the target sequences of both groups of T. cruzi strains. Furthermore, the sequence dimorphism found in this sequence opens the perspective of strain typing simultaneous with parasite detection.

Epidemiology, biochemistry and evolution of Trypanosoma cruzi lineages based on ribosomal RNA sequences

Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, Caixa Postal 26077, 05599-970Sao Paulo, SP, Brasil *Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil**Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina,Universidade Federal de Sao Paulo, Sao Paulo, SP, BrasilKey words: Trypanosoma cruzi - major lineages - ribosomal RNA - molecular epidemiology - phylogeny

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Graces medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.

Biological Parameters and Molecular Markers of Clone CL Brener - The Reference Organism of the Trypanosoma cruzi Genome Project

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28 degrees C is 58 +/- 13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Graces medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37 degrees C; (d) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24S alpha ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed.

Two types of ribosomal RNA genes in hybrid Trypanosoma cruzi strains

Trypanosoma cruzi isolates can be divided into two major phylogenetic lineages-T. cruzi I and T. cruzi II. The population structure is predominantly clonal, with sexuality having no or limited influence on the evolution of the parasite. Isoenzymes and nuclear gene sequences have provided evidence that some T. cruzi strains are hybrids. Previous work of our group has shown that the putative hybrid strains designated as group 1/2 contain two types of rDNA units, corresponding to those found in T. cruzi I and T. cruzi II. In this study, the presence and transcription of the two types of ribosomal RNA (rRNA) cistrons were investigated in epimastigotes, metacyclic and tissue culture trypomastigotes of group 1/2 isolates. PCR and RT-PCR assays indicate that both types of cistrons are present in group 1/2 strains, but only type-2 genes are transcribed in all developmental stages. The structure of the promoter regions of group 1/2 was compared to reference T. cruzi I and T. cruzi II strains. In all cases, the transcription start point was mapped to a conserved A residue located approximately 1800 bp upstream the 18S rRNA gene. The distribution of rDNA clusters in chromosomal bands of group 1/2 was evaluated by pulsed-field gel electrophoresis (PFGE). The majority of type-2 rDNA genes are localized in a 1.5 Mbp band, whereas type-1 cistrons are mostly concentrated in a 1.1 Mbp band. The structural and functional studies of group 1/2 ribosomal cistrons described here may shed light on the evolutionary processes that took place during the generation of such hybrid organisms.

Trypanosoma cruzi: How Many Relevant Phylogenetic Subdivisions are There? Reply

There is a general agreement between our laboratories 1 and that of Dr Tibayrenc 2 (and see Comment article, this issue) on the existence of two major phylogenetic lineages in Trypanosoma cruzi. Both groups also agree that, within each lineage, genetic diversity exists. The difference between the two research groups is the interpretation of the operational value of the two lineages. Tibayrenc’s group finds that the average and maximum genetic distances within each lineage are not much lower than those recorded in the whole species (this issue). Our data indicate molecular, biological and ecoepidemiological features that correlate to the two proposed lineages. It is interesting to note that in order to characterize the two lineages, the two groups have employed different molecular markers. Tibayrenc’s group used isoenzyme and RAPD (randomly amplified polymorphic DNA) analyses 2 . In our case, the first evidence for the two T. cruzi clusters