Populational
Heterogeneity of Brazilian Trypanosoma cruzi Isolates
Revealed by the Mini-exon and Ribosomal Spacers
Suppl. I:
195-197
O
Fernandes/*/+, SS Santos,
ACV Junqueira, AM Jansen**,
E Cupolillo***, DA Campbell****,
B Zingales*****, JR Coura
Departamento de
Medicina Tropical **Departamento de Protozoologia ***Departamento de
Imunologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de
Janeiro, RJ, Brasil *Departamento de Patologia, Uerj, Rio de Janeiro,
RJ, Brasil
****Department of Microbiology & Immunology, School
of Medicine, University of California, Los Angeles, USA
*****Departamento de Bioquímica, Instituto de Química, Universidade de
São Paulo,
São Paulo, SP, Brasil
Key words:
Trypanosoma cruzi - mini-exon - ribosomal DNA - major
phylogenetic lineages
Chagas disease in
humans, a result of infection by the protozoan Trypanosoma
cruzi, shows considerable diversity in clinical manifestations
and chronic pathology of cardiac or digestive alterations. This
variability has been attributed to both variation in the host immune
response and to genomic heterogeneity of the parasite. Although
described as one taxon, T. cruzi shows substantial heterogeneity
in genotype and phenotype perhaps as a result of its clonal method of
propagation, in which it is proposed that mutations accumulate in
different sub-populations of the parasite (M Tibayrenc et al. 1990
Proc Natl Acad Sci USA 87: 2414-2418).
Initial studies
using isoenzyme electrophoresis analysis (MA Miles et al. 1977 Trans
R Soc Trop Med Hyg 71: 217-225, 1978 Nature 272: 819-821,
1980 Trans R Soc Trop Med Hyg 74: 221-237, MA Miles & RE
Cibulski 1986 Parasitol Today 4: 94-97) indicated that T.
cruzi could be classified into three groups (termed zymodemes) based
on six enzyme electrophoretic profiles. Zymodeme I and III were
associated with forest-dwelling (sylvatic) mammals, such as opossums;
zymodeme II was associated with human cases of Chagas disease,
domiciliated mammals and domestic triatomines.
Further analysis
using additional enzyme markers and larger number of isolates indicated
greater diversity within the taxon and 43 zymodemes were defined (M
Tibayrenc et al. 1986 Proc Natl Acad Sci USA 83: 115-119, 1993
Proc Natl Acad Sci USA 90: 1335-1339, M Tibayrenc & FJ Ayala
1988 Evolution 42: 277-292). This increased level of
discrimination between T. cruzi isolates did not reveal obvious
linkages between the zymodemes and aspects of pathology, transmission,
epidemiology, or geographic distribution beyond the correlation of
zymodemes I, II and III and the epidemiological cycles. The complex
structure of T. cruzi population, inspired a major effort to
determine molecular markers that correlate with specific features of the
human-parasite relationship. Similar variability among parasite
populations was also observed in restriction-fragment-length
polymorphism in the mitochondrial DNA (C Morel & L Simpson 1980
Am J Trop Med Hyg 29 Suppl: 1070-1074), nuclear DNA
fingerprinting (AM Macedo et al. 1992 Mol Biochem Parasitol 55:
147-154) and karyotyping studies (J Henriksson et al. 1993 Exp
Parasitol 77: 334-348, J Henriksson 1996 Parasitol Today
12: 108-114). Once again, no correlation with biological features
was observed due to the extreme heterogeneity of the isolates.
In contrast to
the diversity suggested by the above techniques, PCR amplification of
sequences from the 24Sa ribosomal RNA (rRNA) gene and from the
non-transcribed spacer of the mini-exon gene indicated a clear
dimorphism among T. cruzi isolates. This dimorphism allowed the
definition of two lineages that correlated broadly with zymodemes I and
II (O Fernandes et al. 1998 Am J Trop Med Hyg 58: 807-811, 1999
Parasitology 118: 161-166, RP Souto & B Zingales 1993 Mol
Biochem Parasitol 62: 45-52, RP Souto et al. 1996 Mol Biochem
Parasitol 83: 141-152). An examination of 88 T. cruzi stocks
collected from humans, wild mammals and triatomines and originating from
different Countries of South America (Brazil, Argentina, Chile, Bolivia
and Venezuela) by mini-exon gene and 24Sa rRNA typing approach, and
randomly amplified polymorphic DNA (RAPD) analysis further defined two
major parasite lineages that represent substantial phylogenetic
divergence (RP Souto et al. 1996 Mol Biochem Parasitol 83:
141-152).
To verify a
possible association of epidemiological parameters or disease potential
of the isolates with the two T. cruzi lineages that nowadays are
named T. cruzi I and T. cruzi II, we also used the
mini-exon and/or 24Sa rRNA typing method to analyze 86 T. cruzi
field stocks (68 isolated from humans and 18 from triatomines)
derived from four Brazilian geographic areas. These parasite samples
were also clustered into the aforementioned two lineages. The data are
suggestive of a preferential association of to human isolates. T.
cruzi I to the sylvatic cycle of the parasite and T. cruzi
II. Furthermore a clear correlation could be made with the morbidity
of the disease: areas with high morbidity present the circulation of
T. cruzi II; T. cruzi I is evidenced in areas where Chagas
disease is infrequent and the morbidity, as evaluated by the level of
abnormal electrocardiograms, is low (Fernandes et al. 1998 loc.
cit.).
In the wake of
these molecular epidemiological studies, an intriguing question remains.
How could a parasite change its genome during the transition from the
sylvatic (T. cruzi I) to the domestic transmission cycle (T.
cruzi II)? To clarify this specific point, we must re-evaluate the
sylvatic cycle of T. cruzi in the light of the recent division of
this protozoan into the two major lineages. Sixty-eight T. cruzi
isolates collected recently from sylvatic mammals and wild bugs from
different geographical areas in the State of Rio de Janeiro, a Brazilian
region with no cases of autochthonous Chagas disease, were typed. This
study revealed that the sylvatic cycle is more complex than previously
assumed since both T. cruzi lineages were encountered in similar
ecotopes (Fernandes et al. 1999 loc. cit.). Therefore, a new
proposal for the transmission cycles of T. cruzi was elaborated
(B Zingales et al. 1998 Inter J Parasitol 28: 105-112).
Our laboratory
has also adapted a typing approach (E Cupolilo et al. 1995 Mol
Biochem Parasitol 73: 145-155) to be used for T. cruzi
strains using transcribed spacers of the ribosomal gene (Fig. 1).
Ribosomal RNA genes (rDNA) are highly conserved and have proven to be
useful in phylogenetic analysis among trypanosomatids (Cupolilo et al.
1995 loc. cit., O Fernandes et al. 1994 Mol Biochem Parasitol
66: 26221-26271). Typically, in trypanosomatids rDNA is found as
tandemly-repeated units separated by non-transcribed spacers (JL Ramirez
& P Guevara 1987 Mol Biochem Parasitol 66: 261-277, P Guevara
et al. 1992 Mol Biochem Parasitol 56: 15-26). Trypanosomatid rDNA
exhibits an unsual organization where the coding regions for the three
large and five small ribosomal RNA molecules are separated by internal
transcribed spacers (ITS) that show extensive variability. The ITS are
relatively small and flanked by highly conserved segments to which PCR
primers can be designed. In order to produce an amplification product
corresponding to the 5.8S rDNA plus the two flanking ITS, conserved
oligonucleotides were used (5'-GCTGTAGGTGAACCTGC AGCAGCTGGATCATT-3' and
5'-GCGGGTAG TCCTGCCAAACACTCAGGTCTG-3'). Amplification reactions were
performed as previously described (Cupolilo et al. 1995 loc.
cit.) using genomic DNA of 10 T. cruzi strains as templates.
Five of them were from Piauí, where T. cruzi II is the most
frequent and five from Amazonas, where T. cruzi I predominates.
The PCR products were further digested by six restrictions enzymes
(BstUI, EcoRI, HaeIII, RsaI, Sau3AI and TaqI) and submitted to
acrylamide gel electrophoresis (Fig. 2).
The PCR products corresponding to the ITS of the T. cruzi
isolates from Piauí and Amazonas were distinct in size and the resulting
restriction fragment profiles, analyzed by a numerical methodology,
generated a phenetic dendrogram that clusters the isolates into the two
aforementioned lineages with low level of similarity (Fig. 3).
The results show that this approach is also consistent with the
sub-division of the taxon T. cruzi into two phylogenetic
lineages. Considering the ten isolates that were analyzed, T. cruzi
II (Piauí strains _ domestic transmission cycle) is less polymorphic
than T. cruzi I (Amazonas strains- sylvatic transmission cycle).
This finding suggests a possible correlation between the complexity of
the sylvatic transmission cycle and the diversity of the sylvan
parasites (Fernandes et al. 1999 loc. cit.).
Our approaches to
the understanding of genetic dimorphism in T. cruzi have focused
on two genes, the ribosomal RNA and mini-exon loci. It is clear from
karyotype analysis that the same dimorphic nature of the major lineages
is evident in other chromosomes (Henriksson 1996 loc. cit.).
Furthermore, there is evidence for four or five natural clusterings
within T. cruzi II (RP Souto et al. Mol Biochem Parasitol
83: 141-152, S Brisse et al. 1998 Parasitol Today 14:
178-179). The apparent heterozygosity of the rRNA genes within some of
these subdivisions (Souto et al. loc. cit.) underscores the need
for a more detailed analysis of the genetic constitution of parasites
within the taxon T. cruzi. In parallel with the T. cruzi
genome project, our future experiments will be directed towards
defining other dimorphic markers for additional loci in the T.
cruzi genome. Our goal of is to generate markers from all the
chromosomes to measure genetic variability between the two lineages. A
theoretical "variability" map can be applied to any isolate with known
biological/medical history and may eventually yield a correlation to the
outcome of infection by T. cruzi.
Fig.1 |
Fig.2
| Fig.3