Genomic diversity in
Trypanosoma cruzi :
Evolutionary and pathogenetic
implications
Carlos Chagas
1879-1934
Charles Darwin
1809-1882
1909
• A parasitic disease
• One single parasite
• One single form of
transmission (vectorial)
1909
• One single form of
transmission (vectorial)
Ecobiology of Trypanosoma cruzi
1909
• One single parasite
From Buscaglia & DiNoia (2003)
Trypanosoma
cruzi
DTU 1
DTU 2
DTU 2a
(CanIII cl1)
DTU 2b
(Esmeraldo)
DTU 2c
(M6241 &
M5631)
DTU 2d
(Bolivian Z2)
DTU 2e
(CL Brener)
We decided to perform a haplotypic
study of nuclear lineage markers,
microsatellites and mtDNA sequence
variation in 75 clonal strains of
Trypanosoma cruzi and to use the
results to establish the structure of its
genetic diversity and to make
inferences about its evolutionary past
Microsatellites
allele A
allele B
allele C
AA
AB
AC
BB
BC
CC
Microsatellites
• Abundant
• Mutation rates ~10-3-10-4
• Multiallelic
• High heterozygosity
Genoma Anotado CL Brener
Tipo de Repetição Número de loci
Dinucleotídeos
AC
2430
AT
1955
AG
1280
CG
0
Trinucleotídeos
AAT
1830
AAC
1245
AGC
487
ACC
274
AGG
266
AAG
223
ATC
151
ACT
52
ACG
38
CCG
29
Tetranucleotídeos*
AAAT
785
AAAC
371
AAAG
118
ACAT
86
ACAG
68
AGGG
41
AGAT
29
AAGG
28
ACCC
17
AACC
14
ACTC
13
AGGC
11
ACGC
10
ATCC
10
AATT
9
AATG
8
AATC
6
AGCC
3
AAGC
2
ATGC
2
AACT
1
Total
11.892
Aproximately 1% of the T. cruzi
genome is made up of microssatellites
Dinucleotides
47,6%
Trinucleotideo
38,6%
Tetranucleotides
13,7%
etc.
Conversão Gênica
Quanta atividade sexual ocorreu no passado
e ocorre hoje?
Allele sizes
Clone
alleles
84
10100011
84
267/269
115
115
273/279
138
10011101
10011100
138
267/271
150
259/259
150
11010101
167
263/277
167
00001100
182
269/273
182
10101100
FACTOR
MIX
Matrix of genetic distances
75 clones
5 microsatellites
84 115 138 150 167 182
MDS
Visualization of the
distance matrix with MDS
84 0 2
7
3
5 4
115 2 0
4
1
9 3
138 7 4
0
5
6 1
150 3 1
5
0
8 9
167 5 9
6
8
0 3
182 4 3
1
9
3 0
Cluster B
T. cruzi IIc
T. cruzi IIa
Cluster BH
T. cruzi IId + IIe
Cluster C
T. cruzi IIb
Cluster A
T. cruzi I
Transform
multilocal
genotypes
into
haplotypes
Input typing of
microsatellites
and 25alpha
rDNA SNP
141
haplotypes
Construct
haplotypic
network based
on the known
mutation
models of the
markers
Haplogroup Y
Haplogroup Z
Haplogrupo I
Haplogroup X
Hybrids
(DTU 2e)
DTU 2a
T. cruzi I
(DTU 1)
T. cruzi III
(DTU 2c)
Hybrids
(DTU 2d)
T. cruzi II
(DTU 2b)
T. cruzi II
(DTU 2b)
We decided to perform a haplotypic
study of nuclear lineage markers,
microsatellites and mtDNA sequence
variation in 75 clonal strains of
Trypanosoma cruzi and to use the
results to establish the structure of its
genetic diversity and to make
inferences about its evolutionary past
~300pb DNA sequencing
in 41 T. cruzi strains
COII
CytB
Brisse et al., 2003
Machado & Ayala, 2001
NADH1
MDS C
MDS C
MDS C
T. cruzi II
T. Cruzi III /
Hybrids
MDS B/BH
T. cruzi I
MDS A
MDS B/BH
MDS A
MDS B/BH
MDS A
T. cruzi II
Hybrids
T. cruzi III
Westenberger et al, 2006
3 Haplogroups
3 Mitochondrial Haplogroups
Variance of allele size in five microsatellites
Locus MCLG10
T. cruzi I
T. cruzi II
T. cruzi III
Trypanosoma
cruzi
DTU 1
DTU 2
DTU 2a
(CanIII cl1)
DTU 2b
(Esmeraldo)
DTU 2c
(M6241 &
M5631)
DTU 2d
(Bolivian Z2)
DTU 2e
(CL Brener)
Conclusions
• There is concrete evidence for the existence of
a third phylogenetic lineage within the
Trypanosoma cruzi species, which we propose
to call T. cruzi III
• The nomenclature of Brisse et al. (2000) does
not reflect accurately the sistematic
relationship of the lineages and should be
abandoned
• At least according to criteria of genetic
differentiation, there is no evidence that T. cruzi
is more than one single species.
The end
Microssatélite SCLE10
LSSP-PCR
Seleção de novos locos de microssatélites
TcTAC15
TcTAT20
TcAAT8
Pimenta, 2002
TcGAG10
TcCAA10
TcATT14
TcAAAT6
We decided to perform a haplotypic
study of nuclear lineage markers,
microsatellites and mtDNA sequence
variation in 75 clonal strains of
Trypanosoma cruzi and to use the
results to establish the structure of its
genetic diversity and to make
inferences about its evolutionary past
Sensibilidade da full-nested PCR
Iniciadores externos
Iniciadores internos
10fg
100fg
1pg
10pg
100pg
1ng
Branco
10fg
100fg
1pg
10pg
100pg
1ng
Branco
10fg
100fg
1pg
10pg
100pg
1ng
Branco
PM
Locus TcGAG10
Full nested PCR
Enriquecimento de 100 a 1000 vezes na sensibilidade da PCR
Epimastigotes
Sorting of single cells by
FACS
Automatic DNA
sequencer
Electrophoresis
Microplates of 96 wells
Microsatellite PCR
População 1
População 2
T. cruzi II
(90%)
T. cruzi I
(10%)
1909
1979
B
DNA Be-62
parental
A
190
208
1 célula
B
C
D
1B
1C
1D
25B
25C
25D
190
208
DNA Be-62
DNA
Be-78
parental
parental
190
1 célula
208
196
190
205
190
208
208
1 célula
DNA Be-78
parental
181
196
205
190181
DNA Be-78 25B
1 célula
1A
208
TcTAT20
190
208
1 célula
181
DNA Be-78 25B
181
DNA
Be-62 190
parental
190
208
190
1 célula
1 célula
181
208
208
(A)
25A
1 célula
181
DNA Be-78
parental
196
205
190
(A)
70%
190
DNA Be-78 25B
190
(B)
30%
1 célula
181
1 célula
208
1 célula
(B)
181
208
208
1 célula
1 célula
181
190
(A)
208
1 célula
181
(B)
Veloso et al. (2005)
Microsatellite SCLE10
The chaste life of T. cruzi
Mom,
May I go out with
T. odore?
adapted from Juliana R. Pimenta
Go to
your room!
No!
But I have
nothing to
do...
Go clone
yourself!
.
What a pain...
In the room...
Mom,
May I go out with
T. seus?
Cloning,
Cloning
Cloning...
No!
Mãe,
Posso sair com o
T. odoro?
Then...
Next day....
Vá para o
Ok, only
seu quarto!
today
não!
Cloning...
Mas eu não Deixe de ser
tenho nada chata!
.
prá fazer... Vá se clonar
Mom, may I
go outDroga, todo
dia, só clonar...
Pleeeaase!
Later in the night
No quarto...
Moral
Sex is rare in the life of
Trypanosomes,
but now
Saco!
and then it happens!
2 dias depois...
Mãe, posso
Não.
sair com o
Vá clonar!
T.obaldo?
Clonagem,
clonagem,
clonagem ...
Z1 - Miles, 1977
T. cruzi I
Lineage 2 – Souto e cols., 1996
Group 1 - Tibayrenc, 1995
Ribodemes II/III - Clark & Pung, 1994
T. cruzi
Z2 - Miles, 1977
ZA – Romanha, 1979
T. cruzi II
Lineage 1 – Souto e cols., 1996
Group 2 - Tibayrenc, 1995
Ribodeme I - Clark & Pung, 1994
Z3 – Miles, 1978
T. cruzi ?
ZB – Romanha, 1979
Group 1/2 – Souto e cols, 1996
Strains with divergences
T. cruzi is diploid, with
different-sized homologous
chromosome
pairs. Its genome size
(diploid) has been estimated
between 106.4
and 110.7 Mb
20-25% total cell DNA
Few maxicircles ~22Kb
> 104 minicircles
T. cruzi is diploid, with
different-sized homologous
chromosome
pairs. Its genome size
(diploid) has been estimated
between 106.4
and 110.7 Mb
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