genomic diversity in trypanosoma cruzi : evolutionary and pathogenetic implications
DESCRIPTION
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). - PowerPoint PPT PresentationTRANSCRIPT
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
Aproximately 1% of the T. cruzi genome is made up of microssatellites
Dinucleotides
Trinucleotideo
Tetranucleotides
47,6%
38,6%
13,7%
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
MDS
Visualization of the distance matrix with MDS
FACTOR
84
115
138
150
167
182
1 0 1 0 0 0 1 1
1 1 0 1 0 1 0 1
1 0 0 1 1 1 0 11 0 0 1 1 1 0 0
0 0 0 0 1 1 0 0
1 0 1 0 1 1 0 0
MIX
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
84 115 138 150 167 182
Matrix of genetic distances75 clones
5 microsatellites
Allele sizes
Clone alleles
84 267/269
115 273/279
138 267/271
150 259/259
167 263/277
182 269/273
Cluster AT. cruzi I
Cluster CT. cruzi IIb
Cluster BT. cruzi IIc
Cluster BHT. cruzi IId + IIe
T. cruzi IIa
Transform multilocal genotypes
intohaplotypes
Input typing of microsatellitesand 25alpharDNA SNP
Construct haplotypic
network basedon the known
mutation models of the
markers
141haplotypes
T. cruzi I(DTU 1)
T. cruzi II(DTU 2b)
T. cruzi II(DTU 2b)T. cruzi III
(DTU 2c)
DTU 2a
Hybrids(DTU 2e)
Hybrids(DTU 2d)
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
MDS A
MDS A
MDS A
T. cruziT. cruzi I I
MDS C MDS CMDS C
T. cruziT. cruzi II II
MDS B/BH MDS B/BHMDS B/BH
T. Cruzi T. Cruzi IIIIII / /HybridsHybrids
T. cruzi IT. cruzi I
ZZ
I
A
A
T. cruzi IIT. cruzi II
YY
IIb
C
C
T. cruzi IT. cruzi I
ZZ
I
A
A
T. cruzi IIT. cruzi II
YY
IIb
C
C
HibridosHibridos
XY
IId/IIe
B
BH
T. cruzi IIIT. cruzi III
XX
IIc
B
B
T. cruzi IIIT. cruzi III
Doa
dor
Receptor
DNA mitocondrial
DNA mitocondrial
Grupos por MDS
Haplótipos
RAPD / MLEE
DNA mitocondrial
Grupos por MDS
Haplótipos
RAPD / MLEE
XX
IIc
B
B
T. cruzi IT. cruzi I
ZZ
I
A
A
T. cruzi IIT. cruzi II
YY
IIb
C
C
T. cruzi IT. cruzi I
ZZ
I
A
A
T. cruzi IIT. cruzi II
YY
IIb
C
C
HibridosHibridos
XY
IId/IIe
B
BH
T. cruzi IIIT. cruzi III
XX
IIc
B
B
T. cruzi IIIT. cruzi III
Doa
dor
Receptor
DNA mitocondrial
DNA mitocondrial
Grupos por MDS
Haplótipos
RAPD / MLEE
DNA mitocondrial
Grupos por MDS
Haplótipos
RAPD / MLEE
XX
IIc
B
B
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)
T. cruzi IT. cruzi I
ZZ
I
A
A
T. cruzi IIT. cruzi II
YY
IIb
C
C
T. cruzi IT. cruzi I
ZZ
I
A
A
T. cruzi IIT. cruzi II
YY
IIb
C
C
HibridosHibridos
XY
IId/IIe
B
BH
T. cruzi IIIT. cruzi III
XX
IIc
B
B
T. cruzi IIIT. cruzi III
Doa
dor
Receptor
DNA mitocondrial
DNA mitocondrial
Grupos por MDS
Haplótipos
RAPD / MLEE
DNA mitocondrial
Grupos por MDS
Haplótipos
RAPD / MLEE
XX
IIc
B
B
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.
Seleção de novos locos de microssatélites
TcTAC15
TcTAT20
TcAAT8
TcGAG10
TcCAA10
TcATT14
TcAAAT6
Pimenta, 2002
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
Locus TcGAG10
PM 1n
g
100p
g
10p
g
1pg
100f
g
10fg
1ng
100p
g
10p
g
1pg
100f
g
10fg
1ng
100p
g
10p
g
1pg
100f
g
10fg
Bra
nco
Bra
nco
Bra
nco
PM 1n
g
100p
g
10p
g
1pg
100f
g
10fg
1ng
100p
g
10p
g
1pg
100f
g
10fg
1ng
100p
g
10p
g
1pg
100f
g
10fg
Bra
nco
Bra
nco
Bra
nco
Iniciadores externos Iniciadores internos Full nested PCR
Enriquecimento de 100 a 1000 vezes na sensibilidade da PCR
Microplates of 96 wells
Epimastigotes
Microsatellite PCR
Electrophoresis
Automatic DNA sequencer
Sorting of single cells by FACS
A B C D
1A 1B 1C 1D
25A 25B 25C 25D
Veloso et al. (2005)
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
TcTAT20
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
1 célula
1 célula
1 célula
1 célula
208
181
181
190
190
208
DNA Be-62 parental
DNA Be-78 parental
DNA Be-78 25B
190
196 205
190208
181
208
(A)
(B)
70% 30%
But I have nothing to do... .
In the room...
Cloning,Cloning
Cloning...
Mom,May I go out with T. odore?
No!
Go to your room!
The chaste life of T. cruzi adapted from Juliana R. Pimenta
Mom,May I go out with T. seus? No!
Go clone yourself!
What a pain...
Mas eu nãotenho nadaprá fazer...
Deixe de serchata!Vá se clonar.
Droga, tododia, só clonar...
No quarto...
Clonagem,clonagem,
clonagem ...
2 dias depois...
Não.Vá clonar!
Mãe, possosair com o T.obaldo?
Saco!
Mãe,Posso sair com o
T. odoro?
não!
Vá para o
seu quarto!
MoralSex is rare in the life ofTrypanosomes, but now
and then it happens!
Then...
Cloning...
Next day....
Mom, may Igo outPleeeaase!
Ok, onlytoday
Later in the night
Z1 - Miles, 1977
T. cruziT. cruzi
T. cruzi T. cruzi II
T. cruzi T. cruzi IIII
Lineage 2 – Souto e cols., 1996
Group 1 - Tibayrenc, 1995
Ribodemes II/III - Clark & Pung, 1994
ZA – Romanha, 1979
Lineage 1 – Souto e cols., 1996
Z2 - Miles, 1977
Group 2 - Tibayrenc, 1995
Ribodeme I - Clark & Pung, 1994
Z3 – Miles, 1978Z3 – Miles, 1978
Group 1/2 – Souto e cols, 1996Group 1/2 – Souto e cols, 1996T. cruzi T. cruzi ? ? ZB – Romanha, 1979ZB – Romanha, 1979
Strains with divergencesStrains with divergences
T. cruzi is diploid, with different-sized homologous
chromosomepairs. Its genome size
(diploid) has been estimated between 106.4and 110.7 Mb