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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).

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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



Wedecided to perform a haplotypic studyof nuclear lineagemarkers, microsatellitesand mtDNA sequencevariation in 75 clonal strainsofTrypanosomacruziand to use theresults to establishthestructureof its geneticdiversityand to makeinferencesabout its evolutionarypast


Microsatellites

allele A

allele B

allele C

AA

AB

AC

BB

BC

CC


Microsatellites
Microsatellites

  • Abundant

  • Mutation rates ~10-3-10-4

  • Multiallelic

  • High heterozygosity


Dinucleotides

47,6%

Trinucleotideo

38,6%

Tetranucleotides

13,7%

Aproximately 1% of the T. cruzi genome is made up of microssatellites





1 0 1 0 0 0 1 1

84

1 0 0 1 1 1 0 1

115

1 0 0 1 1 1 0 0

138

1 1 0 1 0 1 0 1

150

0 0 0 0 1 1 0 0

167

1 0 1 0 1 1 0 0

182

115

138

150

167

182

84

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

Allele sizes

Clone

alleles

84

267/269

FACTOR

115

273/279

138

267/271

150

259/259

167

263/277

182

269/273

MIX

Matrix of genetic distances

75 clones

5 microsatellites

MDS

VisualizationofthedistancematrixwithMDS


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 microsatellitesand 25alpharDNA SNP

Construct haplotypic network basedon the knownmutation models of themarkers

141haplotypes


Haplogroup Y

Haplogroup Z

Haplogrupo I

Haplogroup X


Hybrids

(DTU2e)

DTU 2a

T. cruzi I

(DTU 1)

T. cruziII(DTU2b)

T. cruziII(DTU2b)

T. cruzi III(DTU 2c)

Hybrids

(DTU2d)


Wedecided to perform a haplotypic studyof nuclear lineagemarkers, microsatellitesandmtDNA sequencevariation in 75 clonal strainsofTrypanosomacruziand to use theresults to establishthestructureof its geneticdiversityand to makeinferencesabout its evolutionarypast


~300pb DNA sequencing

in 41 T. cruzi strains

COII


CytB

Brisse et al., 2003


NADH1

Machado & Ayala, 2001


MDS C

MDS C

MDS C

T. cruzi II

T. CruziIII /

Hybrids

T. cruzi I

MDS B/BH

MDS B/BH

MDS A

MDS B/BH

MDS A

MDS A


Hybrids

T. cruzi II

T. cruzi III



3 Haplogroups

3 Mitochondrial Haplogroups



Locus MCLG10

T. cruzi I

T. cruzi II

T. cruzi III


Conclusions
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.





TcTAC15 TcTAT20

TcAAT8 TcGAG10

TcCAA10 TcATT14

TcAAAT6

Seleção de novos locosde microssatélites

Pimenta, 2002


Wedecided to perform a haplotypic studyof nuclear lineagemarkers, microsatellitesand mtDNA sequencevariation in 75 clonal strainsofTrypanosomacruziand to use theresults to establishthestructureof its geneticdiversityand to makeinferencesabout its evolutionarypast


Sensibilidade da full-nested PCR

Locus TcGAG10

Branco

Branco

Branco

Branco

Branco

Branco

100pg

100pg

100pg

100pg

100pg

100pg

100fg

100fg

100fg

100fg

100fg

100fg

10pg

10pg

10pg

10pg

10pg

10pg

10fg

10fg

10fg

10fg

10fg

10fg

1ng

1ng

1pg

1pg

1ng

1ng

1pg

1pg

1ng

1ng

1pg

1pg

PM

PM

Iniciadores externos

Iniciadores internos

Full nested PCR

Enriquecimento de 100 a 1000 vezes na sensibilidade da PCR


Epimastigotes

Microplates of 96 wells

Sorting of single cells by FACS

Automatic DNA sequencer

Microsatellite PCR

Electrophoresis


População 2

População 1

T. cruzi I (10%)

T. cruzi II (90%)


1909

1979


A

D

B

C

1A

1B

1C

1D

Veloso et al. (2005)

25A

25B

25D

25C

TcTAT20

70% 30%



ThechastelifeofT. cruziadaptedfrom Juliana R. Pimenta

Mom,

Go to

May I go out with

your room!

T. odore?

No!

Go clone yourself!

But I have nothing to do...

What a pain...

.

In the room...

Mom,

May I go out with

No!

T. seus?

Cloning,

Cloning

Cloning...


Mãe,

Next day....

Then...

Vá para o

Ok, onlytoday

Posso sair com o

seu quarto!

T. odoro?

não!

Mom, may Igo out

Pleeeaase!

Deixe de ser

Mas eu não

Droga, todo

chata!

tenho nada

dia, só

clonar

...

se clonar

.

prá

fazer...

Cloning...

Later in the night

Moral

Sex is rare in the life of

Trypanosomes, but now and then it happens!

No quarto...

2 dias depois...

Mãe, posso

Não.

sair com o

clonar

!

T.obaldo?

Saco!

Clonagem,

clonagem,

clonagem ...


Z3 – Miles, 1978

ZB – Romanha, 1979

T. cruzi?

Group 1/2 – Souto e cols, 1996

Strainswithdivergences

Z1 - Miles, 1977

Lineage 2 – Souto e cols., 1996

T. cruziI

Group 1 - Tibayrenc, 1995

Ribodemes II/III - Clark & Pung, 1994

T. cruzi

Z2 - Miles, 1977

ZA – Romanha, 1979

Lineage 1 – Souto e cols., 1996

T. cruziII

Group 2 - Tibayrenc, 1995

Ribodeme I - Clark & Pung, 1994


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 chromosome

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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