Genomic diversity in Trypanosoma cruzi :

Evolutionary and pathogenetic implications

Carlos Chagas1879-1934

Charles Darwin1809-1882

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

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

etc.

Conversão Gênica

Quanta atividade sexual ocorreu no passado e ocorre hoje?

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

Haplogroup Z

Haplogroup Y

Haplogroup X

Haplogrupo I

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

COII

~300pb DNA sequencing in 41 T. cruzi strains

Brisse et al., 2003

CytB

Machado & Ayala, 2001NADH1

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

HybridsHybrids

T. cruziT. cruzi II II

T. cruziT. cruzi III III

Westenberger et al, 2006

3 Mitochondrial Haplogroups

3 Haplogroups

Variance of allele size in five microsatellites

T. cruzi I

T. cruzi II

T. cruzi III

Locus MCLG10

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.

The end

Microssatélite SCLE10

LSSP-PCR

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

População 1

T. cruzi II (90%)

População 2

T. cruzi I (10%)

1909

1979

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%

Microsatellite SCLE10

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

T. cruzi is diploid, with different-sized homologous

chromosomepairs. Its genome size

(diploid) has been estimated between 106.4and 110.7 Mb

20-25% total cell DNAFew maxicircles ~22Kb

> 104 minicircles