Presentation is loading. Please wait.

Presentation is loading. Please wait.

Genomic diversity in Trypanosoma cruzi : Evolutionary and pathogenetic implications.

Similar presentations


Presentation on theme: "Genomic diversity in Trypanosoma cruzi : Evolutionary and pathogenetic implications."— Presentation transcript:

1 Genomic diversity in Trypanosoma cruzi : Evolutionary and pathogenetic implications

2 Carlos Chagas

3 Charles Darwin

4 1909 A parasitic disease One single parasite One single form of transmission (vectorial)

5 1909 One single form of transmission (vectorial)

6 Ecobiology of Trypanosoma cruzi

7 1909 One single parasite

8

9 From Buscaglia & DiNoia (2003)

10

11

12

13

14 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

15 Microsatellites allele A allele B allele C AA ABACBBBCCC

16 Microsatellites Abundant Mutation rates ~ Multiallelic High heterozygosity

17 Aproximately 1% of the T. cruzi genome is made up of microssatellites Dinucleotides Trinucleotideo Tetranucleotides 47,6% 38,6% 13,7%

18

19

20

21 etc.

22 Conversão Gênica

23

24 Quanta atividade sexual ocorreu no passado e ocorre hoje?

25 MDS Visualization of the distance matrix with MDS FACTOR MIX Matrix of genetic distances 75 clones 5 microsatellites Allele sizes Clonealleles / / / / / /273

26 Cluster A T. cruzi I Cluster C T. cruzi IIb Cluster B T. cruzi IIc Cluster BH T. cruzi IId + IIe T. cruzi IIa

27

28

29

30

31

32 Transform multilocal genotypes into haplotypes Input typing of microsatellites and 25alpha rDNA SNP Construct haplotypic network based on the known mutation models of the markers 141 haplotypes

33 Haplogroup Z Haplogroup Y Haplogroup X Haplogrupo I

34 T. cruzi I (DTU 1) T. cruzi II (DTU 2b) T. cruzi III (DTU 2c) DTU 2a Hybrids (DTU 2e) Hybrids (DTU 2d)

35 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

36

37

38 COII ~300pb DNA sequencing in 41 T. cruzi strains

39 Brisse et al., 2003 CytB

40 Machado & Ayala, 2001 NADH1

41 MDS A T. cruzi I MDS C T. cruzi II MDS B/BH T. Cruzi III / Hybrids

42

43 Hybrids T. cruzi II T. cruzi III

44 Westenberger et al, 2006

45 3 Mitochondrial Haplogroups 3 Haplogroups

46 Variance of allele size in five microsatellites

47 T. cruzi I T. cruzi II T. cruzi III Locus MCLG10

48

49

50

51

52

53

54 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.

55 The end

56 Microssatélite SCLE10

57

58

59

60

61 LSSP-PCR

62 Seleção de novos locos de microssatélites TcTAC15 TcTAT20 TcAAT8 TcGAG10 TcCAA10 TcATT14 TcAAAT6 Pimenta, 2002

63 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

64

65

66

67 Sensibilidade da full-nested PCR Locus TcGAG10 PM 1ng100pg 10pg 1pg100fg 10fg 1ng100pg 10pg 1pg100fg 10fg 1ng100pg 10pg 1pg100fg 10fg Branco PM 1ng100pg 10pg 1pg100fg 10fg 1ng100pg 10pg 1pg100fg 10fg 1ng100pg 10pg 1pg100fg 10fg Branco Iniciadores externosIniciadores internosFull nested PCR Enriquecimento de 100 a 1000 vezes na sensibilidade da PCR

68 Microplates of 96 wells Epimastigotes Microsatellite PCR Electrophoresis Automatic DNA sequencer Sorting of single cells by FACS

69 População 1 T. cruzi II (90%) População 2 T. cruzi I (10%)

70

71 A BC D 1A1B1C1D 25A25B 25C 25D Veloso et al. (2005) TcTAT20 70% 30%

72 Microsatellite SCLE10

73 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...

74 Mas eu não tenho nada prá fazer... Deixe de ser chata! Vá se clonar. Droga, todo dia, só clonar... No quarto... Clonagem, clonagem, clonagem... 2 dias depois... Não. Vá clonar! Mãe, posso sair com o T.obaldo? Saco! Mãe, Posso sair com o T. odoro? não! Vá para o seu quarto! Moral Sex is rare in the life of Trypanosomes, but now and then it happens! Then... Cloning... Next day.... Mom, may I go out Pleeeaase! Ok, only today Later in the night

75 Z1 - Miles, 1977 T. cruzi T. cruzi I T. cruzi II 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, 1978 Group 1/2 – Souto e cols, 1996 T. cruzi ? ZB – Romanha, 1979 Strains with divergences

76 T. cruzi is diploid, with different-sized homologous chromosome pairs. Its genome size (diploid) has been estimated between and Mb

77

78 T. cruzi is diploid, with different-sized homologous chromosome pairs. Its genome size (diploid) has been estimated between and Mb 20-25% total cell DNA Few maxicircles ~22Kb > 10 4 minicircles


Download ppt "Genomic diversity in Trypanosoma cruzi : Evolutionary and pathogenetic implications."

Similar presentations


Ads by Google