DO REFERENCE ORGANISMS OF GENOME PROJECTS

COVER THE GENETIC DIVERSITY OF PARASITES ?

Bianca Zingales

Dep. of Biochemistry

Institute of Chemistry

University of São Paulo

zingales@iq.usp.br

This talk has three main goals:

• 1- To describe some characteristics of the genome and molecular biology of parasitic protozoa

• 2 - To show you that most parasites present great genetic diversity

• 3 -To discuss how the information generated in Parasite Genome Projects - that employ Reference Organisms - can be used to approach specific problems of parasite isolates

• 1 - Characteristics of Kinetoplatida

(Leishmania, African and American Trypanosomes)

•Parasites have an asexual reproduction

•Parasites are diploid

• Presence of Kinetoplast

Kinetoplast of T. cruzi

•1 - Characteristics of Kinetoplatida

(Leishmania and African and American Trypanosomes)

•Most of the expressed genes have NO introns

• Promoters of RNA polymerase II have not been found

• Transcription occurs in polycistronic pre-mRNAs

•The conversion of these mRNAs into individual messages occurs by the addition of a

mini-exon sequence (spliced leader) to the 5´end o mRNA (trans-splice process)

•Trans-splicing and addition of a poliA tail to the 3´ end of the mRNA are concomitant

processes

Polycistronic mRNA

Mini-exon genes (100-200 copies)

Exon - 39 bp

Intron ~70 bp

Processing of polycistronic mRNAs

Transcription

Intergenic region

medRNA

Coding genes

A B C

Transcription

A B C

A B C

A CAAA B AAAAAA

Trans-splicing

Mature mRNAs

• 2 - Genetic diversity of Parasites:

Trypanosoma cruzi as a model

• Protozoan causative of Chagas disease

• Affects 16 million people in Latin America

• From Mexico to Chile and Argentina

• There are no available drugs for treatment

•There is no vaccine to prevent infection

- -

SYLVATICSYLVATICCYCLECYCLE

DOMESTICDOMESTICCYCLECYCLE

Populations of T. cruzi circulate in two cycles

STRAIN OR STOCK

Any parasite population isolated from the blood of a mammalian host or digestive tract of the insect

The strains are propagated in the laboratory in liquid medium, in experimental animals, or in tissue culture

Biological, biochemical, immunological and genetic parameters are defined for each strain

HETEROGENEITY OF BIOLOGICAL CHARACTERISTICS OF TRYPANOSOMA CRUZI

STRAINS

• MORPHOLOGY

• COURSE OF INFECTION IN MICE

•TISSUE TROPISM

• SUSCEPTIBILITY TO CHEMOTHERAPEUTIC AGENTS

CLINICAL CHARACTERISTICS

OF CHAGAS DISEASE

CHRONIC AND INCURABLE DISEASE

CLINICAL MANIFESTATIONS IN THE CHRONIC PHASE:

INDETERMINATE FORM - 70 - 80%

CARDIAC - 20 - 30%

DIGESTIVE - 10%

NEUROLOGICAL AND MIXED - 1%

Central question: Is there a correlation between the biologicalvariability of the parasite and the clinical manifestations of Chagas Disease ?

Trypanosoma cruzi invading a mammalian cell Carlos Chagas - 1909

DNA AMOUNT VARIES IN STRAINS OF TRYPANOSOMA CRUZI

Dvorak and co-workers, 80’

MOLECULAR TYPING OF KINETOPLASTIDA

BASED ON GENOMIC OR MITOCHONDRIAL DNA (kDNA)

• RFLP - Restriction fragment length polymorphism

• DNA FINGERPRINTING

• RAPD - Randomly amplified polymorphic DNA

• PCR of specific sequences (rRNA genes, mini-exon genes)

STRAINS OF T.cruzi CAN BECHARACTERIZED BY THE PATTERN OF

RESTRICTION ENDONUCLEASEPRODUCTS OF KINETOPLAST DNA

Morel, Chiari, Camargo, Mattei, Romanha& Simpson. PNAS 77, 6810-6814 (1980)

Variable region

T. cruzi minicircleMinicircle population + Restriction Enzyme

(Schizodeme analysis)

Conserved regionAgarose gel

Macedo, Martins, Chiari & Pena

Mol. Biochem. Parasitol. 55, 147-154 (1992)

DNA FINGERPRINTING OF GENOMIC DNA

•Approach

•Genomic DNA digested with restriction enzymes

• Southern blot

• Hybridization with microsatellite labeled probe

RIBO SO M AL RNA G ENES

p ro ka ryo te s

e uka ryo te s

tryp a no so m a tid s

Our group decided to investigate the genetic diversity of T. cruzi using as target the ribosomal RNA genes (markers for phylogeny)

18S S3 24S S1 24S S2 S6 S4

Cui

ca

cl1

TU18

cl1

MN

cl2

NR

cl3

SC43

cl1

SO3

cl5

Sylv

ioX

10 c

l1P2

09 c

l1C

L Br

ene

r

Esq

uilo

cl1

P11

cl3

OPS

21 c

l11

Bug

2149

cl1

0Es

me

rald

o c

l3

Tula

hue

n

M22

6

- 110-b p - 125-b p

G Y

D71

D72

PC R AM PLIFIC ATIO N O F 24S rRNA G ENE

•Definition of three groups of strains: group 1, 125 bp;

group 2, 110 bp;

group 1/2, 125 and 110 bp

CLB167CA1B147SC43 cl1Bug2149 cl10NR cl3SO3 cl5

Esmeraldo cl3Y

BasileuA1381023115226

GDm28

TulahuenSilvioX10cl1YuYu10171001100410091018

111111111111111

2222222222

ME rDNA

111

1/22

1/21/21/21111

1/21/21/2

2222222222

0.65 0.40 0.20 0.00

T. cruzi II

T. cruzi I

Ribosomal RNA and Mini-Exon gene sequences and RAPD analysis define two major phylogenetic lineages of

T. cruzi

Souto, Fernandes, Macedo, Campbell and Zingales

Mol. Biochem. Parasitol. (1996)

Two Lineages of Trypanosoma cruzi

• Biological Meaning?

• Epidemiological distribution?

• Pathogenesis?

Molecular Molecular epidemiology ofepidemiology ofChagas Chagas disease in Brazildisease in Brazil

Amazonas

Pará Piauí

Paraíba

BahiaGoiás

Espírito Santo

Rio de JaneiroSão Paulo

Rio Grande do Sul

Santa Catarina

Minas Gerais

- -

SILVATIC CYCLESILVATIC CYCLESILVATIC CYCLESILVATIC CYCLE DOMESTIC CYCLEDOMESTIC CYCLEDOMESTIC CYCLEDOMESTIC CYCLE

T. cruzi T. cruzi IIIIT. cruzi T. cruzi IIIIT. cruzi T. cruzi I (and II)I (and II)T. cruzi T. cruzi I (and II)I (and II)

Chagas Disease

Fernandes et al., Am. J. Trop. Med Hyg. 58: 807-811, 1998

Zingales et al., Int. J. Parasitol. 28: 105-112, 1998

ANALYSIS OF 160 STRAINS FROM 12 STATES OF BRAZIL

T. cruzi II

Parasite Genome Projects

Launched by TDR/WHO at FIOCRUZ (Rio de Janeiro) 1994

Reference Organism: T. cruzi CL-Brener

• Origin :– Isolated from Triatoma infestans

• Characteristics : – belongs to T. cruzi II (domestic cycle)– shows clear acute phase in mice (and accidentally infected humans)– shows chronic phase in mice, with preference for heart and muscle cells– is highly susceptible to drugs used against Chagas disease– differentiates efficiently to metacyclics in-vitro– isoenzyme profile, schizodeme and RAPD patterns, and karyotype are stable

for at least 100 generations– haploid genome size: 43.5 Mb

Sequencing of T. cruzi Genome -(CL Brener)

(October 2000)

• 10,000 ESTs

• 12,000 GSS

• 900 other sequences

• partial sequence of chromosome 3

• Approximately 50% of the genes of unknown function

mRNA POPULATION

cDNA LIBRARY CONSTRUCTION

(LIBRARY NORMALIZATION)

RANDOM SELECTION OF CLONES

PARTIAL SEQUENCING OF 5´ ENDS

AUTOMATIC SEQUENCING

EST (EXPRESSED SEQUENCE TAGS)

Sequencing of Chromosome 3 of Trypanosoma cruzi (93.4 kb)

•20 - 30 novel genes and several repeat elements

•Two long clusters , transcribed in opposite directions

•Separated by an ~20-kb long, GC-rich sequence

Analogous situation was found for chromosome 1 of Leishmania major (257 kb)

• 79 protein coding genes

• 29 genes encoded on one strand; 50 genes on the opposite strand

Molecular Karyotype of T. cruzi strains

• Verify chromosome polymorphism among strains

• Establish gene linkage groups

• Compare molecular karyotype of T. cruzi lineages

• Establish molecular markers for chromosome sequencing of CL Brener

3.5-

1.9-

1.6-

1.1-

0.6-

0.4-

Mbp

|T. cruzi II ||Group1/2| |T. cruzi I |

Molecular Karyotype of T. cruzi strainsChromosome separation by PFGE

Molecular Karyotype

• PFGE of chromosomal DNA

• Transfer of DNA to nylon membranes - Southern blot

• Labeling of DNA probes (ESTs) with alpha P32 d-ATP

• Hybridization

• Autoradiography

Methodological Approach

Tc II TcI1/2

MOLECULAR KARYOTYPE OF STRAINS AND CLONES OF Trypanosoma cruzi

Conclusions

•Polymorphism in the molecular karyotype of the strains

• Definition of chromosome markers for genome sequencing

•Homologous chromosomes may have different sizes

• Other non-published observations…..

Central question: Definition of genetic markers of the strains causative of different clinical manifestations of Chagas Disease Application:Prognosis and potential targets for treatment

Trypanosoma cruzi invading a mammalian cell Carlos Chagas - 1909

Microarray Technology

Differential gene expression in T. cruzi strains isolated from patients with different manifestations of Chagas disease

*Pop 1 *Pop 2

(ESTs and cloned genes)

Preparation of target DNAs

• PCR amplification of ESTs

• Purification of the amplified products

Application on the glass slides

Hybridization with Cy5 and Cy3 cDNA populations

Microarray technology can also be employed to investigate the representativeness of genes in the genome of two populations of strains (presence, absence, copy number, etc.)

We hope to have interesting results in the near future !!!!!