DO REFERENCE ORGANISMS OF GENOME PROJECTS COVER THE GENETIC DIVERSITY OF PARASITES ? Bianca Zingales...
Transcript of DO REFERENCE ORGANISMS OF GENOME PROJECTS COVER THE GENETIC DIVERSITY OF PARASITES ? Bianca Zingales...
DO REFERENCE ORGANISMS OF GENOME PROJECTS
COVER THE GENETIC DIVERSITY OF PARASITES ?
Bianca Zingales
Dep. of Biochemistry
Institute of Chemistry
University of São Paulo
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
•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
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
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
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