1. Plasmodium telomeres: a pathogens’ perspective
I’d first of all I’d like to thank you for inviting me here to talk to you today.
I’m going to present my work on the telomere biology of Plasmodium falciparum and the advantages it presents for the parasite.

2. Plasmodium falciparum life cycle
The Plasmodium falciparum life cycle is very complex as I’m sure most of you know. I’d just like to point out one part of the cycle that we’re working on here. This is the asexual, erythrocytic cycle which occurs in the human blood stream and causes disease.
(this part of the cycle has 3 different morphological stages. The ring stage, the trophozoite stage and the the mature schizont stage. The schizont ruptures releasing merozoites which invade new red blood cells and continue the cycle)

3. Knob structure
This is a schematic representation of a knob. You can see the modified surface of the red blood cell
and the knob structures composed of proteins expressed by the parasite.
Here is PfEMP-1, a major player in surface antigenic variation. This molecule is resonsible for adhesion of the infected RBC’s to the capillary vessels.....
Cooke et al., 2OOO

4. sub-telomeric organization
Antigen
genes
Antigen
genes
Telomere
TELOMERE ASSOCIATED REGION
rep 20
Exon I
Exon II
var B
1kb
ATS
cytoplasmic
region
DBL-a
CIDR
DBL-b
DBL-g
DBL-e C
The PfEMP1 molecule, responsible for cytoadherence is encoded by the var gene family
In this chromosome model you can see that the var genes, like other multigene families are
located in the sub telomeric region of the chromosomes.
Shown in more detail: Telomere and the non-coding rep 20 region are indicated
followed by members of gene families like the var, rif and stevor. The order of
gene families seems to be conserved for most if not all chromosome ends.
This is the detailed the var gene structure:
The extra cellular variable domains are encoded by exon I. and the intracellular
domain by exon II. The DBL and CIDR domains have been show to mediate
various adhesive phenotypes during infection in the host.
DBL- can be amplified virtually all var genes using conserved primers.

5. powerful mechanism for generating
Approx. 50 var genes per haploid genome
Tremendous diversity in var gene repertoires
in field isolates
HB3
Dd2
3D7
powerful mechanism for generating
diversity

6. Does the nuclear architecture of P. falciparum have a role in
var gene recombination?

7. Fluorescent In Situ Hybridisation (FISH)
Fluorescent labled
DNA probes (>1-2 kb)
Nucleus
(DAPI)
Slide

8. Telomere Clustering in P. falciparum :
SEXUAL STAGES
TAS
telomere
ectopic
recombination
virulence factor
genes
Recent studies in Pf. Using FISH have given some idea about the 3 dimentional organization of the nucleus.
Here for example FISH has been perfomed using a telomeric probe on a young asexual trophozoite. You can see that the telomeres form clusters, anchoring the chromosomes to the nuclear periphery.
However the same analysis in sexual stages revels majors changes in nuclear arquitecture.
You can see the clusters concentrated at one pole of the elongated nucleus.
However, we don’t know if both chr ends are found in the same cluster or in different clusters.
In order understand this process better we investigated the chromosome organization
in the Pf nuclei.
ASEXUAL STAGES

9. Epigenetic var gene regulation

10. Molecular mechanism of var gene expression and switching
14 linear
chromosomes
Internal region

11. Does the sub-nuclear architecture have a role in var gene regulation ?
var a
var b
var gene expression
var c
var d
var e
Rings
Trophozoites
Schizonts

12. Chromatin architecture in P. falciparum

13. Chromosome 2 painting
130 Kb
170 Kb
Nuclear periphery
10 Kb
150 Kb
The chromosome ends are compact and concentrated at the nuclear perifery forming clusters.

14. Nuclear compartmentalization of
chromosome 2 A B

15. Model of a chromosome end
Rap 1
nuclear
pore
Taz 1
Rif 1
nuclear
membrane
Sir 2-4
Ku 70
Saccharomyces cereviciae is so far the best model for telomere biology.
We know that silencing factors, such as Sir 2,3, and 4 are concentrated at the nuclear perifery.
and that these telomere binding proteins have an important role in epigenetic regulation of gene
expression and nuclear architecture.
Using the Pf data base we found orthologues to S cerevisiae telomere associated proteins.
Gene INactivation experiments are underway to study the specific role of these proteins.
Knockout mutations of these proteins in yeast are non lethal and we hope that this is also the
case in PF
In parallel we have also produced GST fusion proteins of these candidates and made antibodies
against most of them.
Mlp complex
telomere

16. Immunolocalisation Sir 2-13 Sir 2-14 Sir 4 Sir 3 Ku 70 Nop I TELOMERE
FISH
These are the first results using these antibodies. As expected the proteins are found concentrated
in clusters at the nuclear perifery.
This opens new feilds in the studies of Pf biology and in the very near future we hope to
understand the more about the nuclear architecture and it’s role in epigentic regulation of
virulence factor genes.
Sir 3
Ku 70
Nop I

17. Typical chromosome end of Plasmodium falciparum
Sir complex in Saccharomyces cerevisiae
inactive
active
-ac
heterochromatin
Histone hyperacetylation alters chromatin
Typical chromosome end of Plasmodium falciparum

19. Localization of Sir2 by Immuno-Electron
Microscopy A B
The Sir2 protein localizes to the electron-dense heterochromatic region at the nuclear periphery of P. falciparum parasites. Panels (A) and (B) show developing merozoites in a late-schizont-stage parasite. Scale bars in (A) and (B) are 250µm.

20. Sir 2 in P. falciparum

21. Confirm presence of Sir 2 in P. falciparum
FISH +IF IF
Pf-Sir2-13
FISH
Telomere
Merged
+DAPI
Merged

22. Confirm presence of Sir 2 in P. falciparum
CHIP
HRP1
GBP130
Rep 20
Telomere
Input
Anti-Sir2
Anti-rabbit
Anti-acetyl
histone H4
anti-PfSir2 Y Y Y Y
Sonication
Fixation
Telomere
A chromosomal gradient of PfSir2 and acetylated histones

23. Heterochromatin model in P. falciparum
TAREs
antigen genes
Telomere 1 2 3 4 5
6 (rep20)
var gene
10 kb A B C

24. CHIP using Anti-acetyl histone H4
Expressed blood stage gene ON
Single copy at telomeric location
OFF ON
OFF
OFF
22 copies at telomeric location
OFF
Non expressed blood stage gene
OFF
Non expressed blood stage gene

25. Conclusions: We have identified the first telomere binding proteins in
P. falciparum, PfSir2
PfSir 2 binds to the telomere, spreads as far as the rep20
Element and is therefore a neighbour of the var gene promoter.
Specific histone modifications can be targeted to specific gene
promotor regions (activated chromatin domains)

26. Nuclear compartimentalization
DAPI IF
Merged
anti-Pfsir2-13
anti-acetyl histone H3
anti-acetyl histone H4

27. 3D model of P. falciparum nuclei
P. falciparum nucleus is sub compartmentalised.

28. Centro de Investigación y de Estudios
Collaborators
Artur Scherf
Institut Pasteur
France
Stuart Ralph
Spencer Shorte
Centro de Investigación y de Estudios
Avanzados del IPN
México
Rosaura Hernandes