1. Antigen variation is a common theme in the life
of protozoan parasites
Prediction of number of
Number of genes
surface antigens
Baking yeast
Plasmodium falciparum
Humans (60 var genes and others)
‘Ancient’
Protists?
Trypanosoma brucei
(>1000)
Trichomonas vaginalis
(~800)
Giardia lamblia
(Sogin, 1991) (190)

2. Antigen variation is a common theme in the life
of protozoan parasites
Antigen variation is indeed necessary for parasite survival in the
host (ex. Plasmodium falciparum, Trypanosome brucei)
Different names for similar things:
•Plasmodium: surface antigens - var, rifin, stevor and Pfmc-2TM
•Trypanosome: Variant Surface Glycoproteins or VSG.
•Giardia: Variant-specific Surface proteins or VSP.
Mutual exclusive antigen variation is achieved by controlling
gene expression in different ways:
•Plasmodium: transcription factors, epigenetics and nuclear architeture.
•Trypanosome: Recombination of a silent copy into a telomeric expression
site appears to be the major mechanism of switching
•Giardia: Evidence for epigenetics and miRNA.

3. Malaria is a major life threat in the tropical world
http://www.cdc.gov/malaria/
•3.3 billion people (half the world’s population) live in areas at risk of malaria
transmission in 109 countries and territories
•In 2008, malaria caused an estimated 190 - 311 million clinical episodes, and 708,000 -
1,003,000 deaths.
•89% of the malaria deaths worldwide occur in Africa.
•Malaria is the 5th cause of death from infectious diseases worldwide (after respiratory
infections, HIV/AIDS, diarrheal diseases, and tuberculosis) in low-income countries.
•Malaria is the 2nd leading cause of death from infectious diseases in Africa, after
HIV/AIDS.

4. Plasmodium develops a life cycle between the mosquito
and a vertebrate host
P. falciparum is the most virulent specie and it circulates between
Anopheles and humans.
Miller et al., 2002
Plasmodium is an intra-erythrocytic parasite

5. Plasmodium utilizes a very active way of cell invasion
restricted to erythrocytes or red blood cells (RBC)
• Advantages:
1. A RBC is a rigid cell that supports the shear stress exerted by the blood flow.
2. RBCs do not have an active cytoskeleton and are unable to export proteins and
traffic vesicles (very unresponsive).
• Disadvantages:
1. Not a rich-nutrient environment,
2. Cells with a relative short life,
3. Cells that are continuously recycled in the liver/spleen and therefore exposed to
the immune system.
• The parasite modifies the RBC membrane profoundly to enable its own
survival and proliferation:
1. Changes deformability and thermo-resistance of RBCs.
2. Promote formation of new channels for the import and export of nutrients.
3. Place adhesive proteins on the surface of infected RBCs that induce adherence
of infected cells to the endothelium avoiding clearance by the host.

6. PfEMP-1 is an adhesin produced by the multi-copy
gene family var and it is an antigen variant
•There are four multi-copy gene families coding for antigen
variants in P. falciparum: var (60), rifin, stevor and Pfmc-
2TM.
•They are all transmembrane proteins that are placed on
the surface of the erythrocyte.
•PfEMP1 (P. falciparum Erythrocyte Membrane Protein 1) is
by far the best studied case of antigen variation in
Plasmodium falciparum.
•PfEMP1 is encoded by a var gene.
(Scherf et al., 2008)
PfEMP-1 is the most important adhesin implicated in the
pathogenesis of Malaria.

7. The surface of P. falciparum-infected RBCs are highly modified
and PfEMP-1 adhesin sticks out from the knobs
(Maier’s lab, La Trobe University, Australia)
(Maier, 2009)

8. The surface of P. falciparum-infected RBCs are highly modified
and PfEMP-1 adhesin sticks out from the knobs and promotes
adhesion to endothelia
(Maier, 2009)
a. Transmission electron micrograph of a knobby parasite (P)-infected red blood cell
(RBC) adhering to the surface of a microvascular endothelial cell (EN).
b. Detail of the interface between an infected RBC and an endothelial cell showing
strands of electron dense connecting material located at knobs (arrows). Note the
presence of a Maurer's cleft (MC).

9. The surface of P. falciparum-infected RBCs are highly modified
and PfEMP-1 adhesin sticks out from the knobs and promotes
adhesion to endothelia, rosetting, clumping and microvascular
obstruction.
Miller et al., 2002

10. Infection by Plasmodium is characterized by episodes
of regular fever
Mutually exclusive expression of var genes
A-F: only one var is expressed.
Neva and Brown, 1994

11. Antigen multi-copy genes are localized at the end of chromosomes
Var genes (the ‘two-block’ genes) are
preferentially found at the
subtelomeres of chromosomes.
The other colored blocks represent
antigen multi-copy genes other than
var. They immediately follow var genes
in the chromosomes.
Gardner et al. The genome sequence of the human parasite
Plasmodium falciparum. Nature, 2002.

12. The genomic organisation of var genes and nuclear architecture
determine diversification of var genes in populations of Plasmodium
Three genetically distinct isolates of P.
falciparum have little overlap of var
genes in their genomes
All 60 var genes are found clustered in
4-7 foci in the nuclear periphery
including those that are physically
located close to the centromer.
Var genes are surrounded by repeats
(TARE, telomere-associated repeated
elements).

13. Mutual exclusive expression:
only one var gene is expressed at a given time
1. Every var gene is recognized as part of a single family.
2. Switch in expression must be coordinated and low rates of
switch must be guaranteed.
3. It requires a memory: the expression of one gene tends to last
for many cell cycles and switches occur at a relatively low rate.
• PfEMP-1 adhesion avoids clearance and promotes disease
• PfEMP-1 antigen variation extends period of infection by
avoidance of immune recognition

14. Mutual exclusive expression:
only one var gene is expressed at a given time
1. Every var gene is recognized as part of a single family.
They follow a similar gene structure.
•They are flanked by three types of regulatory sequence (upsA, B or C).
•The intron has also promoter activity.
(Dzikowski & Deitsch, 2009)
Mutual exclusive expression is also referred as allelic exclusion
or mono-allelic expression

15. Mutual exclusive expression:
only one var gene is expressed at a given time
2. Switch in expression must be coordinated and low rates of
switch must be guaranteed.
(Maier, 2009)
Up to 18h
Pre-S phase S phase
(ring stage or early (schizont or late
trophozoyte) trophozoyte)

16. Mutual exclusive expression:
only one var gene is expressed at a given time
2. Switch in expression must be coordinated and low rates of
switch must be guaranteed.
(Maier, 2009)
Which var Transient
were we using repression of var:
No before? ‘poised’ state.
Switch or not switch?
It means memory!

17. Mutual exclusive expression:
only one var gene is expressed at a given time
2. Switch in expression must be coordinated and low rates of
switch must be guaranteed.
(Maier, 2009)
Hey guys, Transient
which one are repression of var:
Yes we going to use? ‘poised’ state.
Switch or not switch?
It means coordination!

18. Mutual exclusive expression:
only one var gene is expressed at a given time
2. Switch in expression must be coordinated.
Up to 18h
Ring stage late trophozoyte
UPS + + + -
Intron + + + +

19. Luciferase expression levels showing activity of a var upstream
promoter and an intron promoter at different times in the cell cycle
Calderwood et al. 2003

20. True or False?
1. The var intron causes silencing of a ‘paired’ gene
independently of the phase of cell cycle.
2. The var intron causes repression of a ‘paired’ gene
independently of its orientation.
3. The repression caused by the var intron happens
before S phase.
4. Repression or silencing of the ‘paired’ gene
happens with the var intron independently of the
‘paired’ gene promoter.
5. Repression or silencing of the ‘paired’ gene only
happens if the intron is a var intron.
Deitsch et al. Nature, 2001.