Antigen variation is a common theme in the life of protozoan parasites

Number of genes

‘Ancient’ Protists?

(Sogin, 1991)

Plasmodium falciparum(60 var genes and others)

Trypanosoma brucei (>1000)

Trichomonas vaginalis (~800)

Giardia lamblia (190)

Prediction of number of surface antigens

Humans

Baking yeast

Antigen variation is a common theme in the life of protozoan parasites

Antigen variation is indeed necessary for parasite survival in thehost (ex. Plasmodium falciparum, Trypanosome brucei)

Different names for similar things:

Mutual exclusive antigen variation is achieved by controllinggene expression in different ways:

•Plasmodium: surface antigens - var, rifin, stevor and Pfmc-2TM•Trypanosome: Variant Surface Glycoproteins or VSG.•Giardia: Variant-specific Surface proteins or VSP.

•Plasmodium: transcription factors, epigenetics and nuclear architeture.•Trypanosome: Recombination of a silent copy into a telomeric expressionsite appears to be the major mechanism of switching•Giardia: Evidence for epigenetics and miRNA.

•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.

Malaria is a major life threat in the tropical worldhttp://www.cdc.gov/malaria/

Plasmodium develops a life cycle between the mosquito and a vertebrate host

P. falciparum is the most virulent specie and it circulates betweenAnopheles and humans.

Plasmodium is an intra-erythrocytic parasite

Miller et al., 2002

Plasmodium utilizes a very active way of cell invasionrestricted 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 ownsurvival 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.

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 antigenvariants in P. falciparum: var (60), rifin, stevor and Pfmc-2TM.

•They are all transmembrane proteins that are placed onthe surface of the erythrocyte.

•PfEMP1 (P. falciparum Erythrocyte Membrane Protein 1) isby far the best studied case of antigen variation inPlasmodium 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.

The surface of P. falciparum-infected RBCs are highly modifiedand PfEMP-1 adhesin sticks out from the knobs

(Maier, 2009)

(Maier’s lab, La Trobe University, Australia)

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).

(Maier, 2009)

The surface of P. falciparum-infected RBCs are highly modifiedand PfEMP-1 adhesin sticks out from the knobs and promotesadhesion to endothelia

Miller et al., 2002

The surface of P. falciparum-infected RBCs are highly modifiedand PfEMP-1 adhesin sticks out from the knobs and promotesadhesion to endothelia, rosetting, clumping and microvascularobstruction.

Infection by Plasmodium is characterized by episodes of regular fever

Neva and Brown, 1994

Mutually exclusive expression of var genesA-F: only one var is expressed.

Gardner et al. The genome sequence of the human parasite Plasmodium falciparum. Nature, 2002.

Antigen multi-copy genes are localized at the end of chromosomes

Var genes (the ‘two-block’ genes) arepreferentially found at thesubtelomeres of chromosomes.

The other colored blocks representantigen multi-copy genes other thanvar. They immediately follow var genesin the chromosomes.

The genomic organisation of var genes and nuclear architecture

Three genetically distinct isolates of P.falciparum have little overlap of vargenes in their genomes

All 60 var genes are found clustered in4-7 foci in the nuclear peripheryincluding those that are physicallylocated close to the centromer.

Var genes are surrounded by repeats(TARE, telomere-associated repeatedelements).

determine diversification of var genes in populations of Plasmodium

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 byavoidance of immune recognition

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 are flanked by three types of regulatory sequence (upsA, B or C).

•The intron has also promoter activity.

They follow a similar gene structure.

(Dzikowski & Deitsch, 2009)

Mutual exclusive expression is also referred as allelic exclusion or mono-allelic expression

(Maier, 2009)

Pre-S phase (ring stage or early

trophozoyte)

Up to 18hS phase

(schizont or late trophozoyte)

Mutual exclusive expression: only one var gene is expressed at a given time

2. Switch in expression must be coordinated and low rates ofswitch must be guaranteed.

(Maier, 2009)

Mutual exclusive expression: only one var gene is expressed at a given time

2. Switch in expression must be coordinated and low rates ofswitch must be guaranteed.

Transient repression of var:

‘poised’ state.

Switch or not switch?

No

Which varwere we using before?

It means memory!

(Maier, 2009)

Mutual exclusive expression: only one var gene is expressed at a given time

2. Switch in expression must be coordinated and low rates ofswitch must be guaranteed.

Transient repression of var:

‘poised’ state.

Switch or not switch?

Yes

Hey guys, which one are we going to use?

It means coordination!

Ring stageUp to 18h

late trophozoyte

Mutual exclusive expression: only one var gene is expressed at a given time

2. Switch in expression must be coordinated.

+

+

UPS + +

Intron ++ +

-

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

1. The var intron causes silencing of a ‘paired’ geneindependently of the phase of cell cycle.

2. The var intron causes repression of a ‘paired’ geneindependently of its orientation.

3. The repression caused by the var intron happensbefore S phase.

4. Repression or silencing of the ‘paired’ genehappens with the var intron independently of the‘paired’ gene promoter.

5. Repression or silencing of the ‘paired’ gene onlyhappens if the intron is a var intron.

True or False?

Deitsch et al. Nature, 2001.