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)
Antigen variation is a common theme in the life of protozoan parasitesAntigen 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.
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.
Plasmodium develops a life cycle between the mosquito and a vertebrate hostP. falciparum is the most virulent specie and it circulates betweenAnopheles and humans. Miller et al., 2002 Plasmodium is an intra-erythrocytic parasite
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 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.
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.
The surface of P. falciparum-infected RBCs are highly modifiedand PfEMP-1 adhesin sticks out from the knobs (Maier’s lab, La Trobe University, Australia) (Maier, 2009)
The surface of P. falciparum-infected RBCs are highly modifiedand PfEMP-1 adhesin sticks out from the knobs and promotesadhesion 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 Maurers cleft (MC).
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. Miller et al., 2002
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
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.
The genomic organisation of var genes and nuclear architecturedetermine 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).
Mutual exclusive expression: only one var gene is expressed at a given time1. 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
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
Mutual exclusive expression: only one var gene is expressed at a given time2. 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)
Mutual exclusive expression: only one var gene is expressed at a given time2. 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!
Mutual exclusive expression: only one var gene is expressed at a given time2. 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!
Mutual exclusive expression: only one var gene is expressed at a given time2. Switch in expression must be coordinated. Up to 18h Ring stage late trophozoyte UPS + + + - Intron + + + +
Luciferase expression levels showing activity of a var upstreampromoter and an intron promoter at different times in the cell cycle Calderwood et al. 2003
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.
A particular slide catching your eye?
Clipping is a handy way to collect important slides you want to go back to later.