lecture 17

Question 1

What is ectopic recombination?

Answer 1

• leads to enhanced antigenic diversity
• recombination between heterologous chromosomes
• parasite’s way of generating rapid diversity in var genes
• how does it do it ?

Question 2

How does ectopic recombination occur?

Answer 2

• chromosome ends cluster at the nuclear periphery: physical location of the chromosomes
• four spots: should at least see 14, would expect 28 because they are on either end
• so what we have are aggregated var genes held together by various proteins
• P. falciparum chromosome end clustering promotes recombination between aligned genes , a paper in Nature showed that they were held in perfect alignment
• the result of this is that every life cycle has a lot more recombination occurring than typical meiotic recombination
• shuffling at a frequent rate
• end up with very different var genes
• centrally located var genes also cluster
• that’s why there is so much diversity in the var gene repertoire from parasite to parasite

Question 3

What is the Achilles’ heel in the malaria parasite?

Answer 3

• other exported proteins
• Plasmodium parasites export 100s of proteins into the RBC cytosol: crucial virulence and nutrient uptake roles
• when the parasite invaded the RBC, a second membrane formed around it called the vacuolar membrane
• this was part of RBC membrane that enveloped it
• the parasite wants to get proteins out into the cytosol/PfEMP1 out to plasma membrane of RBC
• turns out this is a very hard thing to do

Question 4

What are crucial functions for other exported proteins?

Answer 4

• e.g. KAHRP is essential for ‘knob’ formation and adherence
• enables proteins/blood cell to hang on to endothelium for dear life
• KAHRP “knockout” parasites don’t adhere to vascular endothelium
• there are like 500 others

Question 5

What allows parasite protein export into the RBC cytosol and beyond?

Answer 5

• all the proteins that were exported that we knew about had a common looking N terminus
• hydrophobic region - embedded signal sequence
• conserved ‘PEXEL’ motif: RXLXE/Q/D
• string of amino acids
• maybe this motif is responsible for export
• if you mutated any of these residues the protein was no longer exported out of the vacuole
• ~5% (maybe up to 10) P. falciparum proteome is predicted to be exported
• a knockout screen of 51 exported proteins (46 PEXEL-containing revealed proteins have many virulence and survival roles
• 25% of exported genes are essential to growth

Question 6

What is important about the PEXEL protein-export translocon?

Answer 6

• race to find this translocon: initially unknown
• nexus through which many if not all of these functions are connected
• protein export is a major point of vulnerability: how do these proteins cross the parasitophorous vacuole

Question 7

Why is translocation more complicated than the PEXEL protein-export translocon?

Answer 7

• step upstream we need to care about
• ER
• all exported proteins have a signal sequence
• they get transported from the cytosol into the ER
• go through secretory pathway
• the PEXEL is a cleavage site that is recognised in the ER by an enzyme known as Plasmepsin V
• RxLxE/Q/D –> RxL + xE/Q/D
• if you got this cleavage event to occur in the ER, proteins that were cleaved in this way went down a very particular vesicular pathway
• dumped their contents into a particular region of the vacuolar space
• Plasmepsin V (aspartyl protease) becomes a major drug target for malaria

Question 8

What is a putative Plasmodium translocon of PEXEL proteins?

Answer 8

• PTEX
• initially only putative
• now real

key criteria:

• plasmodium specific and in the correct location
• essential to blood-stages
• energy source, an un-folding mechanism
• binds transiting cargo PEXEL proteins

Question 9

What do we know of PTEX function?

Answer 9

• sits on the vacuolar membrane
• proteins come along recognise this machinery (unknown how) and and pass through HSP101
• HSP101: ATPase, heat shock protein, has to unfold proteins that have reached this point
• pass through a pore in the membrane (energy dependent)
• refolded by proteins on the other side
• PTEX inhibitors are an approach to block many essential proteins/processes via the one target
• EXP2 forms the pore

Question 10

What are potential malaria vaccines?

Answer 10

3 types:

1) Pre-erythrocytic (e.g. RTS,S, whole parasite)
- RTS,S is in stage III clinical trial - blocks sporozoites entering the liver
- doesn’t work brilliantly but it does work
- already in 14,000 children in Africa
2) transmission blocking
- antibodies for human that get taken up by mosquito and prevent IT from getting infected
- ethically - can’t technically do a trial on someone for whom the vaccine doesn’t benefit directly
- very exciting, way of the future
3) blood-stage (antimerozoite)
- proteins involved in merozoite entering blood cell
- it pulls membrane around it through formation of tight junctions –> energy dependent

Question 11

What do different Plasmodium species prefer?

Answer 11

• different RBCs
• e.g. P. vivax only likes young RBS, reticulocytes
• older RBCs has different surface molecules
• P. falciparum recognises all RBCs

Question 12

What is a limiting factor of anti-merozoite antibodies?

Answer 12

• very little time to act
• up to 1.5 minutes when the merozoite bursts out of the RBC before invading new cell
• 30 seconds to invade
• rationale for an anti-merozoite vaccine remains strong but the most effective approach is not obvious and there is little functional knowledge to help inform vaccine design

Question 13

What are the steps of parasite/RBC interaction and which are vaccine targets?

Answer 13

• primary contact
• signal?
• secondary interactions
• actin-myosin motor
• surface protein shedding
• secondary ligand shedding
• re-sealing
• recovery from echinocytosis
• primary contact and secondary interactions

Question 14

Where are we toward a blood-stage vaccine?

Answer 14

• still some way off - empirical vaccine approaches not working
• most antigens identified (50 - 100) - but which are the best
• very little functional knowledge of individual antigens
• new genomic technologies unraveling function
• rational vaccine design becoming feasible

Question 15

Why is it important that the RBC surface is highly polymorphic?

Answer 15

• different RBCs between different people
• different surface molecules at different stages of cell life
• Duffy glycoprotein - anyone who is endemic to west africa does not express this protein while most of the rest of the world does
• Duffy is essential for P. vivax to invade the cell
• so west africa does not have P. vivax at all
• instead of just one or two secondary ligands responsible for binding RBCs now have at least 8 proteins in P. falciparum - 4 erythrocyte binding antigens and 4 reticulocyte binding homologue proteins
• P. vivax has only one - binds duffy
• P. falciparum has alternate ways of entering the RBC
• multiple red cell receptors and invasion ligands
• causes a problem for vaccine developmont

Question 16

What can alter receptor-ligand usage?

Answer 16

• e.g. the W2mef strain
• uses EBA175 to bind to sialic acid on GlyA
• if you grow them with NA-treated erythrocytes they can’t invade in the first cycle but will eventually switch entry pathway –> same strain but different entry mechanism
• same invasion rates in normal RBCs, different in treated RBCs indicating that they have different invasion mechanisms

Question 17

What happens if you delete the ligand (EBA175) used by W2mef?

Answer 17

• get a transcriptional switch in that parasite
• expresses a different invasion protein
• 10,000 fold upregulation of the RNA encoding the new protein (Rh4)
• the point is that a single parasite, genetically identical, can switch the ligand that it expresses and the receptor that uses to enter the RBC

Question 18

What do alternate invasion pathways mean for parasites?

Answer 18

• allow parasites to invade via a diverse range of RBC receptors and to avoid immunity
• variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies
• part of the reason we have different blood groups is because of coevolution with malaria
• immunity in people recognises parasites differently
• variation in ligand usage is a mechanism of immune evasion
• EBA and Rh proteins are important targets of inhibitory antibodies

Question 19

What are alternate invasion pathways?

Answer 19

• accommodate RBC polymorphism
• immune avoidance
• at least 3 major pathways now identified
• vaccine must cover all major pathways
• (antigenic diversity remains a problem)