lecture 17 Flashcards
What is ectopic recombination?
- leads to enhanced antigenic diversity
- recombination between heterologous chromosomes
- parasite’s way of generating rapid diversity in var genes
- how does it do it ?
How does ectopic recombination occur?
- 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
What is the Achilles’ heel in the malaria parasite?
- 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
What are crucial functions for other exported proteins?
- 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
What allows parasite protein export into the RBC cytosol and beyond?
- 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
What is important about the PEXEL protein-export translocon?
- 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
Why is translocation more complicated than the PEXEL protein-export translocon?
- 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
What is a putative Plasmodium translocon of PEXEL proteins?
- 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
What do we know of PTEX function?
- 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
What are potential malaria vaccines?
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
What do different Plasmodium species prefer?
- different RBCs
- e.g. P. vivax only likes young RBS, reticulocytes
- older RBCs has different surface molecules
- P. falciparum recognises all RBCs
What is a limiting factor of anti-merozoite antibodies?
- 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
What are the steps of parasite/RBC interaction and which are vaccine targets?
- primary contact
- signal?
- secondary interactions
- actin-myosin motor
- surface protein shedding
- secondary ligand shedding
- re-sealing
- recovery from echinocytosis
- primary contact and secondary interactions
Where are we toward a blood-stage vaccine?
- 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
Why is it important that the RBC surface is highly polymorphic?
- 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
