Lecture 17 - Malaria 2 Flashcards Preview

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Flashcards in Lecture 17 - Malaria 2 Deck (39):

Describe variability in var genes.
Why is this so?

Highly polymorphic

• Take isolates from two people form the same village
• These var genes would have had a recent common ancestor
• When sequenced, the isolates are similar, apart from the 60 var genes, which are very different

Due to:
• Ectopic recombination


Describe ectopic recombination

Recombination between heterologous chromosomes
(not the pigeon pair, because these are haploid organisms)
This evolves very rapid diversity

• The ends of the chromosomes cluster together
• The ends are quite similar, despite being different chromosomes
• There is recombination between these aligned genes at the ends


What are the 'Hundreds of proteins'?
Give an example of one

• Hundreds of proteins (500) that are exported out of the parasite to
perform a specific function.
• It doesn’t matter for us what this function is, but we will
look at how they are transported out of the parasite
• Crucial to the parasite (virulence or life cycle)
• This could be a good target for a drug

• Exported protein vital for rough appearance of the infected RBC
• Crucial for infected RBC adherence to the endothelium
• Just one example of 500 exported proteins


Describe the vacuolar membrane

When the parasite is invading an RBC, it pushes through the membrane, which ends up surrounding it inside the RBC


What is KAHRP?

One of the hundreds of exported proteins important for virulence

Essential for knob formation and adherence
• Rough appearance w/ PfEMP1 present
• The knobbly rough structures are what PfEMP1 is embedded into
• Allow the parasite to hang onto to the endothelium under physiological flow pressure
• KHARP is essential to these knobbly structure

• smooth appearance
• can't adhere to vascular endothelium
• PfEMP1 is still there, but it can't hang on to the endothelial walls under physiological flow pressure


What is the PEXEL motif?

Why is it important?


• 5 aa long

Plasmodium export element
• Of the exported proteins that were known, all had a common looking N-terminus.
It had:
• a hydrophobic region (signal sequence
• as well as a motif: PEXEL

Where 'x' is any amino acid

• This motif is responsible for export out the protein out of the vacuolar membrane

• If any of the residues in the PEXEL motif, GFP was not able to be exported out of the vacuolar membrane

• The proteins with the PEXEL motif have important functions for the life cycle of the parasite:
- Cytoadherence
- Waste clearance
- Membrane rigidity
- Nutrient delivery
• Thus, they can not be KO'd
• Thus, PTEX is a good target for a malarial drug


How much of the genome of the malaria parasite has the PEXEL motif?

Around 5%

This means around 5% of the gene product is exported


What was seen in knockout screens of 51 exported proteins?

What is the significance of this?

PTEX is a great drug target

(46 were PEXEL containing)
• The genes have very varied functions.
• A quarter of these genes could not be knocked out
• The genes were essential to the blood stage metabolism.
• This would be a great drug target.
• A drug wants to kill a parasite

NB It is thought that almost all of these proteins have a common transport mechanism


How do the proteins get across the vacuolar membrane?

1. Parasite → Vacuolar space
• In the ER of the parasite
• Plasmepsin V cleaves PEXEL
RxLx(E/Q/D) → RxL + x(E/Q/D)
• Normal vesicular trafficking to the a very particular area of the plasma membrane of the parasite
• Vacuole contents released into vacuolar space

2. Vacuolar space → RBC cytosol

Movement through a translocon: PTEX

• Proteins recognise machinery of translocon (?, still unconfirmed)
• Hsp101 unfolds proteins so that they can pass through
• Passes through EXP2 pore
• Refolded by other subunits on the other side


What are the key criteria of the translocon?

• Plasmodium specific
• Essential to blood stages
• Energy source, because all translocons use ATP or GTP
• Must bind transiting cargo PEXEL proteins


What is the structure of the translocon?

5 protein subunits

• A heat shock protein
• The energy source
• Unfolds proteins

• Forms a pore through the plasma membrane


What is the translocon called?



What are the best targets for a malaria drug?

How about a vaccine?

1. PTEX:
• It plays a vital role in the movement of hundreds of proteins out of the vacuolar space

2. Invasion ligands
• EBA's
• PfRH's

• NB Too variable, vaccine / drug would need to address all

3. PfEMP1
• Too variable


What are the types of vaccine that are being developed?

3 types:

1. Pre-erythrocytic

2. Transmission blocking

3. Blood stage
• Anti-merozoite


Describe transmission blocking vaccines

• Vaccinating people so that Abs are made
• Abs are taken up by mosquitoes when taking a blood meal
• Mosquitoes are immune to infection


Describe pre-erythrocytic vaccines

• RTS.S, in phase 3
• Recombinant protein that blocks the process of sporozoites entering the liver
• Could make the market
• In 14,000 children around Africa
• Works a bit, but is not amazing


Describe briefly Blood stage vaccines

• Anti-merozoite
• Against molecules on the merozoite in the blood stage that are vital for entry into the RBC


Describe merozoite invasion of RBCs

1. Merozoite free in the blood for about 1 minute

2. Primary contact

3. Secondary interactions:
• Reorients so that the apical complex becomes juxtaposed to the parasite

4. Buds into the membrane (creating the vacuolar space)
• Apical complex spews out important ligands
• Actin-myosin motor pulls the membrane around the parasite
• Surface protein shedding

4. Resealing, creating a vacuolar membrane around the parasite

5. Recovery from echinocytosis


Explain 'different Plasmodium species prefer different RBCs'.
Give examples

• Different species invade different RBCs
• First indication that it was a complex process, not one size fits all

• RBCs are phenotypically different at the different stages

• P. vivax only infect young RBCs (won't infect older ones, which has different surface antigens)
• P. falciparum infect all RBCs


What is echinocytosis?

Dehydration of the RBC that occurs rapidly after invasion of the parasite


Which stages of merozoite infection of RBCs are possible vaccine targets?

• Primary contact

• Secondary interactions


How long is the merozoite free in the blood / entering the RBC?
Why is this significant?

Free in the blood for about 1.5 minutes in total

This is a very short time for antibodies to bind


Describe the success of empirical blood stage vaccine approaches

Did not work
→ need a rational approach


Why is rational approach to blood stage vaccines now feasible?

Genomic Knockout technological is now much more widely available
There is the possibility of finding out what all the antigen do, so we know which ones to target


Describe the surface of RBCs.
Give a specific example of a surface compound

• Highly polymorphic and heterogeneous
• Different surface molecules between individuals

e.g. Duffy glycoprotein
• This glycoprotein on the surface is vital for P. vivax invasion
• The entire population of West Africa lacks this glycoprotein on their RBC surfaces
• Thus, there is no P. vivax infection in West Africa


What is the EBA-175 related family?

• This is a family of four surface molecules on P. falciparum that are all related
• These molecules are ligands for RBC invasion
• They all have different ligands


What is the PfRh family?

Plasmodium falciparum reticulocyte homologue family

• Four surface proteins that are ligands for RBC invasion
• All have different receptors


Explain the W2mef story

• A single parasite can have one specific phenotype, and then switch to have another invasion phenotype by gene KO.

• W2mef expressed just one of the binding ligands: sialic acid
• Treat the RBC with neuraminidase, which removes the sialic acid
• W2mef is initially not able to enter the RBC
• After numerous generations
W2mef switched to another
ligand expression that can invade the RBCs quite well (still W2mef)
→ Sialic acid independent W2mef


What is the effect of NA and trypsin on RBCs?

NA: cleaves sialic acid
Trypsin: cleaves proteins


Where is Sialic acid found?

On Gly4, on the surface of RBCs


What happens in the parasite when there is a KO of EBA175 in W2mef?

• W2mef uses EBA175 as the invasion ligand
• After the KO, it is no longer able to invade
• After a short time, it switches its expression to another invasion ligand: Rh4
• Able to invade again via another receptor: Complement receptor-1


What is the receptor on RBCs used by Rh4?

Complement receptor-1


What is the take home message about P. falciparum in terms of invasion ligands?

What does this mean for vaccine development?

A single, genetically identical parasite can switch ligand expression and thus the receptor that it uses to get into the RBC

All these different ligands:
• 4 EBA's
• 4 Rh's
encode for different receptor binding characteristics

This poses a problem for vaccine development.


Why do humans have different blood groups?

Why do malaria parasites (P. falciparum) have so many invasion ligands?

Blood group diversity, to a large extent, due to our co-evolution with malaria.

The reason the parasite has the ability to invade different RBCs is probably an evolutionary one to do with plasticity of RBCs that we have evolved to deal with malaria
It has adapted to have different invasion pathways


Why is it beneficial for the parasite to have different invasion pathways?

1. Deal with the plasticity of RBC surface
2. Escape immunity that develops against one pathway


Describe what was learnt about invasion pathways using inhibitory antibodies

• Antibodies were isolated from many different immune individuals
• The antibodies were tested against two strains:
1. W2mef (EBA175 invasion pathway)
2. W2mef∆175 (Rh4 invasion pathway)
• There was great difference between individuals as to which strain could be controlled:
- some controlled W2mef
- some controlled W2mef∆175

This tells us that the different invasion pathways are beneficial for the parasite, in that it allows them to avoid the immune response, and thus still be able to invade.


How many major invasion pathways have been identified as of yet?

3 major pathways


What must a blood stage vaccine block?

It must block all major invasion pathways


How long is the PEXEL motif?

5 amino acids long:

R x L x (E x Q x D)