Lecture 9: Plasmodium - Intracellular Survival and Modification of the Host Cell Flashcards
What are the advantages and disadvantages of an erythrocyte environment?
Advantages:
- no antigen presentation
- haemoglobin rich environment and source of amino acids
Disadvantages:
- lacks a nucleus
- lacks machinery to transport proteins to subcellular locations
What is the earliest stage of parasite development seen within the red blood cell?
the ring stage
How does the plasmodium digest erythrocyte haemoglobin and why?
digests Hb in a specialised acidic organelle called the digestive vacuole (DV) or food vacuole
why? breakdown of Hb supplies amino acids to synthesise new proteins
Why must plasmodium still import dome amino acids?
requires amino acids that are rare in Hb or completely absent in Hb (such as Ile) so these must be imported
What is the downside of haemoglobin breakdown for the parasite and what is the pharmacological significance of this?
Hb digestion releases toxic waste product heme (which the parasite converts to the chemically inert haemazoin)
The pharmacological significance of this is that Chloroquine works by inhibiting the formation of haemazoin so heme builds up and parasite dies.
Other than acquisition of amino acids, what might be another purpose for the Hb breakdown by plasmodium in the RBC?
As the parasite undergoes intra-erythrocytic development from ring, early trophozoite, late trophozoite and schizont stage (with many merozoites) the size increases and takes up more space in the RBC
- the digestion of Hb may have an additional role in creating space for parasite growth and prevent erythrocyte lysis
Why does plasmodium export proteins from the parasite into the erythrocyte cytosol?
- increase nutrient uptake from blood plasma
- remodel RBC for parasite benefit
- facilitate adhesion of infected cell to endothelial cells
What membranes do parasite proteins need to traverse when moving from the parasite to the erythrocyte cytosol (or vice versa)
- parasite membranes
- parasitophorous vacuole membrane
- erythrocyte membrane in some cases to reach the cell surface (E.g…………………….)
What are Maurer’s clefts and when do they develop?
large, flattened membranous structures for protein trafficking
They bud from the parasitophorous vacuole membrane in early ring-stage development and migrate towards the erythrocyte membrane
They become physically tethered to the RBC membrane as parasite differentiates into trophozoite stage
Why is tethering of the Maurer’s clefts to the RBC membrane during trophozoite stage development important?
helps to deliver parasite protein complexes to RBC membrane and allow insertion into RBC surface
What is thy key protein released immediately post invasion and where are they released from?
RESA (Ring-infected Erythrocyte surface antigen)
Released from dense granules that fuse with the PVM
Describe the remodelling of the RBC in early ring stage
- much more RESA associated with inner surface of RBC membrane
- maurer’s cleft formation that are migrating towards RBC membrane
- PTEX transports proteins across PVM (E.g. KAHRP - Knob associated His rich protein)
At what stage do you get knob formation?
late ring/early trophozoite
Most exported parasite protein contain what motif?
PEXEL motif located downstream of a hydrophobic signal sequence
What is the purpose of the hydrophobic signal sequence of exported parasite proteins?
required for entry into the ER
What five residues make up the PEXEL motif?
RxLxE/Q/D (where x is any uncharged amino acid residue)
What is the function of the PEXEL motif?
- enables identification of PEXEL proteins for export and cleavage
- cleavage of the PEXEL motif uncovers an export signal at the exposed N-terminal end to direct mature protein into host cell
True or false: PEXEL proteins are only present in P. falciparum?
False: they are present in all Plasmodium species but are particularly abundant in P . falciparum and closely related species
How are PEXEL exported proteins processed?
Proteolytic cleavage between the leucine (L) and adjacent uncharged amino acid (RxL — xE/Q/D) by aspartyl protease (Plasmepsin V) in the endoplasmic reticulum
The exposed N-terminal amino acid is then N-acetylated for protein export
What is the name of the aspartyl protease that cleaves the PEXEL motif?
Plasmepsin V
How does Plasmepsin V recognise the PEXEL motif?
recognises the arginine (R) and leucine (L) residues
Which amino acid is important for protein export post-cleavage?
the fifth amino acid (E/Q/D)
What happens if the fifth amino acid in the PEXEL motif is mutated?
the proteins are trapped in the vacuolar space (can get out of the ER but trapped in vacuolar space)
What happens if the arginine or leucine amino acids are mutated to alanine?
the proteins remain trapped in the ER (unable to be recognised by plasmepsin V so not cleaved and the N-terminal export signal is not exposed)
What are plasmodium proteins that lack a PEXEL motif called?
PEXEL-negative exported proteins (PNEPs)
How are PNEPs exported if they do not contain a PEXEL motif or N-terminal hydrophobic signal sequence?
The N-terminus in some cases contain an export signal that is functionally equivalent to a the N-terminus of a protein that has a cleaved PEXEL motif
Give an example of an important PNEP?
PfEMP1 (P. falciparum erythrocyte membrane protein 1)
What is PTEX and what does it do?
Plasmodium translocon of exported proteins
It transports both PEXEL and PNEP proteins across the PVM
What are the three homo-oligomeric components of PTEX?
Heat shock protein 101 (HSP101)
PTEX150
Exported protein 2 (EXP2)
what is the role of the HSP101 component of the PTEX?
an ATP-powered unfoldase that is located on cis side of PVM and acts as the molecular motor of PTEX
what is the role of the PTEX150 component of the PTEX?
plays a structural role and regulate stability of the PTEX
what is the role of the EXP2 component of the PTEX?
forms pore in PVM - protein-conducting channel in the PVM that facilitates protein transport
What are the auxiliary components of the PTEX and what is their proposed role?
PTEX88 and thioredoxin 2 (TRX2)
- these are not essential for parasite survival but presumably optimise translocation of protein cargo
True or false: the PTEX is essential for Plasmodium survival?
True (cannot modify the RBC without it)
How are PNEPs and PEXEL proteins transported to the RBC surface following translocation through PTEX?
proteins secreted from parasite cytoplasm into parasitophorous vacuole and exported into the RBC cytoplasm via PTEX
Associate with Maurer’s clefts via chaperone mediated transport, which migrate towards erythrocyte surface for insertion
Plasmodium infected cells become ___(1)___ and ___(2)___
- Rigid
- Sticky
(either way round is fine)
How do infected RBCs become rigid (giving an example of a parasite protein which causes this rigidity)?
parasite proteins bind the RBC cytoskeleton and reduce elasticity - E.g. RESA stabilises the spectrin tetramer and increases cellular rigidity
How do infected RBCs become sticky (giving an example of a parasite protein that causes this adhesion)?
parasite proteins in erythrocyte membrane bind to host receptors resulting in cytoadherence that sequesters infected RBCs in microvasculature to prevent splenic clearance
E.g. PfEMP1
What is PfEMP1?
Plasmodium falciparum Erythrocyte membrane protein 1
- a multi-domain trans-membrane protein expressed at knob structures on erythrocyte surface
- mediate adhesion of infected erythrocytes (trophozoite and schizont stages) to endothelial cells by interaction with endothelial receptors (cytoadherence)
Which genes encode for pfEMP1?
Var genes
Describe the structure of PfEMP1
- Extracellular domain (ECD) comprised of a number of different domains that allow binding to particular host receptors
- Transmembrane domain (TMD)
- Intracellular acidic terminal segment (ATS) anchored through into protein underlying the knob structures
Give three host receptors that have been shown the mediate cytoadherence
Chondroitin Sulphate A (CSA) - a sulphated glycosaminoglycan that is a major constituent of cartilage but also lines blood vessels
Complement receptor type 1 (CR1) - present on RBC surface
Endothelial protein C receptor (EPCR) - has cytoprotective and anti-inflammatory effects under normal circumstances
Intracellular adhesion molecule 1 (ICAM1)
CD36
Hoe many var genes are transcribes in an individual parasite?
only one (all other var genes are silenced)
How does var gene expression allow parasite survival and immune evasion?
infrequent switches in mutually exclusive var gene expression generates subpopulations of parasite that proliferate in the presence of an immune response targeting previous PfEMP1 variants
What is pregnancy associated malaria?
Contraction of malaria during pregnancy which can result in serious complications for mother (anaemia) and child (still birth, low birth weight)
How does sequestration of infected RBCs occur in PAM and cause clinical manifestation?
the particular PfEMP1 type binds selectively to low-sulphated CSA that lines surface of placental villi resulting in sequestration of infected RBCs in intervillous spaces of placenta
- this causes inflammatory response and reduced blood flow - associated with severe maternal anaemia and low foetal birth weight or still birth
Why is PAM most pronounced for women in their first pregnancy compared to subsequent pregnancies?
in subsequent pregnancies, the women is already primed to respond to parasites that might be expressing different PfEMP1 types so is more able to control the infections better so doesn’t suffer with PAM condition - antibodies present against these PfEMP1 types so parasite cannot sequester and will be cleared by spleen
What is rosetting?
infected red blood cells bind to uninfected red blood cells
What are the proposed functions of rosetting?
- protect infected red blood cells from host immune response
- protect newly released merozoites from invasion-inhibitory antibodies
- enable emerging merozoites to rapidly invade the bound uninfected RBCs
Which parasite ligand and host receptor interaction is known to be one mediator of rosetting?
PfEMP1 binding to CR1 expressed on surface of RBCs
True or false: rosetting has been shown to link with disease severity?
True (bigger complex even harder to pass through microvasculature)
Describe the structure of CR1
Integral membrane glycoprotein composed of complement control protein (CCP) repeats that contain two disulphide binds per CCP
- 7 CCP units are grouped into larger domains called long homologous repeats (LHRs)
How many isoforms of CR1 are there?
4
In addition to having sites to which PfEMP1 can bind, what else is able to bind to CR1 and what does this mean in terms of potential function?
RBLs released from micronemes of merozoite are able to bind to a site on the CR1 glycoprotein
this means that CR1 may be mediate merozoite invasion of RBCs
What is severe malaria characterised by?
severe anaemia
hypoglycaemia
cerebral malaria
True or false: severe malaria caused by P. falciparum is more commonly observed in adults than children?
False: its more commonly observed in children than adults
Which host receptor protein is important in mediating severe malaria?
Endothelial protein C receptor (EPCR)
How does EPCR function under normal conditions?
Protein C binds EPCR resulting in activation of protein C, which has anti-coagulant activity
Activated protein C/EPCR complex also binds to protease activated receptor 1 (PAR1) drives cascade that results in anti-inflammatory cytokines and cytoprotective signalling
What are the consequences of an infected RBC binding via a PfEMP1 subtype to the EPCR?
different PfEMP1 subtypes that bind to EPCR on RBCs obstruct microvasculature but also inhibit the activation of protein C and the associated anti-coagulant activity and PAR1 signalling effect such as cytoprotective signalling and anti-inflammatory cytokines which further contributes to pathogenesis
What are two examples of different PfEMP1 subtypes that are implicated in severe malaria by binding to EPCR?
DC8 and DC13
