Lecture 4 - Plasmodium - Mechanisms of cell invasion Flashcards
Malaria background
Endemic in 83 countries
Stable case rate between 2000 and 2019
Case rate increasing since 2020
~263 million cases in 2023
Malaria cases steadily declining from 861,000 in 2000 to 597,000 in 2023
African regions account for 94% of cases and 95% of deaths (76% of malaria deaths in children <5 years old)
Malaria interventions
Interventions such as insecticide-treated bed nets, indoor residual spraying and combination drug therapy have helped reduce incidence
Plasmodium species
Plasmodium falciparum - ~50% cases - Melignant tertian
Plasmodium vivax - ~43% of cases - benign tertian
Plasmodium malariae - ~7% - Quartan
Plasmodium ovale - ~1% - Quotidian
Plasmodium knowlesi - ? - Quotidian
Two distinct species? Plasmodium ovale curtisi and Plasmodium ovale wallikeri
Zoonotic malaria parasite transmitted to man from non-human primate hosts
What is the most lethal malaria species
P. falciparum - most lethal human malaria species owing to:
sequestration of infected RBCs in microvasculature of brain and other organs
avoidance of splenic clearance by infected RBCs
Plasmodium lifecycle
3 stages adapted for invasion
Ookinete - Mosquito - gut epithelial cells
Sporozoite - Mosquito - salivary glands and hepatocytes
Merozoites - red blood cells
Morphology of merozoites, sporozoites and ookinetes
1.5u - merozoite
10-13u - ookinete
12-15u - Sporozoite
Plasmodium and red blood cells
RBC preference:
- P. falciparum - all erythrocytes
- P. vivax - (Duffy antigen +ve) reticulocytes
- P. ovale - reticulocytes
- P. malariae - mature erythrocytes
Erythrocytes lack phagocytic capability, so parasite has to drive an active invasion process
Erythrocytes lack biosynthetic pathways
Erythrocyte highly ordered cytoskeleton precludes endocytosis
Plasmodium merozoites need to:
Recognise the erythrocyte
Actively invade the erythrocyte
Modify the erythrocyte to support its own development
Erythrocyte membrane
Plasma membrane has ~20 major and ~850 minor protein involved in pritein binding, transport, signal transduction etc
Structural integrity of cytoskeleton provided by vertical interactions with different proteins, forming ankyrin and 4.1R macro protein complexes
RBC cytoskeleton - matrix consisting of spectrin, actin, protein 4.1, ankyrin and actin-associated proteins
Glycophorins are the major sialic acid-containing glycoproteins on RBC surface
Merozoite release and erythrocyte invasion
Outside the erythrocyte, merozoites cannot replicate and only remain infective for a few minutes
Brief extracellular stage - a target for immune responses
Merozoite invasion of erythrocytes takes <1 minute
Invasion
a highly organised, dynamic and multi-step process
involves sequential interaction of parasite ligands discharged from apical organelles with erythrocyte proteins
Time course of organelle secretion and RBC invasion
1 Egress (action of going out, or leaving a place) - secretes exonemes - Red cell rupture, surface proteolysis, calcium release
2 Attachment and reorientation - Micronemes - Reversible attachment
3 Formation of tight junction complex (also known as a moving junction)
4 Ingress (migration of tight junction complex)
5 Vacuole sealing
Merozoite surface protein 1 (MSP1)
> 40 protein on merozoite - trafficked to membrane during schizont stage - tethered by GPI anchors or peripheral associations with GPI-anchored proteins
MSP1 - most abundant merozoite surface protein- synthesized as 200kDa precursor cleaved into 4 fragments held together on surface as a non-covalent complex, and associate with other peripheral proteins e.g. MSP6 and MSP7
MSP1 conserved throughout Plasmodium species and is an essential protein unique to Plasmodium - antibody responses inhibit parasite replication in vitro and protect in vivo – vaccine candidate?
Surface location of MSP1 - speculation that it functions in erythrocyte invasion - MSP1 binds erythrocyte proteins glycophorin A and Band 3
MSP1 processing and PfSUB1
Minutes before egress, a serine protease ‘SUB1’ is discharged from merozoite exonemes into the PV lumen - cleaves MSP1 and partner proteins
PfSUB1 is a calcium-dependent redox switch subtilisin – a labile disulphide switch regulates PfSUB1 catalysis
In the exoneme a reducing environment maintains PfSUB1 in an inactive state preventing autolysis
But the parasitophorous vacuole (PV) is an oxidising environment and so upon discharge from exoneme, there is an oxidative reconstitution of the Cys521-Cys534 disulphide bond – this activates the enzyme
MSP1 complex
Schizont surface -> Merozoite surface -> ‘Ring’ stage surface
MSP1 processing and erythrocyte egress
Resistance of erythrocyte membrane to mechanical shear stress is dependent upon underlying cytoskeleton - in particular lattice of spectrin tetramers
Proposed that SUB1-processed MSP1 perturbs interactions between spectrin and other cytoskeletal components, such as ankyrin
Following breakdown of PV membrane, intracellular merozoites contact inner face of RBC membrane - MSP1 binds to spectrin lattice and produces shear forces that disrupt the cytoskeleton (aided by protease activity e.g. host cell calpain-1)
Erythrocyte egress is impaired if MSP1 is not processed correctly
Parasites expressing inefficiently processed MSP1 – egress delayed
Parasites lacking surface-bound MSP1 - severe egress defect
Parasites become trapped in the partially-ruptured red blood cell
What occurs immediately after egress from erythrocyte
Upon release from an infected erythrocyte merozoite experiences a rise in cytoplasmic calcium (a response to rise in potassium ion concentration encountered in external environment)
Rise in cytoplasmic calcium triggers release of micronemal proteins required to interact with erythrocyte receptors
Receptor-ligand interaction restores cytoplasmic calcium levels and results in release of rhoptry contents
Merozoite erythrocyte invasion involves a cascade of protein-protein interactions
Initial contact - reversible low affinity association – BUT these interactions are specific i.e. merozoites only attach to erythrocytes from susceptible hosts
Initial contact often occurs between long axis of merozoite and erythrocyte – elicits dynamic deformation i.e. erythrocyte surface ‘puckers up’ around merozoite
Molecules important for invasion:
MSP1 (and other MSPs) are GPI-anchored - attach only to outer leaflet of plasma membrane – other ligands span plasma membrane and link directly or indirectly to merozoite cytoskeleton
Erythrocyte Binding Ligands (EBLs; or Erythrocyte Binding Antigens)
Reticulocyte Binding Ligands (RBLs; or reticulocyte-binding family homolog - Rh )
EBLs and RBLs bind to different receptors on erythrocyte surface and irreversibly commit parasite to invasion - proteins may work cooperatively, but can also define alternative routes for initiating invasion
Structure of EBL and Rh proteins
EBA-175
EBA-140/BAEBL
EBA-181/JESEBL
Erythrocyte binding ligand1 (EBL-1)
Mrz adhesive erythrocytic binding protein (MAEBL)
PfRH1, PfRH2a, PfRH2b, PfRH4 and PfRH5 also a pseudogene PfRH3
EBL proteins (erythrocyte binding antigens - EBA) are transmembrane type 1 proteins defined by presence of cysteine-rich Duffy binding-like (DBL) domain (Region II) - named after domain present in P. vivax and P. knowlesiprotein that binds the Duffy antigen
EBL proteins - important for RBC invasion and bind to RBC in a sialic acid-dependent manner
PfRh proteins – high MW, limited sequence conservation and no identifiable domain structures
Host receptors and P. falciparum ligands involved in erythrocyte invasion
Early interactions are mediated by the MSP1 complex
EBA/PfRh ligands released – these bind with higher affinity to a range of receptors
EBAs – sialic acid-dependent pathway
PfRH2, PfRH4, PfRH5, and MSP1 - sialic acid-independent pathway
Duffy antigen aka Duffy Antigen Receptor for Chemokines (DARC) or Fy glycoprotein (FY)
DARC is a 40–45kDa glycoprotein - originally identified as blood group antigen on the surface of erythrocytes – but it is also expressed on endothelial cells
DARC binds with high affinity to some chemokines and may enhance leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium
On erythrocyte surface, DARC may play a ‘scavenging’ role i.e. to eliminate excess toxic chemokines produced in pathological situations
The role on endothelial cells is more important, since expression on endothelial cells is highly conserved - whereas DARC function on RBCs is dispensable
P. vivax and the Duffy antigen
P. vivax is widespread throughout tropics and subtropics but is absent from West Africa where > 95% of the population are Duffy negative - example of how a mutation in the human genome can results in resistance to P. vivax
P. vivax binds to Duffy antigen-positive reticulocytes
Experimental infection of human volunteers with P. vivax showed that individuals who were negative for the Duffy blood group antigen were resistant to blood-stage infection (Miller et al 1976 New Engl. J. Med. 295:302–304)
In vitro invasion studies demonstrated that when P. knowlesi merozoites interact with Duffy-negative human erythrocytes - initial interaction and apical reorientation occurs normally but MJ does not develop and invasion is aborted (Singh et al 2005 Molecular Microbiology 55(6):1925-1934)
Diversity of proteins expressed by P. falciparum
Treatment of erythrocytes with different enzymes - e.g. trypsin, chymotrypsin or neurominidase, then tested different geographical isoaltes of Pf to see if the enzyme treatment made the RBC more or less sensitive to invasion
Neurominidase is a sialidase that cleaves sialic acid (e.g. on glycophorins), and affects different receptors and reveals multiple erythrocyte invasion pathways
Distinct geographical patterns to the invasion pathways used e.g.
Gambian isolates – merozoites invade using receptors that are sensitive to all three enzymes
Kenyan isolates - merozoites invade using neurominidase-resistant receptors (i.e. sialic acid-independent pathway)
Merozoite reorientation
Merozoite reorientates to position apical end to face erythrocyte membrane
Adhesion molecules may induce local membrane curvature - apical end of merozoite point towards erythrocyte
Suggested that a concentration gradient of EBA and PfRh adhesins may increase towards apical end of merozoite and assist in reorientation
Host receptors and P. falciparum ligands involved in invasion
Apical Membrane Antigen 1 (AMA1) [microneme) and Rhoptry neck protein complex (RON) [rhoptry] proteins form a ‘tight junction’ - this is critical for merozoite invasion.
Merozoite invasion is an active process powered by a parasite derived molecular motor complex
Parasite binding to host receptors on erythrocyte surface provides a ‘foothold’ for forward movement
Cytochalasin (blocks assembly/disassembly of actin monomers) inhibits merozoite invasion and implicates the actin-myosin cytoskeleton in parasite invasion
Parasite invasion motor complex - organised around a single headed class XIV myosin (unique to Apicomplexans) which tread along dynamic, short actin filaments lying in membrane of the parasite
Plasmodium expressed both the merozoite ligand AND receptor embedded in erythrocyte membrane
Microneme proteins (AMA1 and MTRAP) on merozoite surface and rhoptry neck protein RON2 inserted into erythrocyte membrane
Intracellular C-termini of AMA1 and MTRAP (adhesins) link to short actin filaments
Actin filaments and adhesins propelled towards parasite posterior by myosin attached to inner membrane complex
Force generated through ATPase activity of myosin - resulting conformational change results in net movement of parasite
Myosin is anchored, and so does not move, transmembrane adhesins pulled through fluid plasma membrane
When adhesins reach posterior end they are cleaved by parasite proteases
Formation of a parasitophorous vacuole
- Upon reaching posterior pole, merozoite proteins at tight junction removed by protease - merozoite does not actually penetrate erythrocyte membrane but creates a parasitophorous vacuole (PV)
- PV separates Plasmodium from the erythrocyte cytoplasm – creates environment hospitable for intra-erythrocytic development
- Newly infected erythrocyte takes on stellate appearance - phenomenon known as echinocytosis - remains like this for 5-10 minutes before returning to biconcave shape
Plasmodium inside the erythrocyte
Erythrocyte remodelling by Plasmodium results in profound structural and morphological changes that are central to parasite survival
BUT remodelling results in changes to physical properties of the erythrocyte - becomes more rigid and adhesive – this results in infected erythrocytes blocking blood flow within microvasculature
Parasite-induced changes to the erythrocyte play an important role in pathogenesis of malaria.
