Lecture 8: Plasmodium - mechanisms of cell invasion

Question 1

How many countries in malaria endemic to?

Answer 1

85

Question 2

What percentage of all malaria deaths in Africa are in children under 5?

Answer 2

80%

Question 3

Which Plasmodium species is most important in humans and why?

Answer 3

Plasmodium falciparum because it accounts for about 50% of cases and causes the most severe illness

Question 4

Why is P. falciparum the most lethal malaria species?

Answer 4

• sequesters infected red blood cells in microvasculature of the brain and other organs (to avoid splenic clearance of infected RBCs)

Question 5

Describe the plasmodium lifecycle

Answer 5

• Transmitted by the bite of mosquito which inoculates sporozoite from salivary glands which travel to the liver and invade hepatocytes
• sporozoites develop into merozoites in hepatocyte which are released and invade RBCs
• erythrocytic cycle: formation of ring stage, trophozoite, schizont, release of merozoites and re-infection of RBCs continues
• some form male and female gametocytes that are taken up by mosquito during blood meal
  –> develop within midgut of mosquito, pass across gut epithelial wall as ookinete, develop into an oocyst that ruptures releasing sporozoites that travel to salivary glands

Question 6

What are the three stages of plasmodium parasite life cycle that are specialised for invasion?

Answer 6

merozoite
sporozoite
ookinete

Question 7

What are the four different apical organelles that may be present in the merozoite, ookinete and sporozoite?

Answer 7

Rhoptry
Exonemes
Micronemes
Dense granules

Question 8

True or false: Different plasmodium species have red blood cell preference?

Answer 8

True (depending on RBC antigen and age/maturity of RBC)

Question 9

Why does merozoite invasion of RBC occur very rapidly?

Answer 9

outside of RBC, merozoites cannot replicate and only remain infective for a few minutes, they are also targets of immune response and risk being cleared when extracellular

Question 10

What are the 5 steps in merozoite release and RBC invasion?

Answer 10

1. Egress
2. Attachment and reorientation
3. Formation of tight junction complex (AKA moving junction)
4. Ingress (migration of tight junction complex)
5. vacuole sealing

Question 11

Describe a protein that plays a key role in merozoite egress

Answer 11

Merozoite surface protein 1 (MSP1)
- GPI anchored
- one of the major proteins of the merozoite surface and most abundant
- synthesised as a large precursor that is cleaved into various fragments that are held together on the surface as a complex and interact with other MSP proteins

Question 12

True or false: MSP1 is only involved in merozoite egress?

Answer 12

False: also involved in recognition and invasion

Question 13

What is released minutes before egress (what is it called, where is it released from and what does it do)?

Answer 13

SUB1

released from merozoite exonemes (an apical organelle) into the parasitophorous vacuole

cleaves MSP1 and partner proteins

Question 14

What is SUB1?

Answer 14

a serine protease with a calcium-dependent redox switch

Question 15

How does the release of SUB1 into the parasitophorous vacuole allow its activation?

Answer 15

It has a calcium-dependent redox switch
- in the exoneme, a reducing environment maintains SUB1 in an inactive state, preventing autolysis
- in the parasitophorous vacuole, an extracellular-like oxidising environment results in oxidative reconstitution of the Cys521-Cys534 disulphide bond which activates the enzyme

Question 16

what is the importance of MSP1 processing by SUB1 in egress?

Answer 16

SUB1 processed MSP1 perturbs interactions between spectrin and other cytoskeletal components such as ankyrin
- merozoite interacts with the cytoskeleton and weakens the cytoskeleton for its egress

Question 17

Explain how the micronemal proteins are released upon release of merozoites from an infected erythrocyte

Answer 17

Upon release from RBC, the merozoites experience a rise in cytoplasmic calcium in response to the rise in K+ concentration encountered in the external environment
- this rise in calcium triggers release of proteins from microneme

Question 18

What is the purpose of microneme proteins?

Answer 18

interact with erythrocyte receptors

Question 19

How does the release of micronemal proteins result in the release of rhoptry contents?

Answer 19

micronemal proteins interact with erythrocyte receptors.
this receptor-ligand interaction restores the cytoplasmic calcium and results in the release of rhoptry contents

Question 20

Other than MSP1, what other two protein families are important for merozoite attachment?

Answer 20

Erythrocyte binding ligands (EBLs)

Reticulocyte binding ligands (RBLs)

Question 21

What causes the irreversible commitment of a merozoite to invasion?

Answer 21

binding of EBLs and RBLs to different receptors on the RBC surface

Question 22

Which RBC antigen is particularly important for the Plasmodium vivax invasion?

Answer 22

Duffy antigen

Question 23

What is the role of the Duffy antigen?

Answer 23

expressed on surface of RBCs and endothelial cells and binds with high affinity to some chemokines

• may play a scavenging role on RBC surface to eliminate excess toxic chemokines produced in pathological situations and dampen down the immune response

Question 24

Why is P. vivax widespread throughout the tropic and subtropics but absent from West Africa?

Answer 24

because more than 95% of the population are Duffy antigen negative

Question 25

What is the importance of P. falciparum expression of an array of different adhesins?

Answer 25

bind to different erythrocyte receptors (10 have been identified to date)
- allows them to counteract the extremely polymorphic nature of RBC surface receptors and the heterogeneity within the human population of expression of different RBC proteins

Question 26

Describe the host receptors and P. falciparum ligands involved in erythrocyte invasion (early and committed interaction)

Answer 26

Early interactions are mediated by the MSP1 complex in a sialic acid-independent pathway

Micronemal proteins released EBLs and RBLs that bind with a higher affinity to a range of receptors

Question 27

What follows the initial attachment to the RBC involving weak binding with MSP1?

Answer 27

Reorientation of the merozoite so the apical end faces the RBC membrane and strong binding involving micronemal EBLs and RBLs.

Question 28

Do EBLs and RBLs both bind to RBC receptors in a sialic acid dependent pathway?

Answer 28

No, only EBLs bind in a sialic acid dependent pathway, RBLs bind in a sialic acid-independent pathway

Question 29

What does sialic acid dependent binding to RBC surface mean?

Answer 29

binds to RBC surface via glycophorins (which are the major sialic acid containing glycoproteins on RBC surface)

Question 30

Describe the formation of the tight junction/ moving junction critical for invasion of the merozoite into the RBC

Answer 30

Apical Membrane Antigen 1 (AMA1) and MTRAPs released from microneme into merozoite surface membrane

Rhoptry neck protein complex (RON) released from rhoptry that are inserted into the red cell membrane

These RON proteins interact with the AMA1 to form a tight junction

Question 31

True or false: merozoite invasion is a passive process? why/why not?

Answer 31

False: merozoite invasion is an active process since RBCs cannot phagocytose

Question 32

What drives the active invasion of merozoites into the RBC?

Answer 32

Powered by myosin derived motor
- parasite binds to host receptors
- motor complex is organised around a single headed class XIV myosin (unique to apicomplexans) that tread along dynamic short actin filaments lying in the membrane of the parasite.

Question 33

How does cytochalasin suggest an actin-myosin role in parasite invasion?

Answer 33

cytochalasin blocks the assembly/disassembly of actin monomers
- was shown to inhibit merozoite invasion

Question 34

Describe the steps involved in merozoite invasion following formation of a tight junction

Answer 34

micronemal proteins AMA1 and MTRAP form tight junction with rhoptry neck proteins RONs
- intracellular C-termini of AMA1 and MTRAP link to short actin filaments in merozoite cytoplasm
- myosin anchored within intracellular environment to the inner membrane complex (that is attached to the merozoite cytoskeleton)
- the force generated through ATPase activity of myosin pulls on the actin filament and the adhesins (AMA1 and MTRAP) moving them through the fluid plasma membrane from the apical surface to the posterior end and forcing the merozoite into the RBC

Question 35

Once the adhesins reach the posterior parasite, what happens to them?

Answer 35

They are cleaved by parasite proteases

Question 36

Describe the formation of the parasitophorous vacuole post-invasion

Answer 36

upon reaching the posterior end, the merozoite proteins at the tight junction (AMA1-RON complex) is cleaved by proteases and the merozoite is contained within a parasitophorous vacuole formed from the RBC membrane

Question 37

What is the purpose of the parasitophorous vacuole?

Answer 37

separate the plasmodium from the erythrocyte cytoplasm and creates an environment hospitable for intra-erythrocytic development

Question 38

What are the results of plasmodium modifying and remodelingl the erythrocyte environment to allow parasite survival?

Answer 38

remodelling results in changes to the physical properties of the erythrocyte that make it:
- more rigid (cytoskeleton)
- more adhesive
–> block blood flow in microvasculature