Lecture 5 - Plasmodium - Intracellular survival and modification of the host cell

Question 1

Erythrocytes

Answer 1

• No antigen presentation
• Haemoglobin rich environment - source of amino acids

Mature erythrocytes are terminally differentiated and lack:
- Nucleus
- Machinery to trasnport proteins to subcellular locations

Parasite has high nutritional demand - can’t be satisfied by host erythrocyte

Plasmodium remodels erythrocyte to ensure its own survival

Question 2

Development of plasmodium (intra-erythrocytic)

Answer 2

Ring - 0-5 hours (parasitophorous vacuole membrane and Parasitophorous vacuole form)

Early trophozoite - 5-10 hours (food vacuole, tubulovesicular network, and maurer’s clefts form)

Mid-late trophozoite - 10-20 hours (Knobs form)

Schizont - >40 hours

Question 3

Plasmodium digestive vacuole and haemoglobin

Answer 3

Haemoglobin ~95% of erythrocyte cytosolic protein

Plasmodium digests 60-80% of Hb in specialised acidic organelle - digestive vacuole

Hb breakdown releases amino acids for Plasmodium to synthesize new proteins

Plasmodium has limited capacirt for de novo amino acid synthesis - requires importation of rare (e.g. met, cys, gln, glu) or absent amino acid (Ile)

Hb digestion releases the toxic waste product heme - this is neutralized by formation of hematin dimers that biocrystallise to chemically inert haemozoin

[mode of action of chloroquine – prevents haemozoin formation]

Question 4

Haemoglobin digestion

Answer 4

~16% of amino acid generated by haemoglobin breakdown are used by Plasmodium to make parasite proteins

Uptake and digestion of haemoglobin by Plasmodium could serve another purpose e.g. prevent erythrocyte lysis

Plasmodium increases in volume (~25-fold) during intra-erythrocytic development - degradation of cytoplasm necessary to create space for parasite growth

Diffusion of haemoglobin-derived amino acids into host cell cytoplasm might help regulate intracellular osmolarity of infected erythrocyte during parasite development

Question 5

Protein export into infected erythrocyte

Answer 5

~5% plasmodium proteins exported to erythrocyte cytosol
-> Increases nutrient uptake from blood plasma
-> Remodels red blood cell for parasite’s benefit
-> Facilitates adhesion of infected cell to endothelial cells

Question 6

How do parasite proteins reach final destination?

Answer 6

Must transverse:

• Parasite membranes
• Parasitophorous vacuole membrane (PVM)
• (In some cases) erythrocyte membrane reaches cell surface

Question 7

Export of protein into erythrocyte

Answer 7

• Parasite establishes trafficking network in host cell e.g. Maurer’s clefts
• Large, flattened membraneous structures - bud from PVM in early ring-stage development - migrate towards erythrocyte membrane - physically tether as ring -> trophozoite

Question 8

Plasmodium export element (PEXEL)

Answer 8

• PEXEL motif in most exported proteins
• 15-30 residues downstream of hydrophobic signal sequence
• Particularly abundant in P. falciparum

PEXEL motif is set of 5 residues - RxLxE/Q/D (x=any uncharged amino acid)

Question 9

Function of PEXEL

Answer 9

Twofold function

1. Enables identification of PEXEL proteins for export and cleavage
2. Uncovers export signal at ‘new’ N terminus to direct mature protein to host cell

Question 10

Processing of PEXEL exported proteins

Answer 10

1. Proteolytically cleaved - RxL↓ xE/D/Q
2. N-acetylated (i.e. addition of acetyl group) - Ac-xE/Q/D

• PEXEL motifs recognised for cleavage by aspartyl protease (plasmepsin V) - occurs in ER and uncovers export signal for trafficking

PEXEL arginine (R) and leucine (L) residues required for recognition and cleavage by plasmepsin V and fifth amino acid (E/Q/D) important for protein export (post-cleavage)

If arginine (R) or leucine (L) residues are mutated to alanine, proteins remain trapped in the ER (cleavage required to release N terminus from ER membrane); mutation of fifth residue (E/Q/D) proteins are trapped within vacuolar space

Question 11

PEXEL-negative exported proteins (PNEPs)

Answer 11

• No N-terminal hydrophobic signal sequence
• No PEXEL motif’
• No conserved export sequences

Question 12

How are PNEPs exported?

Answer 12

Export signal that functionally equivalent to N-terminus present on plasmepsin V-cleaved PEXEL proteins

Possible to replace the N-terminus of (some) PNEPs with the N- terminus of a cleaved PEXEL protein

Question 13

Plasmodium translocon of exported proteins (PTEX)

Answer 13

~1.2 mDa complex

• Located in discrete regions of parasitophorous vascuole membrane - transports PEXEL and PNEP proteins

Comprised of homo-oligomers of three components:
Heat shock protein 101 (HSP101) - located on cisside of PVM and the molecular motor of the PTEX
PTEX150 - plays structural role and regulates stability of the PTEX
Exported protein 2 (EXP2) - proposed protein-conducting channel in PVM that facilitates protein transport