parasite-vector molecular interactions

Question 1

malaria lifecycle

Answer 1

• bloodstream gametocytes in bloodmeal
• exit RBCs and flagellate → gametes
• male and female gametes fertilised → diploid zygote
• → ookinete → invade midgut epithelium
• reach basal lamina → oocyst → rounds of replication
• cell division → bursting → sporozoite release
• migrate to salivary glands → inject into human
• infect liver cell → schizont → rupture and merozoite release
• invasion of RBC → trophozoite
• reinfects RBCs or forms gametocytes

Question 2

incompatible vectors

Answer 2

• some stages of plasmodium life cycle lived
• no invasion of midgut epithelium
  
  • specific receptor-ligand interaction

Question 3

vaccine targets

Answer 3

• TBVs only prevent transmission
  
  • provides herd immunity but not individual immunity
  • theoretically can target any stage
• best stage to target is pre-midgut invasion
  
  • higher levels of parasite targets (if using antibodies/immune based cells)
  • more susceptible before reaching midgut

Question 4

TBV targets

Answer 4

• gametocyte to ookinete transmission
  
  • lower numbers
  • little antigenic variation of plasmodium in mosquito
    
    • only innate immunity - less selection pressure

Question 5

drawbacks of TBVs

Answer 5

• lack of host immune boosting
• antibody titres wane over time
  
  • anitgens specific to mosquito stage of parasite
  • human will not encounter these again after vaccination
• titres may become insufficient

Question 6

events required for full malaria transmission

Answer 6

• parasite devlopment
  
  • gametogenesis, sporogonic development
• cell adhesion, entry, exit
  
  • midgut entry
  • salivary gland entry
• evasion of mosquito innate immune system

Question 7

male plasmodium gametogenesis

Answer 7

• rounding up (condensation)
• emergence from RBC membrane
• DNA replication (x3)
• mitosis (x3)
• axoneme assembly
• exflagellation

Question 8

female plasmodium gametogenesis

Answer 8

• rounding up
• emergence
• expression of P25/P28
• fewer morphological changes but slower

Question 9

P28 transcripts in females

Answer 9

• strong localisation to ookinete
• transcripts found from gametocyte to ookinete stages
  
  • FISH
• but protein found gamete stage onwards
  
  • western blotting
• parasite stores up transcript until needed on the surface
• immunisation with P28 produces potent TB antibodies

Question 10

control of gametogenesis

Answer 10

• strict control by mosquito midgut conditions
  
  • maximal efficiency of sexual reproduction and transmission
  • prevent TB immune response to prematurely produced mosquito stage antigen (in human host)
• parasite won’t risk exposing antigen to human host immune system if not needed at this stage

Question 11

factors regulating gametogenesis

Answer 11

• originally thought to be:
  
  • temperature drop (5 degrees)
  • pH increase in midgut to pH8
    
    • pH alone actually not enough
• HPLC identifies chemicals present to induce exflagellation and gametocyte activation
  
  • xanthurenic acid
• probably 2nd messengers and protein kinase cascades involved in triggering gametogenesis
  
  • potentia targets

Question 12

xanthurenic acid

Answer 12

• sufficient to produce 100% exflagellation in male parasites in combination with pH increase
• specific mosquito-derived molecule
• triggers gametogenesis
• highly specific downstream signalling pathways likely
  
  • potential for intervention

Question 13

protein kinases in gametogenesis induction

Answer 13

• identify differential expression in presence of XA
• knockout
• MAP2K knockout downstream of XA
  
  • DNA aligns at equator, axoneme separation, cell polarisation but no exflagellation
• CDPK4 knockout
  
  • almost no activity
  • plant-like → potential herbicide use
• 2 kinases acting at different stages of the pathway
  
  • activated by second messengers

Question 14

antibodies in bloodmeal

Answer 14

• can block transmission
• can agglutinate microgametes with e.g. ANTI-230

Question 15

male gamete targets

Answer 15

• P45 and P48
• target for blocking fertilisation and zygote formation
• knockout stuides identified as likely receptor for TBVs

Question 16

other intervention stages

Answer 16

• ookinete differentiation
  
  • mitochondrial formation, cytoskeleton
• traversal of midgut epithelium
  
  • cell-specific epithelial-receptor interaction
  • requires recognition, attachment, gliding apical orientation, membrane disruption, entry, traversal, egress on basal side
  • each step requires specific molecules that could be targets

Question 17

peritrophic membrane

Answer 17

• secreted by midut epithelium
• encapsulates bloodmeal
• chitin
• parasite must escape this before reaching midgut
• chitinase 1 needed
  
  • knockout → infectivity reduced up to 90%

Question 18

gliding motility

Answer 18

• actin and myosin filaents tetherered by membrane-spanning protein (CTRP in ookinete)
  
  • recognises host cell receptors
  • provides traction on host cell epithelia
• actin/myosin motor moves cell forward
• CTRP cleaved externally and left behind allowing movement
• CTRP knockout → no invasion
• midgut epithelium receptors unknown

Question 19

current model for midgut invasion by ookinetes

Answer 19

• triggers several innate immune reactions in first invaded cell
• ROS/RNS production
• cell undergoes apoptosis and is extruded
• parasite has already spread to other epithelial cells
• ookinete leaves trail of P28 across midgut epithelium

Question 20

basal lamina contact

Answer 20

• ookinete becomes dormant → oocyst
• several rounds of asexual reproduction
• some trigger results in downstream signalling in ookinete
  
  • potentially laminin
• release of sporozoites into haemocoel

Question 21

summary of plasmodium invasion

Answer 21

• early development key for transmission
• huge losses along the way with 2 severe bottlenecks
  
  • before sexual reproduction
  • before sporozoites develop
• outcome dependent on interaction between human bloodmeal, mosquito and parasite

Question 22

salivary gland invasion

Answer 22

• specific interaction between parasite and salivary gland receptors
• salivary gland from refractory mosquito
  
  • put in susceptible mosquito haemocoel
  • no infection
• susceptible salivary gland in refractory parasite → infection

Question 23

sporozoite infectivity

Answer 23

• inject sporozoites from crushed oocysts into hemocoel
• 41/41 mosquitoes → infected salivary glands
• remove sporozoites from infected glands
  
  • inject into hemocoel of another mosquito
  • no infection
• some change in sporozoites has made them less infectious
• differential expression needed to identify genes responsible

Question 24

TRAP

Answer 24

• required for gliding motility in sporozoites of salivary gland instead of CTRP
  
  • only expressed in sporozoites
  • localised to micronemes and sporozoite surfaec
  • interacts with saglin receptor on gland
• knockout → sporogonic cycle arrest

Question 25

circumsporozoite protein

Answer 25

• specifically expressed in sporozoites
• localised to micronemes and sporozoite surface
• selectively binds slaivary glands
  
  • one particular lobe
• knockout → arrest of sporogonic cycle