Module 3: MALARIA

Question 1

Describe the life cycle of malaria

Answer 1

3 stages of malaria:

1. mosquito stage (sexual reproduction= definitive host)
2. liver stage (asexual reproduction)
3. blood stage (asexual reproduction, major amplification stage)

mosquito stage: mos bites human, ingests gametocytes, microgametes penetrate macrogamete producing zygotes –> zygotes become motile & elongate (ookinetes) –> become oocytes –> grow & meiosis –> sporozoites –> sporozoites penetrate gut wall –> salivary glands –> mos bites another human injects sporozoites

liver :sporozoites infect macrophage-like liver cells –> become schizonts –> release merozoites (can lay dormant in liver up to weeks/years, then release when conditions are right ) –> asexual reproduction

blood: asexual reproduction, major amplification.
merozoite infects rbc –> rbc sticks to blood vessel wall (cytoadherence), moves out of circulation –> asexual reproduction produces more merozoite that busts out to infect more rbc

some parasites differentiate into gametocytes –> injested by mosquito again

Question 2

why is malarial infection delayed?

Answer 2

when mos bites- there are few sporozoites injected into humans. in the liver, the parasite can lay dormant for up to weeks-years, will only get symptoms when it invades the bloodstream (rbc)
DISEASE only occurs in the bloodstage

Question 3

what are the symptoms of malaria

Answer 3

• fever/ chills/ anemia (might not get symptoms up to 2 weeks after bitten)
• most deaths result from P.falciparum
  1. cerebral anemia
  2. severe anemia
  3. placental anemia
• P.vivax= high morbidity, low mortality (high disease, low death)
  why? because it releases hypnozoites (latent form that can live in the liver for years- explains why some peoplekeep getting malaria)

Question 4

what is the purpose of cytoadherence/ sequestration of malaria

Answer 4

to avoid splenic clearance
- usually blood moving through spleen filtered through fenestrations that remove old/damaged cells by macrophage like clearance system

but in malaria- merozoites infect rbc and cuase it to stick to blood vessel wall- doesnt move to spleen to get cleared & can replicate

Question 5

what is hemozoin and what is its function

Answer 5

Hemozoin is the disposal product of heme, formed when parasite infects rbc & digests heme)

Parasite infects rbc (needs nutreints & space to grow) so it digests Hb  releases digested haem, becomes hemozoin ( since haem is very toxic, digested haem polymerised into crystal called hemozoin)

Question 6

why doesnt every child infected with malaria die?

Answer 6

different patterns of cytoadherence. PfEMP1 will cause rbc to adhere to blood vessel of different sites

because the PfEMP1 is specific. 60 different var genes determine the shape of PfEMP1 = infected rbc will express specific PfEMP1 that targets specific rececptor on microvascular endothelium (Blood vessel wall) of different organs

Question 7

what is responsible for the WAVES of parasitemia/ infection in malaria

Answer 7

PfEMP1 specificity is constantly changing to escape the body’s immune system.

specific var gene encodes for specific PfEMP1 gene that dictates which receptor on the blood vessel of which site the infected rbc will bind to. first wave may be a PfEMP1 that causes rbc to bind to skeletal muscle blood walls –> body builds immunity to this (eg. might make antibodies that target & destroy this specific PfEMP1) –> then the parasite can mutate = change var gene of PfEMP1 –> new PfEMP1 now targets diff site (brain) = a new wave of infection that body has not yet made an immune response to

immune system is always a step behind

Question 8

what is the purpose of P.falciparum continously changing the PfEMP1 var genes

Answer 8

since P. falciparum does not have a latent form (like P.vivax does to persist in host until conditions are right). IN order to increase its survival it needs to persist in the host for as long as possible to incrase chance it can transmit to ANOTHER person

Question 9

What are the var genes ?

Answer 9

var genes encode the PfEMPs

• there are approx 60 var genes per genome
• they are located on 14 chromosomes, particularly concentrated at the ends

Question 10

how does parasite change the var genes?

Answer 10

Clonal antigenic variation= allows parasite to switch to use different genes

• these genes are epigenetically modified (histone modification + nuclear location)
• by changing gene expression, can switch on / off genes to change PfEMP1

Question 11

Explain why placental malaria causes reinfection of previously recovered patients

Answer 11

women who are infected as kids, still have parasite in blood dont get sick anymore because their bodies have developed immunity against the previous pfemp1. When a female gets pregnant, the placenta- new organ- forms and the parasite induces a new type of PfEMP1 that targets the CSA on syncitiotrophoblasts in the placenta. This means the patinet will get sick again because previously there had not been the selective force present to cause the body to build immunity against this specific pfemp1 that targets csa

Question 12

how are var genes silenced while allowing only 1 to be expressed?

Answer 12

the Pfemp protein is encoded by 60 different var genes, located on 14 chr, particularly concentrated at the ends.

each time a pfemp protein is expressed, 1 var gene is switched on whilst the other 59 are silenced by HETEROCHROMATIN.
- limits Transcription factors accessibility to the DNA in order to activate gene transcription

1. epigenetic control of transcription
2. histone modification /heterochromatin at the nuclear periphery = genes are inaccessible to transcription factors
3. a particular LOCALISATION is assocaited with new histone modification that permits transcription= NEEDS CSA SELECTION:

on the condition of CSA SELECTION: the silenced var2csa gene will physically move to the TRANSCRIPTIONALLY ACTIVE SITE where TFs are able to access the DNA, transcribe those gene, making a new pfemp1 protein that speicifcally targets the csa on placental blood walls

Question 13

why is there more heterogeneity in var genes compared to other genes

Answer 13

ectopic recombination

- end of chr align , recombination of NON-HOMOLOGOUS chromsomes

Question 14

what is the purpose of gene clustering at the end of p.falciparum chr

Answer 14

1. heterochromatin formation= to silence the 59 /60 genes so to express a specific type of pfemp1
2. to allow for RECOMBINATION BETWEEN HETEROLOGOUS CHROMSOMES= ectopic recombination= where NON-homologous chr align to produce high heterogeneity (variance) among var genes

Question 15

describe the function of KAHRP & what happens when KAHRP is knocked out

Answer 15

kahrp gene codes for knob protein

• knob is essential for cytoadherence- it stabilises cytoadherence of Pfemp1 on parasite on blood wall
• knob knockout = parasite infected rbc isnt able to stick of blood walls = no cytoadherence

Question 16

how do malarial parasites export proteins

Answer 16

10% of the parasites protein genome is exported to the surface, along with pfemp1 protein.

• these proteins have many functions includes:
  1. getting the pfemp1 to surface
  2. proteins also help export other proteins - this information is contained in a sequence at the end terminal region of exported proteins (ER SIGNAL SEQUENCE + PEXEL MOTIF)
  3. exported proteins have virulence + SURVIVAL / GROWTH FUNCTIONS

Question 17

how are the proteins that the malarial parasite code for exported?

Answer 17

through the pexel protein-export translocon

• links many functions together
• block pexel protein translocon= block proteins from eliciting their function (impeded survival, growth, virulence of parasite)

2 IMPORTANT SITES IN EXPORT:

1. ER MEMBRANE
2. PARASITOPHOROUS VACUOLE MEMBRANE

Question 18

what is the function of plasmepsin v enzyme

Answer 18

ER signal sequence –> PEXEL motif

• on the PEXEL motif is a cleavage site
• plasmepsin v enzyme recognises & cleaves this PEXEL cleavage sit in the ER –> makes the end terminus of all exported proteins have the sequence:

x (any amino acid), E, Q, D

–> this guides the proteins via vesicular traffic –> moves to a place with the pexel translocon = where PTEX (a pexel translocon) is present –> which allows proteins to be translocated across membrane

must have plasmepsin enzyme to cleave the pexel motif and give the end terminus of exported proteins that sequence- the sequence is essential to guide them to the ptex/pexel translocon

Question 19

what is the ptex. what is the key criteria (4)

Answer 19

ptex is the putative (possible) pexel translocon

• responisble for translocating exported proteins
• ptex protein is essential for growth (reduced exp of ptex protein by conditional knockdown- found that the more the gene was knocked down, the faster and larger no. of parasites died)

key criteria:

1. plasmodium specific & in the correct location
2. essential to blood-stages
3. has an energy source- an unfolding mechanism
4. binds to transiting cargo PEXEL proteins

Question 20

what is conditional knockdown and why is it useful

Answer 20

Conditional knockdown is used instead of knocking out essential genes in an unspecific manner (where it could just cause the parasite to die all together). Which means that

1. yes it probably means Ptex protein is important
2. but you cant assign a fucntion to it since the parasite is dead before u can see how absence of ptex would affect parasite

rather, you introduced a compound like GlcN to destabilise the RNA –> cause decreased expression of PTEX protein

1. can confirm ptex’s function
2. see how reduced ptex can change the parasite

• found that the more u knocked down the gene coding for pxel protein, the faster and larger no. of parasites died
• useful for drug design

Question 21

function of pexel motif

Answer 21

PEXEL= TAG on most exported proteins that alloes it to move to the TRANSLOCON (which allows export across parasitophorous vacuole membrane into the rbc cytosol)
Block pexel protein-export= block function of proteins involved in survival + virulence of parasite

Question 22

how might understanding PTEX allow for drug development?

Answer 22

ptex inhibitors = can prevent the exported protein from translocating across the parasitophorous vac membrane into the rbc cytosol = prevent the paraistes proteins from inseting to the rbc’s surface

Question 23

list 3 types of potential malaria vaccines

Answer 23

1. pre-erythrocyte = block parasite from entering/maturing in the liver
   - vaccine against RTS,S (surface protein on parasites)
   - vaccine against whole protein (targets all proteins expressed on parasite surface)
2. transmission blocking
   - vaccine prevents transmission in the mosquito
   - make recombinant protein that mimics parasite –> inject in human –> human makes antibodies against parasite –> parasite bites human & injests antibodies –> mosquito cant develop the parasite so it wont pass it on
   - controversy = not actually protecting infected human
3. blood stage (anti-merozoite)

Question 24

why is the blood stage of malaria good target for vaccination

Answer 24

1. because we know that to develop immunity against malaria- humans develop antibodies against the ligands expressed on parasite surface
2. blood stage is the major amplification stage
3. we know that rbc are highly polymorphic (each type of rbc expresses a different set of proteins/receptors)

Question 25

what is the process that plasmodium falciparum infects cell & export proteins

Answer 25

- rbc is highly polymorphic - P.falciparum has 2 major gene families = expresses different ligands on surface in response to SELECTION PRESSURE (immune pressure) 1. parasite binds to rbc (ligand-receptor interaction : eg, EBA175 ligand on parasite binds sialic acid on Glycophorin A receptor of rbc) PRIMARY CONTACT= parasite has polarity SIGNAL = when parasite touches rbc, there are signals that tell proteins to come out SECONDARY INTERACTIONS= parasite rolls around rbc surface until apical side touches rbc & forms TIGHT JUNCTIONS ACTIN MYOSIN MOTOR IN PARASITE = draws parasite in resealing= grey surface coat of proteins are removed parasite enters rbc --> takes some membrane lipid with it --> wraps itself in parasitophorous membrane 2. it expresses on the surface of rbc: 1) specific Pfemp1 protein (according to var genes transcirbed) 2) 10% of parasites genome = these proteins are exported protein roles include: 1. survival / growth (nutrient uptake) 2. virulence factors 3. proteins that help export pfemp1 4. found that on the end-terminal region of these genes that encode exported proteins - has information encoded that helps with parasite protein export (like PEXEL + ER signal sequence) 3. plasmepsin v enzyme recgonsises & cleaves pexel cleavage site in ER RxLxE/Q/D --> RxL + x/E/Q/D --> pexel is now ready to move to the ptex translocon 4. protein (pexel) moves through ptex --> ptex binds cargo pexel protein --> unfolding it, moves through pore, then refolds on the other side - -> ptex moves it across the PV membrane into the cytosol of rbc

Question 26

why is P.falciparum able to persist despite highly polymoprhic rbc ? or how does it cope with rbc polymorphism

Answer 26

P.falciparum are able to express different LIGANDS to bind to receptors on rbc surface - parasite switches ligands according to DIFFERENT SELECTION PRESSURES - diff P.falciparum in different places have 99% same genome, but that 1% genome encodes for ligands are different (different genome for var genes, eba genes + RH genes = enode diff pfemp1, eba, rh ligands)

Question 27

what are the primary & secondary actions that are vaccine targets

Answer 27

Primary contact – merozoite has polarity= apical + distal end 2) Signal = when parasite touches rbc, tells proteins to come out (most likely signal transduction cascade) 3) Secondary interactions - parasite rolls around surface until apical end touching  forms tight junction 4) Actin -myosin motor in parasite draws parasite in 5) Resealing: Grey surface coat of proteins are removed

Question 28

how does immunity to malaria develop

Answer 28

- younger you are = the more malaria episodes u have per person per year - immunity to severe malaria develops quickly - BROAD + robust immunity to malaria develops SLOWLY - placental women return to susceptible state (placenta)

Question 29

what are the 2 types of gene families that parasite encodes for

Answer 29

1. eba - 5 EBA ligands - EBA 175 on parasite binds sialic acid on GLyA of rbc 2. pfRH - 5 RH ligands all together = 5EBA + 5Rh ligands = express 10 different ligands

Question 30

what drives evolution of different parasite ligands?

Answer 30

1) rbc polymorphism = diversity of receptors on its surface | 2) immunity = body developing antibodies against those ligands

Question 31

how is plasmodium falciparum able to avoid the immune system

Answer 31

1. by switching on/off var genes = to code for different PFEMP1 gene / protein = adhere to blood walls in different tissues - there are 60 var genes, coded in 14 chrs at ends - var genes switch on/off according to: 1) epigenetic control (histone modificartion 2) histone modification 3) HETEROCHROMATIN = winds dna up into tight bundles = disable tf access to dna to transcribe genes 4) a particular localisation associated with NEW histone modification that permits transcription - in the presence of CSA selection = silenced var gene physically moves to transcriptionally acitve site 2. by changing the ligands expressed - EBA or PFRH gene family

Question 32

describe similarities and differences between a W2mef parasite and M2mefdelta175 parasite

Answer 32

similarity of w2mef + w2mefdelta175 serum - similar genome overall excpet for surface ligands: EBA and Rh different: - W2mef = sialic acid dependant. parasite expresses eba175 to bind to sialic acid of glyA on rbc surface - -> selection pressure = body does not have immunity agaisnt EBA175, but has immunity against RH4 pathway - W2mefdelta175= sialic acid independant doesnt use eba175, cos the body has developed immunity agaisnt it - instead expresses rh4 family

Question 33

Explain how a single drug could possibly target hundreds of different proteins in the malarial parasite.

Answer 33

1) the drug would ideally neutralise a conserved/common region of the protein that is present in all types of malarial strains. For example: the PEXEL motif sequence is the tag that allows all exported proteins to move through PTEX. So by targeting PEXEL = could block lots of these exported proteins from moving through the PTEX pore across the PV membrane. 2) The drug could block a COMMON pathway of invasion used by multiple dif

Question 34

what are the issues with blood stage vaccine?

Answer 34

antigenic diversity & different invasion pathways - pfemp1 - eba - pfrh

Question 35

what are 2 types of immunity against malarial infection

Answer 35

anti-severe immunity develops quickly, anti-parasitemia immunity (braod + robust) immunity develops slowly 1) anti sever immunity = production of antibodies against PfEMP1 = allows infection with less severe phenotype, parasite still remains in body, but unable for infected rbc to bind to blood walls - developed first few years of children life - immunity depends on pfemp1 expression = child can still be infected by new pfemp1 (either from new infection or from pfemp1 mutation within the body) - malaria parasites still exist in body, less severe phenotye, not enough to cause severe illness 2) anti-parasitemia immunity = when body makes antibodies against blood stage= against merozoites - immunity develops later in life after anti-pfemp1 ab - but provides more strong and robust immunity = antibodies are able to target many different strains of malaria = as it targets a universally conserved pathway of malarial infection = which is to blockk the primary + secondary interactions bw merozite + rbc --> prevent rbc from getting infected - malarial parasites still in blood, but decreases infection of rbc = - provides evidence that blood-stage vaccines are important = as this is where the pathogenesis occurs

Question 36

where are we towards a blood stage vaccine?

Answer 36

1. still some way off: empirical vaccines dont always work (for eg. recombinant form of human antibodies doesnt always work) 2. most antigens on merozoite surface are identified (but dont know which one is best - 50-100) 3. little functional knowledge known about the individual antigens 4. new genomic technologies are unravelling function 5. rational vaccine design becomes feasible

Question 37

WHY IS IS SO HARD TO MAKE VACCINE?

Answer 37

(1) ANTIGENIC DIVERSITY - Malaria able to switch its use of surface ligands due to selection pressures (immune system makes ab against eba175… / gene knockouts) (2) Need vaccine that is able to target multiple pathways, others if ab can only bind 1 surface ligand= no use (3) POINT MUTATIONS = make EBA175 in different isolate slightly different, even though they bind to the same GLY A Hard to make vaccine = bc of selection pressures that cause malaria to switch it’s use of surface ligands