Malaria

Question 1

What are the 3 stages in the life cycle of a malaria parasite?

Mosquito (Female Anopheles)

Answer 1

1. Mosquito stage - sexual reproduction
   – Stages: Gamate → Zygote → Ookinete
2. Liver stage - asexual reproduction)
   – Hepatocyte stage → Sporozoites infect hepatocytes → Merozoites form & exit hepatocytes
3. Blood stage - asexual reproduction, major amplification stage
   – disease occurs only in this stage
   – Merozoites infect erythrocytes = divide rapidly → Gametocytes form
4. Mosquito ingests gametocytes from human host, sexual reproduction in mosquito

Question 2

What is PfEMP1?

Answer 2

a parasite protein

Question 3

What is cytoadherence/sequestration - mechanism of PfEMP1?

Answer 3

When an infected cell sticks itself to the vascular endothelial wall of a blood vessel in order to avoid splenic clearance

• spleen can recognise the altered red blood cell = remove it through reticular endothelial clearance mechanism
• altered red blood cell: merozoit form that gets released to infect the red blood cell

Question 4

What is the concequences of PfEMP1?

consequence of its mechanism

Answer 4

As parasite digests haemoglobin (thats full of iron) = crystallises it into gold metal

• enters brain = cerebral malaraia

Question 5

What are the possible places the parasite can go to?

Answer 5

• Brain = cerebral malaria
• Placenta = placental malaria
  *

Question 6

What gene encodes the PfEMP? How many chromosomes?

Answer 6

var gene
14 chromosomes
ALSO: around 60 copies per genome but only ONE is expressed at any one time

Question 7

How many promoters drive the var genes?

Answer 7

3 different promotors

upsA, upsB, upsC

Question 8

Explain why previously immune women in endemic malarial regions experience malarial disease during their first pregnancy

Answer 8

Placental Vulnerability

• women were not exposed to placental parasites before

var2CSA gene

• gene allows parasite to stick to placenta, rich in sugar (CSA)

Selective Pressure

• non-pregnant women = parasite primarily infects red blood cell with less CSA
• placental infection = new, ideal environment for placenta with var2CSA

CSA = Chondroitin Sulfate A

Question 9

How are most var gene ‘silenced’ while allowing just one to be expressed?

Answer 9

• access to transcription factors which allows var gene to be expressed or silenced
• parasite uses combination of physical location & chemical modifications (epigenetics) to control var gene expression

SILENCED:

• Heterochromatic = DNA physically wrapped up tightly = transcription factors hard to access
• = chromosome ends cluster at nuclear periphery = in a ‘silent’ heterochromatic state
• ALSO: histone modification = genes inaccessible to transcription factors

EXPRESSED:

• Epigenetic switch = parasite can activate specific var gene === loosening chromatin structure OR changing histone modification
• Antigenic variation = switch allows different expression of PfEMP1 protein on parasites surface = allows evasion to immune system = recrudescence (reappearance of symptoms)

Question 10

Describe the process Plasmodium falciparum uses to export proteins

Answer 10

1. Signal Peptide & ER entry
2. Pexel Motif Cleavage
3. Vesicular Pathway
4. PTEX Translocon

Question 11

What occurs during the ‘Signal Peptide & ER entry’ step.

STEP 1

Answer 11

• proteins for export have signal sequence at N-terminus
• sequence helps them enter ER through standard cellular mechanism

Question 12

What occurs during the ‘Pexel Motif Cleavage’ step?

STEP 2

Answer 12

• ER = contains specific protease = Plasmepsin 5 = cleaves protein at “pexel motif” (RxLxEQD)
• This cleavage useful for targeting protein for export

Question 13

What occurs during the ‘Vesicular Pathway’ step?

STEP 3

Answer 13

• the cleaved proteins = directed to specific vesicular pathway for transport
• exact detail not clear BUT maybe a licensing mechanism = based on presence of pexel motif cleavage

Question 14

What occurs during the ‘PTEX Translocon’ step?

STEP 4

Answer 14

• PVM = membrane surrounding parasite within host red blood cell
• proteins arrive at PVM
• PTEX acts as a translocon = channel that allows protein to cross membrane & enter exported space between parasite and red blood cell
• PTEX subunits:
  – EXB2: A channel-forming protein.
  Hitchhock Protein 101: An AAA+
  – ATPase that provides energy for the translocation process.
  – Accessory proteins: PTX150, PTX88, and thioredoxin (functions not fully understood)

PVM = Parasitophorous Vacuole Membrane

Question 15

What is KAHRP useful for?

Answer 15

a export protein that is essential for ‘knob’ formation and adherence

Question 16

What is the KAHRP ‘knockout’?

Describe the process

Answer 16

1. plasmid replicates in parasite
2. goes to targeting gene (KAHRP) and squeezes in the plasmid
3. causing invasion into red blood cell
4. infection = red blood cell surface does not have knobs on it
5. when in circulation = instead of binding it bounces off

Question 17

What are the ‘knob’ structure useful for?

Answer 17

To be an anchor point for PfEMP1
* otherwise it just gets ripped off easily

Question 18

Why is the Protein Export Machinary a target for malaria drug development?

Answer 18

Essential for many proteins

• disrupt export as 10% of parasite genome encodes exported proteins

Plasmepsin 5

• protease = cleaves pexel motif of protential drug target
• inhibit to prevent proteins from being properly licensed for export

PTEX Complex

• drugs that block = prevent exported proteins from reaching destination

Disrupting Multiple Functions

• disrupt function of many exported proteins = parasite unable to develop resistance

Question 19

What is the pexel motif ‘RxLxEQD’?

Answer 19

predicts other exported proteins

• presents a downstream of signal sequences in many exported proteins

Question 20

What are the function of exported proteins?

Answer 20

Various functions within exported space between parasite and red blood cell

• Nutrient uptake and waste removal
• Protein folding
• Cytoadherence (sticking to red blood cells)
• Structural modification of the infected red blood cell

Question 21

What is the conditional knockout technique?

Answer 21

creating a parasite that can only survive in the presence of a specific compound (e.g. glucosamine)

Question 22

List & Describe the three types of potential malaria vaccines

Answer 22

1. Pre-erythrocytic (eg, RTS,S, whole parasite)
   – targets liver stage
   – aims to prevent parasite from establishing infection in liver
2. Transmission blocking
   – targets sexual stage of parasites life cycle
   – aims to prevent transmission of parasite from human to mosquito
3. Blood-stage (anti-merozoite)
   – targets stage where parasite infects red blood cells and multiples
   – aims to prevent parasite from causing disease and symptoms

Question 23

Why is the malarial blood stage a good target for vaccination?

Answer 23

Amplification & Disease

• stage where parasites multiple rapidly

Vaccine Target Accessibility

• parasite accessible to host immune system
• any immune response against parasite = directly interact with them = neutralise impact

Invasion Process

• interaction between parasite ligands & receptors on red blood cell surface

Speed of Invasion

• fast = small window for parasite to invade red blood cells
• interfering with process = reduce parasite load

Question 24

Describe the process by which merozoites invade red blood cells

Answer 24

1. merozoites makes contact with RBC
   – involves surface proteins (GPI-anchored surface proteins)
2. merozoites reorients itself AT
   – apical end (specialised for invation) is positioned correctly - RBC membrane
3. merozoite forms tight junction with RBC membrane
4. parasite secretes ligands from apical organelles = interacts with specific receptors on surface of RBC
5. merozoite injects ligand into RBC = tight junction = parasite-encoded proteases removes surface coat of RBC
6. upon entry, infected RBC = changes = becomes speculated & dehydrated
   – deformation = calcium flux induced by parasite
7. merozoite is completely internalised within RBL, enclosed within parasitophorous vacuole = multiples further and cause further infection

RBC = red blood cells

Question 25

What are the selective pressure that can alter receptor-ligand expression in Plasmodium?

Answer 25

Selective pressure can alter receptor-ligand expression in Plasmodium through a process of evolutionary adaptation driven by the host immune response. Here’s how it happens:

Polymorphism of Red Blood Cells:
Red blood cells exhibit polymorphism, including variations in surface proteins and receptors.
Interaction with Plasmodium:
Plasmodium parasites interact with these polymorphic receptors on the surface of red blood cells during invasion.
Selective Pressure:
The host’s immune system exerts selective pressure on Plasmodium parasites, favoring those parasites that can effectively invade specific red blood cell types.
Host immunity targets parasite ligands, which are involved in receptor-ligand interactions during invasion.
Evolutionary Response:
Plasmodium parasites evolve to adapt to this selective pressure by altering the expression of their receptor-ligand pairs.
Variation in Ligands:
Parasites may switch between different ligands, allowing them to invade different types of red blood cells.
Example - EBAs and RHs:
For instance, Plasmodium falciparum can use erythrocyte binding antigens (EBAs) or reticulocyte binding proteins (RHs) for invasion.
EBAs and RHs interact with different receptors on the surface of red blood cells.
Switching Ligand Expression:
Under selective pressure, the parasite can switch between using EBAs and RHs, depending on the availability of specific receptor types on the surface of red blood cells.
In summary, the interaction between Plasmodium parasites and host red blood cells is dynamic and subject to evolutionary adaptation. Selective pressure from the host immune system can drive changes in receptor-ligand expression in Plasmodium, allowing the parasite to adapt to different host environments and evade immune responses.

Question 26

Describe the basis of the most common rapid diagnostic test for malaria & explain why new tests are required

Answer 26

Detection of Parasite Antigens:
The RDT detects specific antigens produced by the malaria parasite, typically histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH), or aldolase.
Principle of the Test:
A small amount of blood is collected from the patient via finger prick.
The blood sample is applied to the sample pad of the RDT device.
If the sample contains malaria antigens, they bind to specific antibodies on the test strip.
This antigen-antibody binding results in the appearance of colored lines on the test strip, indicating a positive result for malaria infection.
Interpretation of Results:
A positive test result is indicated by the appearance of colored lines in the test and control regions of the test strip.
A negative test result is indicated by the appearance of a colored line only in the control region.
The absence of any colored lines indicates an invalid test and necessitates retesting.
Why New Tests are Required:

Emergence of Antigenic Variation:
Some strains of Plasmodium falciparum, the most deadly malaria parasite, have evolved to produce reduced or undetectable levels of HRP2.
This antigenic variation compromises the sensitivity of HRP2-based RDTs, leading to false-negative results.
Impact on Malaria Diagnosis:
False-negative results can delay appropriate treatment, leading to increased morbidity and mortality.
In areas where HRP2-based RDTs are commonly used, false-negative results can underestimate the true burden of malaria and compromise disease surveillance efforts.
Need for Alternative Tests:
New diagnostic tests are required to overcome the limitations of HRP2-based RDTs.
Alternative tests should be capable of detecting a broader range of parasite antigens, including those not affected by antigenic variation.
Ideally, new tests should be affordable, easy to use in resource-limited settings, and provide rapid and accurate diagnosis of malaria infections.
In conclusion, while HRP2-based RDTs have been valuable tools for malaria diagnosis, the emergence of antigenic variation in Plasmodium parasites underscores the need for new diagnostic tests capable of detecting a broader range of parasite antigens. These tests should address the limitations of current RDTs and contribute to more accurate and timely diagnosis of malaria infections.