Lecture 10: Plasmodium - resistance and susceptibility to infection

Question 1

True or false: malaria has lead to very strong selective pressure for erythrocyte polymorphisms?

Answer 1

True (E.g. SCA and sickle-cell trait)

Question 2

What causes SCA?

Answer 2

Point mutation (glutamic acid to valine at codon 6) in beta chain of haemoglobin means that under low oxygen tension, RBCs with HbS distort into sickle shapes

Question 3

What is sickle-cell trait?

Answer 3

heterozygous individual (one HbS allele and one Hb allele) - generally asymptomatic

Question 4

What is the relationship between the HbS allele and malaria endemicity?

Answer 4

There is a higher frequency of the HbS allele in populations where there is high malaria endemicity

Question 5

True or false: HbS homozygotes are resistant to plasmodium infection?

Answer 5

True but they experience severe sickling of RBCs under normal condition and anaemia (high morbidity and mortality)

Question 6

Why is sickle-cell trait protective against plasmodium infection?

Answer 6

1. host microRNAs post-transcriptionally regulate expression of parasite genes -abnormal transcription by the intraerythrocytic parasite (potentially affects Maurer’s cleft formation) - impair ability of parasite to present proteins like PfEMP1 on RBC surface (reduce cytoadherence and increase splenic clearance)
2. Haemichromes (oxidised, denatureed Hb) accumulates in HbS- containing RBCs and results in formation of Bnad 3 aggregates and inhibition of knob formation resulting in increased removal of infected RBC from circulation

Question 7

Describe the structure of the Duffy antigen

Answer 7

Extracellular N-terminal domain with binding site for Duffy binding protein ligand (DBP)
7 transmembrane domains
Intracellular C-terminal domain

Question 8

Which cells have the highest Duffy antigen expression?

Answer 8

Young reticulocytes

Question 9

For which two plasmodium species is binding to Duffy antigen particular important?

Answer 9

P. vivax

P. knowlesi

Question 10

What is the importance of the Tyr41 amino acid?

Answer 10

It can get sulphated which is required for the high-affinity binding of BDP to the Duffy antigen

Question 11

What is the purpose of the Duffy antigen (DARC)?

Answer 11

Originally identified as a blood group antigen but also expressed on endothelial cells and binds with high affinity to 11 different chemokines
- facilitates transport of chemokines across endothelium (endothelial cells)
- scavenges excess toxic chemokines (RBC)

Question 12

Why is DARC considered a silent chemokine receptor?

Answer 12

it is unable to support intracellular signaling since it lacks a G-protein motif

Question 13

How do we know that the function of DARC is more important on endothelial cells than RBCs?

Answer 13

Expression is conserved on endothelial cells but not on all RBCs so function on erythrocytes appears dispensable

Question 14

True or false: humans negative for the Duffy blood group antigen show some resistance to P. falciparum infection?

Answer 14

False:
- resistant to blood-stage infection with P. vivax
- interaction and reorientation but moving junction does not develop and invasion is aborted with P. knowlesi

Question 15

How can DARC expression be absent on erythrocytes whilst still being present on endothelial cells (null phenotype (Fy(a-b-)))?

Answer 15

the Duffy locus (FY) has two exons and an upstream promoter specific for erythroid expression
- point mutation in this erythroid promoter region within the binding site for the transcription factor (GATA-1)
- GATA-1 unable to bind to Duffy gene promoter meaning Duffy protein is absent from RBC surface and results in null ‘erythrocyte silent’ phenotype

Question 16

True or false: DARC negative individuals are resistant to hepatic and blood-stage P.vivax infection?

Answer 16

False: only resistant to blood-stage infection but are still susceptible to hepatic-stage infection

Question 17

What are the two major co-dominant Duffy alleles and how do they differ?

Answer 17

FYA and FYB
- they differ by a single amino acid at residue 42 (Fyb = aspartic acid, Fya = glycine) - residue 42 is the last amino acid in the DBP binding site

Question 18

Why does expression of the Fy(a) DARC allele also cause reduced susceptibility to P.vivax (three theories)?

Answer 18

1. Fyb allele encodes DARC with aspartic acid which is negatively charged and helps to coordinate binding of the positively charged PvDBP, where as the glycine of the Fy(a) DARC is neutral and does not contribute to binding to the DBP
2. Expression of the Fy(a) allele has been seen to increased susceptbility of the Trr41 to arylsulfatase and loss of sulphate groups necessary for PvDBP binding
3. Fy(a) individuals are more susceptible to antibody inhibition - antibodies block PvDBP binding to red blood cells

Question 19

What are Glycophorins A and B (GPA/GPB)?

Answer 19

Trans-membrane sialoglycoproteins with the N-terminus orientated outside of the RBC that carries the N-terminal MNS blood group
GPA and GPB N-terminal domains carry O-glycans
GPA also carries an N-glycan

Question 20

What contributes most of the carbohydrate present on the RBC surface?

Answer 20

GPA and GPB

Question 21

What is the consequence of the Dantu hybrid rearrangement of the GPA and GPB exons?

Answer 21

Dantu hybrid blood group rearrangement is taking some exons of GPA and GPB
- individuals expressing the Dantu blood group results in decreased ability of the antigen to be glycosylated resulting in 40% reduced risk of severe malaria in these individuals

Question 22

Why might the Dantu blood group be absent (or at very low frequency) outside of east Africa given the strong protective effective of the Dantu antigen against malaria (why is it not more widespread)?

Answer 22

• the variant may have only recently risen (recent evolution in the human genome) so hasn’t had time to disperse

-It may only be protective against strains of P. falciparum specific to east Africa

Question 23

What are Glycophorins C and D (GPC/GPD)?

Answer 23

both products of the single GYPC gene and play an important role in maintaining the mechanical stability of erythrocytes
GPD uses an alternative methionine start codon in exon 2 so lacks exon 1 and several glycosylation sites including the N-glycan

Question 24

What is the Gerbich negative blood group?

Answer 24

deletion of exon 3 of the GYPC gene that affects the extracellular components of both GPC and GPD

Question 25

What is the effect of Gerbich negative RBCs?

Answer 25

increased resistance to P. falciparum invasion in vitro

Question 26

How does GPC mediate binding of EBA-140 ligand (a P. falciparum EBL needed for invasion)?

Answer 26

EBA-140 binding is dependent on sialic acid (requries the single N-glycan near the N-terminus (exon 1) with contributions from sialylated O-linked glycans and the GPC protein backbone)

Question 27

Why is the Gerbich phenotype associated with P. falciparum resistance when no exon 1 glycosylation sites are lost?

Answer 27

Lack of mature processing of the glycans within the golgi so the protein being expressed lacks the ability to be glycosylated in the normal way even though the protein backbone in exon 1 is unaltered - so you lose the ability to be recognised by the P. falciparum EBA-140 because no longer glycosylating and producing the recognition signal in exon 1.

Question 28

What is the Helgesson phenotype?

Answer 28

Extremely low expression of CR1 and associated Knops antigen = increased potential for host recognition as parasite no longer able to form rosettes and sequester as effectively resulting splenic clearance

Question 29

What is the Knops blood group antigen?

Answer 29

result from polymorphisms in CCP25 and CCP26 of complement receptor 1.

Question 30

Band 3 protein is associated with which blood group antigen?

Answer 30

the Diego blood group

Question 31

Describe the structure of the Band 3 protein

Answer 31

Two distinct functional domains: - Transmembrane domain - anion exchange - responsible for gas transport and acid-base equilibrium - cytoplasmic domain - interacts with cytoskeletal proteins (spectrin, ankyrin, proteins 4.1 and 4.2) - involved in maintenance of erythrocyte structural integrity

Question 32

What causes Southeast Asian Ovalocytosis (SAO)?

Answer 32

autosomal dominant mutation in Band 3 gene that results in an in-frame deletion that removes amino acids at the junction between the membrane transport region and the N-terminal cytoplasmic domain

Question 33

What are the effects of S. E. Asian Ovalocytosis?

Answer 33

oval shaped RBCs with: - defective anion transport - reduced expression of different RBC antigens - abnormalities in morphological and mechanical properites (increased membrane rigidity) - mild haemolytic anaemia.

Question 34

What have studies in Papua New Guinea shown about SAO and P. vivax resistance?

Answer 34

reduced risk of clinical P. vivax episodes in infants and reduced P. vivax re-infection --> protection against P. vivax may have contributed to the heterozygote advantage of SAO observed in Papua New Guinea

Question 35

Why is SAO protective?

Answer 35

- decrease anion transport may inhibit parasite growth - impaired ability of malarial parasite to develop within erythrocyte - stable incorporation of GPA into RBC membrane requires Band 3 protein (reduce stability of GPA may impact parasite that require GPA for RBC invasion - sialic acid dependent invasion) - Affect deformabiity of infected RBCs - impair transit through capillaries

Question 36

What is the relationship between glucose-6-phosphate dehydrogenase and malaria endemicity?

Answer 36

high prevalence of G6PD deficiency overlaps with regions of malaria endemicity

Question 37

what is the role of G6PD in RBCs?

Answer 37

acts as enzyme to control oxidative damage in RBCs - works within the pentose phosphate pathway and catalyses the conversion of G6P to 6-phosphogluconate, generating NADPH - NADPH reduces oxidised glutathione to protect RBCs against oxidative damage

Question 38

True or false: G6PD deficiency is a common human genetic condition?

Answer 38

True (many allelic variants cause different levels of enzyme deficiency)

Question 39

Which Plasmodium species is G6PD deficiency believed to confer protection against?

Answer 39

P. falciparum

Question 40

what is the significance of G6PD gene being located on the X-chromosome?

Answer 40

Females can be homozygously deficient or heterozygously deficient (who would have mixed populations of erythrocytes due to random X-inactivation) Males are hemizygously deficient

Question 41

what types of mutations are common in G6PD deficiency and what do they cause?

Answer 41

point mutations and small deletions cause structural defects in the enzyme cause instability or altered activity and decreased affinity of G6PD for substrates degraded more rapidly due to instability

Question 42

How does G6PD deficient RBCs affect plasmodium proliferation?

Answer 42

Under normal conditions, the G6PD deficient RBCs cope with low levels of available NADPH - infection with highly active metabolising plasmodium parasite causes damage to the red blood cell - parasite causes increase in oxidative damage (break down of Hb results in toxic by-products (particularly iron) that are a source of oxidative stress), red cell cannot cope with this due to low NADPH - RBC cannot be protected from the oxidative damage leading to lysis and haemolytic anaemia - plasmodium proliferates less efficiently

Question 43

What did a large multi-centre case-control study report about the G6PD deficiency in terms of clinical malaria outcomes?

Answer 43

Increasing levels of G6PD deficiency were associated with a decreased risk of cerebral malaria but an increased risk of severe malarial anaemia (concluded than an evolutionary trade-off existed between different clinical outcomes of P. falciparum infection in human populations)

Question 44

ER: thalassemias and pyruvate kinase deficiency providing protection against malaria