Lecture 3: Innate Immunity and Trypanosome Lytic Factor (TLF)

Question 1

What are the two morphological T. brucei subspecies found in Africa that cause distinct disease patterns in humans?

Answer 1

T. brucei gambiense (chronic infection)

T. brucei rhondesiense (acute infection)

Question 2

What is Nagana and what causes this disease?

Answer 2

Cattle disease caused by T. b. bruci, T. congolense and T. vivax

Question 3

Which trypanosome species causes the disease surra?

Answer 3

T. evansi

Question 4

Which trypanosome parasites are humans susceptible and resistant to?

Answer 4

Susceptible to infection by T. b. gambiense and T. b rhodesiense

Resistant to infection by T. b. brucei, T. congolense, T. vivax and T. evansi

Question 5

Why are humans resistant to infection by T. b. brucei, T. congolense, T. vivax and T. evansi?

Answer 5

These trypanosomes are susepctible to lysis by Trypanosome lytic factors (TLFs)

Question 6

True or false: TLFs are innate immune effectors?

Answer 6

True (they are not complement proteins or antibodies though)

Question 7

Why are humans susceptible to infection by T. b. gambiense and T. b rhodesiense?

Answer 7

They are resistant to killing by TLFs (have developed mechanisms that counteract the lytic effect of TLF)

Question 8

What is needed for the lytic activity of TLFs?

Answer 8

uptake by endocytosis and trafficking to the lysosomal compartment within the trypanosome

Question 9

what effect does normal human serum (NHS) have on T. b. brucei?

Answer 9

cell swelling and lysis

Question 10

which trypanosoma brucei subspecies can be killed by serum from humans and some primates?

Answer 10

Trypanosoma brucei brucei

(not rhodesience or gambiense)

Question 11

How was it identified that the TLF was a high density lipoprotein?

Answer 11

serum from patients with Tangier disease (characterised by deficiency in HDL) had no trypanocidal activity due to absence of TLFs

Question 12

What are the two distinct lipoprotein complexes present in human blood that exhibit trypanosome lytic activity?

Answer 12

TLF1 and TLF2

Question 13

What are the components of TLF1?

Answer 13

• Apo-lipoprotein A-1 (APOA1)
• Haptoglobin-related protein (Hpr)
• Apo-lipoprotein L-1 (APOL1)
• Trace amounts of apo-lipoprotein A-II and paraoxonase

Question 14

What are the components of TLF2?

Answer 14

• Apo-lipoprotein A-1 (APOA1)
• Haptoglobin-related protein (Hpr)
• Apo-lipoprotein L-1 (APOL1)
• IgM

Question 15

What is APOL1?

Answer 15

a key component of HDLs and is a cofactor of lecithin cholesterol acyltransferase (which recruits free cholesterol to the lipid core of the HDL particles and transport cholesterol to the liver for secretion or re-use)

Question 16

Which human TLF is described as a lipid-poor protein complex?

Answer 16

TLF2

Question 17

What is the role of the haptoglobin-related protein in the TLFs?

Answer 17

binds Hb but is not removed from blood circulation via receptor-mediated process in liver (not recognised by receptors) and plays an important role in promoting TLF1 uptake by binding of Hpr-Hb to T. b. brucei TbHpHbR receptor

Question 18

Which TLF component is necessary for the lysis of the parasites (the lytic component)?

Answer 18

APOL1

Question 19

What was the result of TbHpHbR gene knockout parasites?

Answer 19

more resistant to lysis - hence the receptor is required for efficient lysis by mediating the uptake of TLF

Question 20

What are two unique characteristics of APOL1?

Answer 20

has N-terminal signal sequence meaning it is secreted from cells

it is exclusively associated with HDL particles int he circulation

Question 21

What are the three main domains of the APOL1 protein?

Answer 21

• N-terminal pore-forming domain (contains10 helices homologous to bacterial colicins that forms pores in the membrane)
• Membrane-addressing domain (inserts into membrane)
• C-terminal SRA-interacting domain

Question 22

Describe the mechanism of APOL1 induced lysis of T. b. brucei

Answer 22

• endocytosis of APOL1 contaning HDL particles by TbHpHbR
• low endosomal pH promotes conformational change in APOL1 that exposes the pore forming domain and allows membrane insertion
• this leads to influx of chloride ions and osmotic imbalance that cause lysosome swelling and cell death

Question 23

How may the fact that T. b. brucei is deficient in haem biosynthesis result in TLF arming mechanism?

Answer 23

parasite requires haem from Hb so causes erythrocyte damage to release haem into the plasma which is then taken up into the parasite by binding to Hpr and endocytosis via TbHpHbR receptor

Question 24

What is the role of TbKIFC1?

Answer 24

has cholesterol-trafficking activity that contributes to high plasma membrane fluidity

Question 25

what is the effect of KIFC1 knockdown in cells on Ab-VSG clearance and TLF2 internalisation?

Answer 25

- depletion of KIFC1 = cell lysis occurred at a slower rate that WT cells --> Ab-VSG complexes moved slower to flagellar pocket --> clearance of Ab-VSG complexes is likely a driving force for TLF2 internalisation and trypanosome killing (TLF2 bound IgMs bind to VSG and are internalised at the flagellar pocket to introduce APOL1 into the parasite for killing)

Question 26

What is SRA?

Answer 26

Serum resistance Associated gene expressed by T. b. rhodesiense that is a truncated VSG protein lacking surface-exposed loops and is not expressed on the surface of parasite

Question 27

What is the relationship between the location of TLF and SRA in parasite?

Answer 27

TLF and SRA were shown to co-localise in endosomes and lysosomes

Question 28

how is it believes that SRA expression in T. b. rhodesiense confers resistance to lysis by APOL1?

Answer 28

TLF internalised through flagellar pocket - SRA is able to block the inserting of APOL1 into the endosomal membrane (blocking swelling of parasite and subsequent death)

Question 29

What was the result of SRA overexpression in the TLF1 sensitive T. b. brucei parasite?

Answer 29

reduction in lysis compared to the parasites without the SRA protein

Question 30

Where was the T. b. rhodesiense SRA gene found in the parasite genome?

Answer 30

It is an expression-site associated gene (ESAG) found in the expression site of a truncated VSG

Question 31

What three mechanisms does T. b. gambiense use to evade lysis by TLF?

Answer 31

mutation in the HpHb receptor results in inactivation and reduced uptake of APOL1 increase degradation of APOL1 due to lower pH in early endosome and earlier endosomal protease activity = faster degradation of APOL1 Prevention of APOL1 membrane insertion - TgsGP (T. b. gambeinse specific glycoprotein) a shortened VSG that is GPI anchored in lysosomes causing stiffening and increasing curvature of membrane to prevent APOL1 insertion

Question 32

What was found in the African genome population that conferred protection against T. b. rhodesiense infection?

Answer 32

Two genetic polymorphisms in the human APOL1 gene: - G1 allele (2 amino acid substitutions) - G2 allele (2 amino acid deletion at C-terminus)

Question 33

How was it identified that APOL1 deficiency is linked to T. evansi infection?

Answer 33

T. evansi does not normally cause infection in humans since sensitive to TLF but patient infected with T. evansi was found to have a frameshift mutation in APOL1 gene that meant they were not producing functional APOL1 and were therefore susceptible to T. evansi infection

Question 34

True or false: T. b. gambiense is resistant to TLF by expression of SRA?

Answer 34

False: T. gambeinse does not express SRA (unlike T. rhodesiense) but has 3 independent complementary mechanisms of TLF lysis evasion