Lecture 5: Parasite glycobiology

Question 1

True or false: trypanosomatids all have a kinetoplast?

Answer 1

True

Question 2

Which trypanosoma species is Leishmania clade closely related to?

Answer 2

Trypanosoma cruzi and Trypanosoma brucei

Question 3

What disease is caused by T. brucei?

Answer 3

Human African Trypanosomiasis (HAT) or Sleeping sickness

Question 4

What is the vector for the following parasites:
1) T. brucei
2) T. cruzi
3) Leishmania sp.

Answer 4

1) Tsetse fly

2) Triatomine bug

3) Sandfly

Question 5

Which form of the parasite is taken up by the Tsetse fly and which form of the parasite is injected by the Tsetse fly into the human?

Answer 5

Taken up: stumpy form that rapidly differentiates to procyclic form
Injects: metacyclic form

Question 6

True or false: T. brucei is strictly extracellular?

Answer 6

True

Question 7

True or false: Leishmania sp. is strictly extracellular?

Answer 7

False, they are intracellular (phagocytosed by macrophages and proliferate within parasitophorous vacuole as amastigotes)

Question 8

How does the triatomine bug transmit the T.cruzi parasite to the human host?

Answer 8

Bites around eyes and mouth (soft tissue parts), defecates parasite close to wound site, which is irritated and the parasite is rubbed into the wound

Question 9

True or false: T. cruzi is strictly extracellular?

Answer 9

False: it is intracellular - cross epithelial barrier so can enter tissues (mainly smooth muscles particularly of the heart, skeleton and oesophagus)

Question 10

What is the name of the disease caused by T. cruzi?

Answer 10

Chagas disease

Question 11

what does the surface coat of the T. brucei parasite contain in the human host?

Answer 11

mainly VSGs

Question 12

what does the surface coat of the Leishmania parasite contain in the human host?

Answer 12

LPG
Surface proteases (PSP)
GIPLs

Question 13

what does the surface coat of the T. cruzi parasite contain in the human host?

Answer 13

Mucins
Trans- sialidases
GIPLs

Question 14

What are the T. brucei glycoconjugates and what is the structure of each?

Answer 14

VSG = homodimer, each monomer is N-glycoslyated and GPI anchored to the plasma membrane

Transferrin receptor = heterodimer, both monomers are N-glycosylated but only one monomer is GPI anchored to the plasma membrane

Question 15

True or false: the T. brucei transferrin receptor (TfR) is structurally related to VSG?

Answer 15

True, expressed form the same expression site (ES) by RNA pol1

Question 16

What is the role of the T. brucei TfR?

Answer 16

heterodimer that recognises and internalises host transferrin

Question 17

What is the role of VSG and TfR glycosylation in T. brucei?

Answer 17

shield the plasma membrane from complement and prevent exposure of conserved GPI anchor

Question 18

What glycoproteins are expressed in the surface of the T.brucei parasite in the Tsetse fly mid-gut?

Answer 18

Procyclins (Aka procyclic acidic repetitive proteins (PARPs)

Question 19

Describe the structure of procyclins in T brucei

Answer 19

GPI-anchor that is heavily modified by addition of long glycan chain that masks the surface
Contains a single N-glycosylation site for GlcNAc2Man5 glycan
- pack together to form a dense surface coat.
- multiple procyclin isoforms that are classified as either EP (containing EP repeats) or GPEET (containing GPEEpT repeats) procyclins.

Question 20

How does the expression of procyclin isoforms differ during tsetse transmission?

Answer 20

Developmentally expressed
- GPEET expressed in early infection
- EP expressed in late infection

Question 21

How do procyclins promote survival of parasite in tsetse fly midgut?

Answer 21

N-terminal domain is cleaved by fly proteases and the remaining glycoconjugate is protease-resistant and provides a tough coat against gut hydrolases

Question 22

How does procyclin expression allow the T. brucei parasite to be injected into the host?

Answer 22

Once in midgut, the parasite needs to migrate across the peritrophic matrix into the salivary glands to be injected back out
- procyclin-null mutants are not as able to escape from the mid-hut into the salivary glands

Question 23

Describe the structure and function of the Leishmania LPG?

Answer 23

GPI-anchored phosphoglycan that dominates the cell surface of the Leishmania parasite

• GPI-anchor (conserved)
• glycan core (conserved)
• repeat unit made of mannose and phosphorylated galactose which may or may not be bound by different sugars (hexoses or pentoses) - variation
• cap (variation)

Function:
- required during sandfly transmission
- prevent lysosome-phagosome fusion during metacyclic-to-amastigote differentiation

Question 24

Describe the structure and function of the Leishmania PSP?

Answer 24

GPI-anchored glycoprotein and is a protease widely known as GP63

important for the phagocytosis of metacyclic Leishmania by host macrophages - part of invasion process

Question 25

What are GIPLs on the Leishmania parasite surface and what is their role?

Answer 25

Small glycoinositolphospholipids have role in protection for the otherwise exposed plasma membrane

Question 26

Where does variation in the Leishmania LPG phosphoglycan arise?

Answer 26

variation in glycosylation pattern and length of the repeat unit between species, lifecycle stages and strains

Question 27

What is the function of Leishmania LPG in the promastigote stage?

Answer 27

required for attachment to mid-gut epithelium in the sandfly, which is mediated by the insect lectin (galectin) as the receptor for the LPG ligand (recognises surface galactose) - if attachment does not occur, parasite would be excreted

Question 28

How does the Leishmania LPG structure change as they differentiate form the procyclic to the metacyclic promastigote form in the sandfly midgut?

Answer 28

- cap the galactose residues with different sugars so they are no longer able to attach to the galectin in the sandfly midgut --> this allows them to be injected with the blood meal into the human host

Question 29

The difference in Leishmania LPG strucute is thought to define what in terms of sand fly species and the human host?

Answer 29

Thought to define: - the interaction of Leishmania species with different sandflies - the interaction and severity of disease within the human host

Question 30

What is Leishmania PSG?

Answer 30

Promastigote secretory gel - a mix of extracellular glycoconjugates with a mucin-like filamentous proteophosphoglycan (fPPG) as a major component - secreted in the anterior mid-gut to form a gel-like substance within which infectious metacyclic promastigotes accumulate

Question 31

Which form is the Leishmania parasite in when it is uptaken by the sandfly?

Answer 31

Amastigote (then differentiates to procyclic promastigote that attached to mid-gut, then differentiates to metacyclic promastigote that allows it injection again into the mammalian host)

Question 32

What is the major component of Leishmania PSG?

Answer 32

filamentous proteophosphoglycan (fPPG)

Question 33

What is the function and role of Leishmania PSG?

Answer 33

secreted in anterior mid-gut by metacyclic promastigotes which forms a plug that blocks flies from feeding effectively --> alters the feeding behaviour of the sandfly - results in continued re-probing of the host as it cannot take a full blood meal so thinks it hasn't been able to inject into somewhere where it can feed - eventually regurgitates the PSG plug containing metacyclic promastigotes in order to increase feeding --> result in increased potential for parasite transmission

Question 34

What role does Leishmania PSG play in infectivity within the mammalian host?

Answer 34

role in the infectivity within the mammalian host - inoculation of mice with parasite with addition of PSG results in increased size of lesion and infectivity - glycan moieties on glycosylated fPPG is sufficient for lesion exacerbation

Question 35

What component of the fPPG is responsible for increased lesion size and infectivity in mammalian host?

Answer 35

the glycan moieties on glycosylated fPPG (but not eh peptide backbone of fPPG)

Question 36

What is the major component of the T. cruzi surface coat and what is their structure?

Answer 36

Mucins - glycoproteins rich in Ser/Thr with multiple O-linked glycans

Question 37

Explain the expression of mucins in T. cruzi

Answer 37

Mucins are encoded by an expanded multi-gene family formed of 800 genes - the different classes of mucins are developmentally regulated (different classes expressed in the epimastigotes and mammalian forms)

Question 38

True or false: Trans-sialidases in T. cruzi are encoded by an expanded multi-gene family?

Answer 38

True

Question 39

What are the three main processes in which T. cruzi mucins are involved in?

Answer 39

1. Protection 2. Attachment 3. Host cell invasion

Question 40

How are T. cruzi mucins modified?

Answer 40

Addition of host sialic acid by the parasite derived trans-sialidase bound to cell surface - trans-sialidase transfers sialic acid from host serum sialoglycoconjugates to the mucins on their surface

Question 41

What is the purpose of sialic acid modification of T. cruzi mucins?

Answer 41

sialic acid is quite specific to mammalian cells so addition of sialic acid to mucins allows evasion of immune system and recognition by host immune system as 'self'

Question 42

What are the three different trans-sialidases of T.cruzi and how do they differ?

Answer 42

aTS = active trans-sialidases iTS = inactive trans-sialidases soluble aTS aTS transfers sialic acid from host serum sialoglycoconjugates iTS recognises and binds sialic acid but does not transfer it - this is part of the recognition of host cells for invasion process to occur soluble aTS is used to cause confusion - transfer sialic acid from host serum glycoproteins and remodel the host glycophenotype to cause immune response to self cells - can not distinguish between normal glycophenotype and the parasite. - escape detection from immune system whilst it becomes intracellular