Lecture 6

Question 1

What type of leishmaniasis is usually caused by L. major?

Answer 1

Old world simple cutaneous leishmaniases

Self resolving with good immunity

Question 2

What type of leishmaniasis is usually caused by L. braziliensis?

Answer 2

Mucocutaneous leishmaniasis

Non-resolinvg , destructive immune-mediated pethology; usually to the soft palate

Question 3

What type of leishmaniasis is usually caused by L. ethiopica?

Answer 3

Diffuse cutaneous leishmaniasis

Non-resolving, failure of immunity

Question 4

What type of leishmaniasis is usually caused by L. donovani/inafntu(chagasi)?

Answer 4

Visceral leishmaniasis
Fatal, failure of immunity, immune suppression and resistant to reinfection
L. donoavni can also go on to cause post kala azar dermal leishmaniasis in individuals who survive the VL

Question 5

Describe the promastigote

Answer 5

Kinetoplast anterior to nucleus, flagellum emerges form anterior and has no undulating membrane

Question 6

What are the four types of promastigote?

Answer 6

• Procyclic; short flagellum, replicative
• Nectomonad; non-replicative, adherent
• Leptomonad; dividing
• Metacyclic; flagellum>body length, highly motile, non-replicative, infective form

Question 7

What are axenic amastigotes?

Answer 7

Amastigotes grown in vitro outside of cells. Have some gene expression in common with amastigotes but also some in common with promastigotes and some of their own.

Question 8

What are the key surface molecules of Leishmania?

Answer 8

• gp63
• lipophosphoglycan
• proteophosphoglycan
• glycosylinositolphospholipid (GIPL)

Question 9

What challanges does Leishamania face during its time in sandfly?

Answer 9

1) Escape from the peritrophic membrane
2) Proliferation
3) Attachment to mid-gut epithelium
4) Anterior migration to stomodeal valve
5) Pre-adaptation to life in mammal

Question 10

What does Leishmania use for retention in sandfly?

Answer 10

Galectins expressed in the fly mid-gut. The type of galectin is fly species specific - explaining why Leishmanis parasite species have different invertebrate vectors

Question 11

What does the parasite use to bind to Galectin?

Answer 11

Lipophosphoglycan - which is GPI anchored into the membrane, has a glycan core, a phosphoryltated disaccharide backbone and a terminal cap. Galactose residues on the backbone allow binding to the galectin

Question 12

What other role does the Lipophosphoglycan have?

Answer 12

Following metacyclogenesis the backbone increases in length and has more arabinose residues which increases the resistance of metacyclic against lysis in serum, in particular complement mediated lysis. It achieves this by making the distance between its cap and the membrane attack complex longer than the complex

Question 13

What is the outcome of complement attack that has failed?

Answer 13

The parasite is opsonized and hence targeted to a phagocyte - the cell it needs to infect

Question 14

Apart from differentiation into metacyclic what else is required for optimal infectivity?

Answer 14

• Secreted parasite products

- Vector derived factors

Question 15

Give an example secreted parasite product that is important for infectivity

Answer 15

Filamentous proteophosphoglycan - clogs the sandfly throat so the fly ‘coughs’ to clear its throat when having blood meal, and regurgitates the parasite and the proteophosphoglycan gel onto the site of the blood meal. The gel has been shown to help the parasite infect

Question 16

How do sandflies feed?

Answer 16

They are pool feeders; will macerate local tissue to gain access to dermal capillaries. Have anti-coagulants, immunomodulators and antigens in their saliva to help with feeding.

Question 17

Why is the sandfly feeding method important?

Answer 17

Some factors released in the saliva help the parasite but some reduce its infectivity. Delayed hypersensitivity responses induced by repeated bites can make the site inhospitable for the parasite. targeting vaccine against sandfly saliva antigen might be useful in killing parasite as bystander

Question 18

Which receptors are implicated in Leishmania uptake?

Answer 18

• Complement receptor
• Mannose receptor
• DC-SIGN
• Cell bound proteoglycans
• FcR

Question 19

What complement receptor recognise which convertases?

Answer 19

CR1 : C3b

CR3 : C3bi

Question 20

What part of the promastigote makes the initial contact with the phagocyte?
Then what happens?

Answer 20

The flagella

The parasite turns around and uses the flagella to push against the phagocyte causing damage and recruiting lysosomes

Question 21

What type of compartment holds the parasite in macrophages, dendritic cells and neutrophils?

Answer 21

Both macrophage and dendritic cells form phagolysosome

Neutrophil will form a non-leishmanicidal vacuole

Question 22

What effect has LPG been shown to have during phaholysosome formation?

Answer 22

Appears to inhibit lysosome fusion - C3PO mutant lacks ability to make disaccharide phosphate repeats and hence cannot synthesis LPG and lysosome fusion happens

Question 23

What is commonly targeted by intracellular pathogens to stop acidification?

Answer 23

The vacuolar ATPase - done by promastigote but not the amastigote

Question 24

Why does leishmania release virulence factors when inside the phagocytic cell?

Answer 24

To live in the cell and divide and then go on and infect new cells; need to stop the phagocytic reaching high level of killing ability

Question 25

How might Leishmania release virulence factors?

Answer 25

- Exosomes - Lipid rafts - Translocases/flipases

Question 26

When are promastigote exosomes released? | What is in the exosome?

Answer 26

While the parasite is still being phagocytized to bung up the acitivity of the cell GP63 and EF-1α; access host cell cytosol and activate multiple host protein-tyrosine phosphatases Gp63 is a protease that cuts up molecules involved in signal transduction; in particular the interferon pathway Overall attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α) while enhancing production of the anti-inflammatory cytokine IL-10.

Question 27

Where does Leishmania use exosomes?

Answer 27

In the sand fly mid gut, they are co-egested with the blood meal. Exacerbate cutaneous leishmaniasis pathology. Also released when entering host and increased infection

Question 28

What happens to the flagella during amastigotogenesis?

Answer 28

9+2 flagellum becomes a 9+0 flagella. One daughter makes de novo 9+0 while the other reabsorbs the flagella to make 9+0. The 9+0 then collapses to form a cilium

Question 29

What might the Cilium be important for?

Answer 29

- Integrity of flagella pocket - Facilitate kinetoplast division - Tether the amastigote to the Parasitophorous vacuole (PV) membrane - Act as sensory/signalling organelle (with the PV)

Question 30

What happens to the surface during amastigotogenesis?

Answer 30

Loss of LPGs, GPI-proteins and PPGs. Keeping only the GIPLs. May be important in ability to survive in phagolysosome

Question 31

What nutrient does Leishmania need to acquire?

Answer 31

Iron; which comes to the phagolysosome and the cell tries to remove while the parasite tries to keep them (battle)

Question 32

What parasite evasion strategies exist to target antigenic presenting cell function?

Answer 32

- Minimise expression of PAMPS - Metabolic - Inhibition of APC-T cell synapse - Inhibition of co stimulation - Aberrant cytokine production - Induction of regulatory cytokines (IL-10)

Question 33

Why is neutrophil ingestion of Leishmania different?

Answer 33

The neutrophil is not able to kill the amastigote itself, instead the neutrophil undergoes apoptosis and then is taken up by macrophages. Sometimes with the parasite still inside but sometimes without a parasite. Without parasite still helps the parasite because will act to minimise inflammation and the parasite may also have gained new features by being in the neutrophil which now make it a better invader.