Leishmania

Leishmania: a large group of kinetoplastid parasites causing a variety of syndromes

Phosphoglycans: important molecules for parasite development and pathogenesis

Classic model for polarized T-cell response (Th1/Th2, will be covered later in the immunology part of this course)

Leishmania belong to the order kinetoplastida

Group of flagellates at the basis of the eukaryotic tree

Harbor name-giving mitochondrion with large genome which is always associated with the basal body of the single flagellum

Trypanosoma & Leishmania are the medically important and best studied genera in this group

trypomastigote

epimastigote

promastigote

amastigote

Leishmania parasites exist as pro- and amastigotes

The parasite lives in the digestive tract of sand flies as promastigote

In the mammalian host parasites multiply as intracellular amastiogotes

Leishmania infects and thrives in macrophages

How do they get in and how to the avoid being killed?

Leishmania infects and thrives in macrophages

Uptake of Leishmania amazonensis metacyclic promastigote by a mouse macrophage. The parasite is phagocytosed with the cell body entering first and through the formation of a long tubular pseudopod. Images were captured every 0.5 seconds over the course of 367 seconds. Courret et al. 2002 http://jcs.biologists.org/cgi/content/full/115/11/2303

Leishmania infects and thrives in macrophages

Phagocytosis of a Leishmania amazonensis metacyclic promastigote by a mouse macrophage. The parasite binds to the macrophage plasma membrane by the tip of the flagellum. It then turns around and is finally ingested via the cell body. Images were

captured every 0.5 seconds over the course of 125 seconds. Courret et al. 2002 http://jcs.biologists.org/cgi/content/full/115/11/2303

Leishmania infects and thrives in macrophages

Leishmania stimulates this process by binding elements of the complement system to its surface

Binding of complement can destroy pathogens but also tags them for phagocytosis (opsonization: pathogen bound 3Cb is a potent ‘eat me’ signal for macrophages & neutrophils)

However, the parasite prevents the formation of the fully functional membrane attack complex

A molecule on the surface of the parasite seems to be responsible both for complement activation and prevention of the final attack

Leishmania infects and thrives in macrophages

Macrophages are important “microbe killers”, however several pathogens have found ways to escape killing

Trypansoma cruzi -- induces phagocytosis but then escapes into the cytoplasm

Toxoplasma -- active invasion, parasitophorous vacuole is never part of the endocytic pathway

Mycobacterium tuberculosis -- induce phagocytosis and block lysosomal maturation

Leishmania ...

Leishmania infects and thrives in macrophages

… Leishmania just doesn’t seem to care

Amastigotes thrive in what looks like a fully matured lysosome with acidic pH and abundant lysosomal hydrolases

Amastigotes rapidly divide and will infect new macrophages after rupture of host cell

The dense surface coat covering Leishmania seems to protect the parasite from the action of the lytic enzymes

However, with help from T cells macrophages can be stimulated to kill the parasite

A TH1 response is required for parasite control and healing

Stimmulation with different cytokines leads to the development of two types of T-cells specialized for different immune responses

Th1 and Th2 strongly downregulate each other

This polarization has important consequences for the downstream response and can spell life or death

Non healing Leishmania infections are characterized by a strong TH2 response (remember this was the response useful to get rid of worms by antibody and hypersensitivity)

Healing infections are characterized by TH1 The parasites seems to manipulate this

balance in his favor, we don’t understand yet how that is done

Leishmania is transmitted by sand flies (Phlebotomidae)

Sand flies are minute diptera (only females bite)

They do not fly well and stay close to the ground

In the wild sand flies often breed in rodent burrows

Old world Phlebotomus, new world Lutzomya

Can also transmit Bartonella bacilliformis and Papatsi virus

A variety of species and species complexes causes disease in humans

Kala Azar - Visceral Leishmaniasis

Caused by the L. donovani complex General infection of macrophages in

the entire RES Weeks to months incubation period Abdominal swelling (hepato- and

splenomegaly Often but not always fever occurs in

two daily peaks Progressive weight loss Darkening of the skin Mortality of untreated disease 75-

95%

Cutaneous Leishmaniasis is usually self-limiting

Old world oriental sore is caused by parasites of the L. tropica complex. (similar disease in the new world is caused by L. mexicana)

A chronic but self-limiting dry ulceration at the site of the bite

Ulceration start months after infection

Parasites are not found outside the lesion

Nearly absolute resistance to reinfection (however, there is long term persistence and persistence is required for resistance)

Espundia -- Mucocutaenous Leishmaniasis

Caused by L. braziliensis ~20% of infected patients

develop ulcers of the oral and nasal mucosa

Progression of the ulceration is slow but steady, ultimately destroying all soft parts of the nose, the lips, the soft and the soft palate

Death occurs usually through secondary bacterial infection

Leishmania produce a unique glycoconjugate

Leishmania parasites can be labeled at surprizing efficiency with radioactive sugars and inositol

Label is incorporated into a large glycoconjugate which is acid labile and has a lipid anchor

This lipophosphoglycan (LPG) completely covers the surface of the the promastigote

Lipophosphoglycans share structural features with GPIs

Lipophosphoglycan

Major surface molecule of Leishmania Four domains: 1-O-alkyl-2-lyso-phosphatidylinositol anchor Glycan core Disaccharide phosphate repeat units Oligosaccharide cap

LPG shows structural variation among Leishmania species

Biosynthesis of LPG

What does LPG “not” do?

LPG protects parasites in the sand fly midgut LPG attaches parasites to the sand fly midgut

epithelium LPG protects against complement attack LPG enhances uptake into macrophages LPG interferes with macrophage signaling

preventing oxidative burst LPG protect from toxic macrophage products

Is LPG a pathogenesis factor?Stan Falkow’s virulence postulates

Pathogenesis is reasonably associated with the expression of a certain virulence factor

Inactivation of the gene should result in loss of virulence

Restoration of the gene should fully restore virulence

Identification of LPG genes by genetic complementation

Mutants in LPG biosynthesis as tools to study LPG enzymology & function

To proof the third postulate has been a challenge

Lpg- mutants show significant loss in virulence both in in vitro macrophage infections as well as in in vivo experiments

But: Leishmania tends to loose virulence in culture anyway, and expression of the WT gene did not always fully restore virulence

Chemical mutants might be ‘over-mutated’ and carry multiple mutations (some might not be related to LPG but affect virulence)

Presence of several LPG related molecules further complicate the issue

A number of Leishmania glycoconjugates share structural features with LPG

Knock outs by gene targeting as a cleaner way to obtain mutants

Lpg1 k.o. in L. major has an effect on virulence

http://www.pnas.org/content/97/16/9258.full

Lpg1 k.o. in L. major has an effect on virulence

http://www.pnas.org/content/97/16/9258.full

Lpg1 k.o. in L. major has an effect on virulence

http://www.pnas.org/content/97/16/9258.full

In Leishmania mexicana LPG seems dispensable for infection of mice

lpg1 knock outs infect macrophages in vitro and mice in vivo as well as wt suggesting LPG is not needed

lpg2 knock outs do the same suggesting that PG repeats in general are not needed

Overall, it appears that the main role for LPG might lie in the interaction with the sandfly and not the mammalian host

procyclics and metacyclics

Promastigotes attach to and metacyclics detach from the midgut epithelium

procyclics and metacyclics

Infected macrophages are taken up with the blood meal and amastigotes released by digestion transform into procyclic promastigotes which attach to the midgut epithelium

Attached promastigotes divide rapidly

Metacylcic promastigote detach and pass forward into the pharynx from where they are regurgitated into the bite site

Structural modification of LPG during the sand fly cycle

LPG is structurally modified during metacylogenesis

LPG in metacylics has 2-3 times the number of repeat units

Side chains with terminal galactose are down-regulated in favor of chains with terminal arabino-pyranose

Only phosphoglycans from procyclics attach to the midgut

Opened midguts were incubated with PG from procyclics (A/B) and metacyclics (C/D) and detected with an antibody

Pimenta et al., Science. 256:1812-5.

Phosphoglycan binds to the microvilli of the epithelium

Pimenta et al., Science. 256:1812-5.

Phosphoglycan repeats inhibit attachment of procyclics

Opened midguts were incubated with radiolabeled procyclic promastigotes

(A) 2-5 procyclic PG, 6+7 metacyclic PG (lipid removed)

(B) 1, control, 2, pPG, 3 no Gal, 4 with Gal, 5, second Gal, 6 ara

(C) commercial saccharides: 2 free Gal, 3-5 and digalactosides, 6 similar Man disaccharide

Pimenta et al., Science. 256:1812-5.

LPG is not essential for early survival but for retention

(A + B) number of promastigotes present in midgut upon disection (in B 5x higher inoculum is used)

Sacks et al., PNAS 97: 406-411

LPG binds to a species specific galectin in the sandfly midgut

A gene for an abundantly expressed galactose binding protein or lectin (galectin) was identified in a sandfly sequencing project

A specific antibody against the protein encoded by this gene reacts with the midgut of the sandfly species from which it was isolated (but not from other species)

High resolution microscopy shows that the protein(red in lower panel) is found on the luminal side of the midgut epithelium

Cell 119:329-41

LPG binds to a species specific galectin in the sandfly midgut

Galectin (labeled with a fluorescent dye) binds specifically to procyclic Leishmania major parasites

However little binding is detected when incubated with metacyclic parasites (the stage that detaches and moves to the the proboscis to infect the mammalian host, V1met)

There is little binding to a mutant parasite (Spock) which lacks LPG

(B lower) anti-galectin also blocks binding of procyclic parasites to the midgut epithelium

Cell 119:329-41

Modification of LPG modulate interaction of the parasite and midgut galectin

Summary

Leishmania species cause three clinical syndromes depending on the spread of the infection in the body

Leishmania ‘provoke’ phagocytosis by macrophages and develop intracellular in an fully acidified lysosome

LPG-galectin interaction and modification of LPG regulate attachment and detachment of parasites in the sandfly host