Leishmania

www.sanger.ac.uk/Info/Press/2007/070617.shtml

Leishmania

Life cycle and transmission ecology of Leishmania

Three clinical syndromes caused by these parasites

Understanding host-pathogen interaction at the molecular level - LPG and its role for survival in the insect vector

The phylum Kinetoplastida

Group of protozoans at the basis of the eukaryotic tree

Move using a single flagellum Harbor name-giving

mitochondrion with large genome which is always associated with the basal body of the single flagellum

All kinetoplastids are parasites Trypanosoma & Leishmania are

the medically important and best studied genera in this group (but a huge variety of animals and even plants harbor kinetoplastid parasites)

Leishmania belong to the order kinetoplastida

Different life cycle stages in this group show characteristic morphological differences and are distinguished by the position of the basal body within the cell

However, the differences are not limited to morphology

Different stages express different set of genes from their genomes which adapt their surface, internal structure and metabolism to survival in insect or mammalian hosts

trypomastigote

epimastigote

promastigote

amastigote

Leishmania parasites exist as pro- and amastigotes

The parasite lives in the digestive tract of sandflies as extracellular promastigote

In the mammalian host parasites multiply as intracellular amastiogotes

procyclics and metacyclics

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

Attached promastigotes divide rapidly (procyclics are not infective to mammals)

Metacyclic (infective) promastigotes cease replication, detach and pass forward into the pharynx from where they are regurgitated into the bite site

(attached)

(detached)

Leishmania infects and thrives in macrophages

Within its mammalian host Leishmania infects macrophages and replicates as intracellular amastigoe

Macrophages are “professional” phagocytic cells and an important part of the innate immune system (they also play a role in initiating the adaptive response by antigen presentation)

The parasite invades its host cell passively by triggering phagocytosis

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 (opsonisation: 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

Once a pathogen is taken up by phagocytosis the phagosome usually fuses with primary lysosomes to form secondary lysosomes

Within secondary lysosomes pathogens are killed and digested by the action of lytic enzymes and an acidic environment produced by membrane proton pumps

Additional mechanisms of killing include the generation of oxygen radicals (‘biological bleach’)

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 distribution (all species)

American soldiers contracted Leishmaniasis in Iraq & Afghanistan

A REGION INFLAMED: Hundreds of U.S. Troops Infected by Parasite Borne by Sand Flies, Army SaysBy DONALD G. MCNEIL JR.Published: December 6, 2003Hundreds of American troops in Iraq have been infected with a parasite spread by biting sand flies, and the long-term consequences are still unknown, Army doctors said Friday …

Skin Disease Strikes Iraqi ChildrenThursday, February 14, 2008By MARIA CHENG, AP Medical Writer

LONDON — At least 275 children in southern Iraq have been infected with a disfiguring skin disease, an outbreak some health officials are blaming on the war's devastating effect on the public health system.

Leishmania is transmitted by sand flies (Phlebotomidae)

Sand flies are minute diptera (they are more closely related to mosquitoes than “real” flies and only females bite)

Only mosquito nets with fine meshwork hold them off

In the wild sand flies often breed in rodent burrows

Old world Phlebotomus, new world Lutzomya

Can also transmit Bartonella bacilliformis and Papatsi virus

Ecology of old world Leismaniasis

Close contact of humans and their domestic animals can provide optimal conditions for sand flies and Leishmania transmission (stables provide good breeding ground for larvae)

In urban environments infection is mostly human to human

In rural areas Leishmaniasis can be a zoonosis

Infection in dogs is quite frequent in the Mediterranean

Ecology of new world leishmaniasis

In the new world most people get infected while working or hunting in the forest

Here wild animals including rodents, monkeys and sloths provide a reservoir for the parasite

A transmission pattern within a population of wild animals that result in occasional infection of humans is called sylvatic

A variety of species and species complexes causes disease in humans

Visceral LeishmaniasisLeishmania donovani

Leishmania infantum

Leishmania chagasi

Cutaneous LeishmaniasisLeishmania tropica

Leishmania major

Leishmania aethiopica

Mucocutaneous LeishmaniasisLeishmania brazieliensis

Leishmania mexicana

Leishmania amazoniensis

Three syndromes associated with Leishmania infection in humans

Visceral Leishmaniasis or Kala Azar

Systemic infection of reticulo-entdothelial cells (mostly macrophages) throughout multiple internal organs and the blood

Visceral Leishmaniasis

http://www.who.int/tdr/media/video/b-rolls.htm

Kala Azar - Visceral Leishmaniasis

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

the entire RES Weeks to months incubation period The lead symptom is abdominal

swelling due to hepato- and splenomegaly

High fever. Fever often oscillates with a peak every second day

Progressive drastic weight loss (kachexia)

Darkening of the skin Mortality of untreated disease 75-

95%

Post Kala-Azar Dermal Leishmaniasis

Sequel of visceral leishmaniasis which may manifest years after successful treatment and resolution of Kala Azar.

Dermal lesions may contain parasites in great numbers

Cutaneous Leishmaniasis

Infection remains restricted to the initial site of infection (the bite site)

Cutaneous Leishmaniasis

http://www.who.int/tdr/media/video/b-rolls.htm

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 starts months after infection Parasites are not found outside the lesion A granuloma is formed which finally leads

to healing leaving a depressed scar Nearly absolute resistance to reinfection Inoculation to vaccinate has long been

practiced in the middle east

Espundia or mucocutaenous leishmaniasis

Caused by L. braziliensis ~20% of infected patients develop ulcers of the

oral and nasal mucosa

Espundia or 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, and the soft palate

Death can occur through secondary bacterial infection

Diagnosis & Treatment

Gold standard of diagnosisis demonstration of parasites in the blood or lymph (kala azar) or in scrapings of the ulcer (cutaneous versions)

Treatment for all forms of Leishmaniasis is not satisfactory at the moment

Visceral Leishmaniasis (pentavalent antimonials) cutaneous (amphotheracin B, a liposome formulation is better tolerated but much more expensive)

The available drugs have significant side effects and resistance has emerged (pentavalent antimonials)

HIV coinfection further complicates treatment Better and more affordable drugs are urgently needed

A not-for-profit drug company? New hope for better treatment includes paramomycin an old off-

patent antibiotic which has shown promising activity in studies conducted by the Institute of One World Health

Paramomycin interferes with the growth of a number of bacteria and protozoa (the mode of action is not fully understood)

Intravenous injection has shown significant cure in kala azar patients in India (this was the first large scale clinical trial for this disease)

One World Health develops this drug for kala azar together with an indian drug maker and the indian health authorities

(http://www.oneworldhealth.org)

procyclics and metacyclics

Parasite interacts with the sand fly midgut

Promastigotes attach and detach to the midgut epithelium

Attachment helps them to remain in the insect gut when the blood meal is passed

However, later they need to detach to move to the pharynx and proboscis for infection

How is this accomplished at the molecular level?

Lipophosphoglycans play important roles in Leishmania pathogenesis

Lipophosphglycan or LPG is the dominant molecule on the surface of Leishmania

LPG is not a protein but a glycolipid

The molecule is anchored in the membrane by a lipid to which a long chain of highly hydrophilic sugar-phosphate repeats are attached

The structure of LPG changes over the life cycle to adapt to various functions

LPG is a pathogenesis factor in the mammalian host and important for the life cycle within the sandfly

Structural modification of LPG during the sand fly cycle

LPG is structurally modified during the developmental process of metacylogenesis (parasites decide to stop replicating and begin to pre-adjust to the mammalian host)

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

Sidechains with terminal galactose (squares) are downregulated and the remaining galactose residues are capped with another terminal sugar (arabinopyranose)

Are these chemical changes responsible for the pathogens attachment phenotype?

Only LPG from procyclics attaches to the midgut

Phosphoglycans were isolated from procyclics (those that attach) and metacyclics (those that detach and infect) and were labeled with a dye (resulting in fluorescence seen as ‘white’ in the lower panels)

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

Only procyclic phosphoglycan binds to the midgut

LPG binds to the microvilli of the sandfly midgut epithelium

OK, LPG appears to be the molecule on the parasite responsible forthe attachment of parasites to the sandfly midgut, but what kind ofhost molecule does it bind to?

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 galectin 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 epitheliumCell 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

New studies show that differences in galectins and LPG in different sandfly and parasite species might govern host specificity

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