Leishmania

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Leishmania
www.sanger.ac.uk/Info/Press/2007/070617.shtml
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First exam
Total number of points 70 (we used 68 as
total) Point average 52 First exam is 30 of
grade (we will use total points over all exams
(not letter grade) to calculate the final grade.
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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

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(No Transcript)
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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)

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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
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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

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

(detached)
(attached)
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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

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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/11
5/11/2303
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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
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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

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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)

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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 ...

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Leishmania infects and thrives in macrophages

• Leishmania just doesnt 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

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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 dont understand yet how that is
  done

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Leishmania distribution (all species)
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American soldiers contracted Leishmaniasis in
Iraq Afghanistan
A REGION INFLAMED Hundreds of U.S. Troops
Infected by Parasite Borne by Sand Flies, Army
Says By DONALD G. MCNEIL JR. Published December
6, 2003 Hundreds 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 Children Thursday,
February 14, 2008 By 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.
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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

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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

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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

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A variety of species and species complexes causes
disease in humans
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Three syndromes associated with Leishmania
infection in humans
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Visceral Leishmaniasis or Kala Azar

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

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

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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

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Cutaneous Leishmaniasis

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

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

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Espundia or mucocutaenous leishmaniasis

• Caused by L. braziliensis
• 20 of infected patients develop ulcers of the
  oral and nasal mucosa

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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

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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)
• The available drugs have significant side effects
  and resistance has emerged (pentavalent
  antimonials)
• HIV coinfection further complicates treatment
• New drugs are urgently needed
• One of the new hopes includes paramomycin an old
  antibiotic which has shown promising activity in
  clinical studies done by the Institute of One
  World Health (http//www.oneworldhealth.org)

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procyclics and metacyclics
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LPG and its interactions 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?

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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

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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 are
  downregulated and the remaining galactose
  residues are capped with another terminal sugar
  (arabinopyranose)
• Are these chemical changes responsible for the
  pathogens attachment phenotype?

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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 to the right)
• 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

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LPG binds to the microvilli of the sandfly midgut
epithelium
OK, LPG appears to be the molecule on the
parasite responsible for the attachment of
parasites to the sandfly midgut, but what kind
of host molecule does it bind to?
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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 119329-41
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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 119329-41
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Modification of LPG modulate interaction of the
parasite and midgut galectin
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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