Leishmania

1
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)

2
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
3
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

4
Leishmania infects and thrives in macrophages

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

5
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
6
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
7
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

8
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 ...

9
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

10
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

11
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

12
A variety of species and species complexes causes
disease in humans
13
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

14
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)

15
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

16
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

17
Lipophosphoglycans share structural features with
GPIs
18
Lipophosphoglycan

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

19
LPG shows structural variation among Leishmania
species
20
Biosynthesis of LPG
21
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

22
Is LPG a pathogenesis factor?Stan Falkows
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

23
Identification of LPG genes by genetic
complementation
24
Mutants in LPG biosynthesis as tools to study LPG
enzymology function
25
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

26
A number of Leishmania glycoconjugates share
structural features with LPG
27
Knock outs by gene targeting as a cleaner way to
obtain mutants
28
Lpg1 k.o. in L. major has an effect on virulence
http//www.pnas.org/content/97/16/9258.full
29
Lpg1 k.o. in L. major has an effect on virulence
http//www.pnas.org/content/97/16/9258.full
30
Lpg1 k.o. in L. major has an effect on virulence
http//www.pnas.org/content/97/16/9258.full
31
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

32
procyclics and metacyclics

• Promastigotes attach to and metacyclics detach
  from the midgut epithelium

33
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

34
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

35
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. 2561812-5.
36
Phosphoglycan binds to the microvilli of the
epithelium
Pimenta et al., Science. 2561812-5.
37
Phosphoglycan repeats inhibit attachment of
procyclics

• Opened midguts were incubated with radiolabeled
  procyclic promastigotes
• (A) 2-5 procyclic PG, 67 metacyclic PG (lipid
  removed)
• (B) 1, control, 2, pPG, 3 no bGal, 4 with bGal,
  5, second bGal, 6 ara
• (C) commercial saccharides 2 free Gal, 3-5 b
  and a digalactosides, 6 similar Man disaccharide

Pimenta et al., Science. 2561812-5.
38
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
39
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
40
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
41
Modification of LPG modulate interaction of the
parasite and midgut galectin
42
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