Leishmaniasis
Silvia Reni B. Uliana
Leishmania
Family Trypanosomatidae
Genus Leishmania
Sub-genera Leishmania
Viannia
More than 30 species already
described
cutaneous leishmaniasis
visceral leishmaniasis
Geographical Distribution
cutaneous leishmaniasis
visceral leishmaniasis
14 million cases each year, distributed in 88 countries
(350 million people at risk).
http://www.who.int/emc-documents/surveillance/docs/whocdscsrisr2001.html
Visceral leishmaniasis / AIDS
Life cycle
Amastigote
Reservoir
(MAN)
Mammal
bite
bite
Insect
Assexual
Promastigote
Promastigotes
insect
Extracellular forms living in
the intestine of the mosquito
In the mosquito’s gut they go
through a differentiation process
called metacyclogenesis.
Amastigote
Intracellular forms
“Ideal” host cell: macrophage
Amastigotes are found inside macrophages
at various sites: skin, lymph nodes, spleen,
liver, bone marrow.
Mecanismo de infecção
O hospedeiro vertebrado
se infecta pela picada do
vetor.
Os amastigotas são liberados no
intestino do vetor e transformamse em promastigotas procíclicos.
Estes se reproduzem inicialmente
por divisão binária e
posteriormente se diferenciam em
promastigotas metacíclicos.
Os promastigotas invadem
macrófagos circulantes ou
teciduais e se reproduzem
como amastigotas.
O vetor ingere macrófagos
infectados com o sangue
ingerido
O macrófago parasitado
eventualmente se rompe,
liberando amastigotas que
passam a infectar outros
macrófagos, circulantes ou
teciduais.
Clinical syndromes
Cutaneous leishmaniasis – L. braziliensis,
L. amazonensis, L. guyanensis
L. major
Diffuse leishmaniasis – L. amazonensis
Mucocutaneous leishmaniasis – L. braziliensis
Visceral leishmaniasis – L. chagasi, L. donovani
New World species
Old World species
Cutaneous leishmaniasis
L. amazonensis
L. mexicana
L. guyanensis
L. braziliensis
Reservoir: wild
rodents, marsupials
(opossum), sloth,
anteater
Diffuse leishmaniasis
L. amazonensis
Reservoir: wild rodents
Mucocutaneous leishmaniasis
L. braziliensis
Reservoir: ?
Wild rodents
Visceral leishmaniasis
L. chagasi
L. donovani
L. infantum
Reservoir: fox, dog,
humans
Some tricks performed by Leishmania
S.C. Ilgoutz, M.J. McConville / International Journal for Parasitology 31 (2001) 899±908 906
Some tricks performed by Leishmania -LPG
form McConville MJ in Molecular Biology of Parasitic Protozoa, ed. DF Smith, M Parsons
multiplication inside the gut – adherence - procyclic
being “available” to take the next ride - metacyclic
Immune system
Complement
C3bi
Lysis complex
O’Garra and Arai (2000) Trends Cell Biol.
the innate defense mechanisms…
LPG and complement
lysis complex
“getting home…” - the macrophage!
Interaction with C3bi receptor via complement and gp63
inhibition of the respiratory burst
burst
“shall we make it cosy?…” – the
parasitophorous vacuole
Fusion with lisossomes: gp63 is a
protease (other cysteino-proteases)
“the macrophage army is ready…”
LPG and other GL induce the
production of TGF- and IL-10 AND…
inhibit the production of NO
DISEASE
Diagnosis
•
Biopsy – low sensitivity
which species ?
• Culture – slow
isoenzymes, monoclonal antibodies
PCR
ELISA – cross-reaction, sensitivity
Treatment
• pentavalent antimonials
• anphotericin B
TOXICITY
TIME
RESISTANCE
VECTOR – partially effective in
urban areas
CONTROL
RESERVOIR - wild
PATIENT - treatment
VACCINE
Clinical syndromes
Cutaneous leishmaniasis – L. braziliensis,
L. amazonensis, L. guyanensis
L. major
Diffuse leishmaniasis – L. amazonensis
Mucocutaneous leishmaniasis – L. braziliensis
Visceral leishmaniasis – L. chagasi, L. donovani
New World species
Old World species
What is the difference ?
Why do they sometimes behave “badly” ?
L. donovani
L. tarentolae
C. fasciculata
T. cruzi
T. brucei
Crithidia fasciculata
E. gracilis
Briones et al 1992, MBP
99 60
L. tarentolae 100
98 96
95
L. (L.) amazonensis
L. (L.) donovani L. (L.) chagasi
0.1
Orlando et al 2002 MIOC
Immune-deficiency associated
Primary infection
(with or without symptoms)
Persistence ?
AIDS
Transplantation
“reactivation”
(atypical clinical picture)
Where ? How ?
VIRULENCE
Ilgoutz and McConville (2001) Int J Parasitol
How do they do all this ?
Characterisation of the meta 1 gene in L. major
L S
•Single copy gene
•RNA expression upregulated in metacyclic
promastigotes
2.1 kb
rRNA
•Gene conserved in both
Old and New World
Leishmania species
L.maj
L.don
L.ama
10
20
30
40
MEMKNLLGKH KIVSVNGKPA PAGVTVEFKA SENSGSVYMH
....H..... .V.L...R.. .......... G......H..
....S.I..R RVL....R.. .......... .....TIQ..
L.maj
L.don
L.ama
50
60
70
80
AKVANIMNGP LKLANRKLSG ALVSTMMLGS DDLMNIENAL
.R.......Q .R.E...... .......... ..........
.N...F...Q ...G...... T......... ..........
L.maj
L.don
L.ama
90
100
110
SQGFMEGMTY TVKDGGKLTL QSKTHIIMLV PA*
.......... .......... .......K.. ..*
I....D.... ..H....... K.N..T.K.. ..*
Deduced meta 1 aminoacid sequence
• No significant aminoacid or nucleotide
identity with any known protein.
• No glycosilation sites.
• Hydrophilicity plot with no distinguishing
features.
• Predicted molecular mass 11.95 kDa.
What is the function of the meta 1 protein in
Leishmania ?
A- Lines with double replacement of the meta 1
gene were not viable
ESSENTIAL GENE
B- Overexpressing the meta 1 protein
• High level expression of
the L. major meta 1
protein was obtained in
L. amazonensis
promastigotes, by stable
maintenance of an
episome.
• The levels of expression
were high in both log
phase and stationary
phase promastigotes.
L.maj
L
kDa
18.4 _
14.3 _
6.2 _
S
A
LaHE
L
S
A
AI
M
C- Testing the recombinant lines for a
phenotype
• No change in growth curves
• No differences in in vitro infection of
macrophages
A
Lesion Index
Meta 1 overexpressor in vivo
BALB/c
107 promastigotes
20
WT
ARHyg
Hyg3’
HE
15
10
5
4
6
8
10
12
14
16
B
Lesion Index
Weeks
BALC/c
106 amastigotes
25
20
WT
HE
15
10
5
4
6
8
10
12
14
16
C
Lesion index
Weeks
6
C57Bl/6
106 promastigotes
5
WT
HE
4
3
2
1
6
12
24
Weeks
L. major Chromosome 16